Test Kits for Research & Routine

Gastrointestinal Diseases

1. Colorectal Cancer: prevention and early detection 2. I nflammatory bowel diseases 3. Exocrine pancreatic function 4. Food Intolerance 5. Infectious diseases

Content 

1. C  olorectal Cancer: prevention and early detection................................3

2. I nflammatory bowel diseases..................................5

3. E  xocrine pancreatic function.................................23

4. Food intolerances............. 27

5. Infectious diseases........... 36

Appendix: Stool sample preparation systems................ 43

Albumin..........................................................................................................................................................................................6 a1-Antitrypsin / a1-Antitrypsin Clearance..................................................................................................................7 D-Arabinitol...............................................................................................................................................................................41 Calprotectin (PhiCal® Calprotectin).................................................................................................................................9 CRP (C-reactive Protein).....................................................................................................................................................12 Chymotrypsin..........................................................................................................................................................................24 b-Defensin 2..............................................................................................................................................................................13 Diamine Oxidase (DAO).....................................................................................................................................................34 EDN (EPX)....................................................................................................................................................................................28 Enterovirus.................................................................................................................................................................................39 Ferritin...........................................................................................................................................................................................14 Gliadin...........................................................................................................................................................................................29 Gliadorphin (Gliadomorphin).........................................................................................................................................31 Hemoglobin.................................................................................................................................................................................4 Hemoglobin-Haptoglobin-Complex............................................................................................................................4 Helicobacter pylori................................................................................................................................................................37 Histamin......................................................................................................................................................................................35 Lysozyme....................................................................................................................................................................................15 MutaGEL® Aldolase B .........................................................................................................................................................33 MutaGEL® HLA-DQ 2+8.....................................................................................................................................................30 MutaGEL® Laktase (AS)......................................................................................................................................................32 Norovirus....................................................................................................................................................................................38 Myeloperoxidase (MPO)....................................................................................................................................................16 Pancreatic Amylase...............................................................................................................................................................25 Pancreatic Lipase....................................................................................................................................................................26 PMN-Elastase............................................................................................................................................................................17 S100A8/A9 (MRP 8/14, Calprotectin).........................................................................................................................10 S100A12 (Calgranulin C, EN-RAGE).............................................................................................................................11 Salmonella sp...........................................................................................................................................................................40 Secretory IgA (sIgA)..............................................................................................................................................................18 TNFa ............................................................................................................................................................................................19 TNFa -Blocker Therapy Monitoring............................................................................................................................20 Transglutaminase Antibodies........................................................................................................................................29 Zonulin.........................................................................................................................................................................................22

1. Colorectal cancer: prevention and early detection Colorectal cancer includes cancerous growths in the colon, rectum and appendix. With more than 900,000 deaths worldwide per year, colorectal cancer is the fourth most common form of cancer in the United States and the third leading cause of cancerrelated death in the Western world. Most colon cancers arise sporadically but about 10% of afflicted patients have a familiar genetic disposition. During pathogenesis, adenomatous polyps in the colon arise and are usually benign at the beginning. Some polyps however develop into cancer over years. This time window offers the chance for prevention because colon cancer has a good healing prognosis - if diagnosed early. The risk for polyps increases significantly beyond age 50. Hence, most public health care services offer preventive programmes for people above 50 that include colonoscopy and screening tests for faecal occult blood. Immunological assays specifically detect human hemoglobin in stool and have demonstrated superior performance in the past years in comparison to guaiac-based chemical occult blood tests. Fig.: International colorectal cancer incidence rates by gender. Worldwide, there are 945,000 cases of colorectal cancer per year and 492,000 deaths (Parkin et al.,2005).

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1. Colorectal cancer: prevention and early detection

Hemoglobin Hemoglobin-Haptoglobin-Complex Higher sensitivities in comparison to guaiac based occult blood tests Detection of Hb-Hp complex increases detection rate of adenomas in the upper intestinal tract Highest sensitivity through combination of both parameters (Sieg A et al. 1999 Int J Colorectal Dis 14:267-271; Lüthgens K et al. 1988 Clin Lab 44:543-51)

New: both ELISAs easy to combine One sample collection tube and one dilution step for both parameters

In contrast to commercially available rapid tests based on guaiac dye for the determination of hemoglobin, this hemoglobin ELISA does not require previous adherance to a diet (no raw meat etc.) and recognises human hemoglobin in 100-fold lower concentrations. This avoids false-negative results. Due to the choice of antibodies, false-positive results are practically absent. Recent data show that when determining the hemoglobin-haptoglobincomplexes (Hb-Hp complexes), the clinical specificity and sensitivity can be increased in cases of polyps and carcinoma.

Indications

• • • •

Occult blood in stool Crohn’s disease, ulcerative colitis Suspicion of colon carcinoma Polyps in the colorectum

Reference Values Hemoglobin

Reference Values Hemoglobin-Haptoglobin-Complex

Stool: < 2 µg/ml

Stool: < 2 µg/ml

Hemoglobin (ELISA) Sample volume Matrix Detection limit Calibrators Incubation time Tests Cat. No.

Hemoglobin-Haptoglobin-Complex (ELISA) 100 µl Stool 0.447 µg/ml 0.21 - 50 µg/g 1 h; 1 h; 15 min 96 K 7816D

Sample volume Matrix Detection limit Calibrators Incubation time Tests Cat. No.

100 µl Stool 0.081 µg/ml 0.21 - 50 µg/g 1 h; 1 h; 15 min 96 K 7817D

1-Point-Calibration is possible for both assays (Cat. No. K 7836D, K 7837D)

References Hoepffner N et al. (2006) Aliment Pharmacol Ther 23:145-54 Schirrmacher S et al. (2003) Abstract P4.54 of EUREGIO CCLM, 08.-10.10.2003, Aachen Trojan J et al. (2002) Z Gastroenterol 40(11):921-24 Sieg A et al. (1999) Int J Colorectal Dis 14 : 267-27 Schmidt-Gayk H et al. (1994) Clin Lab 40:77-81

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2. Inflammatory bowel diseases Chronic diarrhea combined with gastrointestinal symptoms without infectious causes is indicative for a chronic, functional bowel disease. In irritable bowel syndrome (IBS), digestion is impaired but the gut is not pathologically altered. The treatment of IBS differs individually and includes motility-modulating agents or even anti-depression therapy. In contrast to IBS, inflammatory bowel diseases (IBD) like Morbus Crohn or Ulcerative Colitis are caused by inflammatory pathologies of the gut mucosa and pose a severe health threat. IBD therapy therefore relies on inhibition of inflammation. Since the symptoms for IBD and IBS overlap to a large degree, Immundiagnostik offers a broad portfolio of user-friendly ELISAs for the differential diagnosis of the diseases. Furthermore, we provide exclusive assays for monitoring and individual modulation of IBD-therapy which enable a comprehensive patient management. The gut mucosa is an important immunocompetent system which performs local and systemic defense and control functions. To protect the organism, the mucosa establishes a powerful mechanic and immunological barrier - a complex system of immune and epithelial cells as well as a bacterial shield, the physiological intestinal flora. Disruptions of the intestinal barrier, e.g. by mucosa irritations can increase the intestinal permeability and can lead to a "leaky gut syndrome", an overactivation of the immune system with elevated release of pro-inflammatory cytokines. As a result, food intolerances, susceptibility to infections or nutrient deficiencies can occur. A pro-inflammatory alteration of the intestinal mucosa has also been observed in IBD patients. The status of the intestinal barrier and of the intestinal immune system can be interpreted by an evaluation of marker proteins with the Immundiagnostik ELISA portofolio.

Indications • • • • • •

Exclusion of intestinal infections Differentiation of IBS and IBD Diagnosis of Morbus Crohn, Colitis ulcerosa Therapy monitoring of IBD patients Integrity of the intestinal barrier Analysis of the intestinal immune system

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2. Inflammatory bowel diseases

Albumin Sandwich ELISA technology with two polyclonal antibodies against human albumin Short incubation time (2.5 hours) High sensitivity Designed to be suitable for processing small amounts of specimen also

Changes in the albumin concentration in plasma, urine und stool are mainly caused by distribution defects, rather than meatbolistic defects. In the case of nutrition withdrawal, the concentration of albumin falls below the lower reference range after one week at the earliest. In the case of protein deficient nutrition, the extent of edemas slightly correlates with the albumin concentration. Higher losses of albumin, e.g. nephrotic syndrome, leads to an increased synthesis. Increased albumin as well as increased haemoglobin concentrations in the stool are not only found in cases of colorectal carcinoma, but also in patients with polyps and chronic inflammatory bowel diseases (Crohn’s disease, ulcerative colitis).

Indications

• • • •

Detection of bleeding in the lower gastrointestinal tract Identification of colorectal carcinomas Crohn’s disease, ulcerative colitis Examination of risk groups

Reference Values Albumin Plasma, Serum

35 – 55 g/l

Urine

5 – 16 mg/l

Stool

< 9,2 mg/l

Albumin (ELISA) Sample volume 100 µl Matrix Urine Stool (100 mg) Detection limit 12.5 µg/l Calibrators 12.5 - 800 µg/l Incubation time 1 h; 1 h; 15 min Tests 96 Cat. No. K 6330

References Trasch und Bloch (1993) Clin Lab 39: 479-484 John et al. (1994) Clin Lab 40:77-81 Berlet S, Armbruster FP, Aker I, Peters J, Schmidt-Gayk H (1986) J Clin Chem Biochem 24(10):771-772

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2. Inflammatory bowel diseases

a1-Antitrypsin

(Alpha-1-Proteinase-Inhibitor) The a1-antitrypsin acts as a primary inhibitor of elastase from polymorphnuclear neutrophilic granulocytes (PMN) and is released during inflammatory processes in order to reduce the proteolytic activity of the PMN elastase in the inflammation region. In addition, it inhibits, via complex formation, a series of serin proteinase such as blood-clotting proteinases, trypsin, chymotrypsin, etc. Thus, the a1-antitrypsin plays an important regulatory as well as antiinflammatory role. The a1-antitrypsin is a linear glycoprotein with a molecular weight of ca. 52 kDa (394 amino acid residues), a free cysteine residue and three carbohydrate side chains. It is predominantly synthesized in the liver but also by intestinal macrophages, monocytes and epithelial cells. Although a1-antitrypsin is the main serine proteinase inhibitor in human plasma, the proof of fecal a1-antitrypsin has become an important marker for intestinal protein loss and permeability as it is able to resist degradation in the gut due to its anti-proteolytic activity. It will, therefore, stay intact and it is possible to detect it in the feces using an immunoassay. Moreover, the measurement of fecal a1-antitrypsin concentration is used to evaluate and monitor chronic inflammatory intestinal diseases. In the clinical routine, the a1-antitrypsin-clearence (ratio of the a1-antitrypsin-ELISA-values of stool and serum samples) has been established along with the sole determination of the 24ha1-antitrypsin-secretion in stool. Thus, the group of J. S. Fordtran (Strygler et al. 1990) reports that the sole determination of the a1-antitrypsin-concentration in stool yielded false positive or false negative results in 21% of the patients compared to the a1-antitrypsin clearance measurements. In a comparative study with the radial immune diffusion (RID), routinely used in the clinical diagnostics, the a1-antitrypsin-ELISA developed by Immundiagnostik demonstrated significant advantages in the analysis of serum, stool and Caco-2-cell culture supernatants (Faust et al. 2001): • The a1-antitrypsin concentrations obtained by the ELISA were on average about 30 % higher than the corresponding values from the radial immuno- diffusion measurements. • Only the ELISA-system detected a1-antitrypsin in the cell culture superna- tants. The results clearly demonstrate that our a1-antitrypsin-ELISA-test is more sensitive than other routinely used methods and that it recognizes the hepatic as well as the enteral a1-antitrypsin form. This newly developed test represents a promising alternative to the use of current clinical routine methods. It is superior to the radial immune diffusion especially in cases with extremely high protein loss through the intestine. The combination of two specific antibodies eliminates, to a large extent, the possibility of false negative results guaranteeing reliable diagnoses. This newly developed test is a non-invasive, simple test for the detection of protein loss through the intestine.

Indications

• • • •

Enteric protein loss and intestinal permeability Morbus Crohn Necrotic enterocolitis Viral, bacterial or allergic inflammation

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Sandwich ELISA It recognizes the a1-Antitrypsin form in stool. Short incubation time (2 hours) Designed to be suitable for small series of specimen also

2. Inflammatory bowel diseases

Reference values a1-Antitrypsin Stool (100mg)

16.1 ± 10.7 mg/dl

Serum

90 - 180 mg/dl



a1-Antitrypsin (ELISA) Sample volume Matrix Detection limit Calibrators Incubation time Tests Cat. No.

100 mg Stool 1.5 µg/l 3.3 – 90 µg/l 1 h; 1 h ; 15 min 96 K 6750

a1-Antitrypsin Clearance (ELISA) Sample volume Matrix Calibrators Incubation time Tests Cat. No.

ELISA

100 µl Serum, Plasma, Stool (100 mg) 3.3 – 90 µg/l 1 h; 1 h ; 15 min 96 K 6752

RID

mg/dl alpha1-PI

1000

500

0 Stuhl

Serum

Figure: Comparison ELISA versus RID for stool and serum samples of alpha1-proteinase inhibitor (Faust D et al. 2001, Z Gastroenterol 39: 769-74)

References Faust D et al. (2002) Clin Exp Immunol. 128(2):279-84. Faust et al. (2001) Z Gastroenterol 39 :769-774 Arndt et al. (1992) The Lancet Vol 340 OCT 24 Strygler B et al. (1990) Gastroenterology 99(5):1380-7. Karbach et al. (1989) Gastroenterol 27:362

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2. Infl ammatory bowel diseases

PhiCal® Calprotectin Calprotectin (MRP 8/14) is a heterodimer of two calcium-binding proteins present in the cytoplasm of neutrophils and expressed by the membranes of monocytes. It constitutes nearly 60% of the soluble cytosol proteins in neutrophils and plays a central role in neutrophil defense. Upon neutrophil activation or endothelial adhesion of monocytes, calprotectin is released and may be detected in serum, body fluids or stool as a potentially useful clinical inflammatory marker. The acute phase protein shows a high stability in faeces (stable for one week at room temperature) and has been established as a faecal marker of inflammatory bowel diseases (IBD). It allows a reliable differentiation between organic intestinal diseases (e.g. chronic inflammatory diseases, infectious diseases, polyps, colon cancer) and functional intestinal diseases (e.g. irritable bowel syndrome). Calprotectin is ideal for monitoring disease activity (e.g. of M. Crohn or after polyp resection) and early detection of relapse. It shows high sensitivity in the detection of colorectal carcinoma (CC) and polyps (CRC: sensitivity 100%, polyps: sensitivity 88%). Due to the highly specific monoclonalantibodies that the immunoassay is based on, the test detects only human calprotectin. The linear part of the calobration curve was optimised for easier differentiation between negative values, poorly increased values and high calprotectin values. This differentiation is important for excluding functional intestinal diseases (e.g. irritable bowel syndrome) and for the diagnosis and monitoring of organic intestinal diseases. The better performance of the monoclonal test system in comparison to the polyclonal system has been shown in a study about prospective validation of faecal leucocyte markers in the differential diagnosis of chronic diarrhoea. There our monoclonal test system has proven to have a better performance than the polyclonal system which was used. Naumann et al. (2004) also found that of the investigated stool parameters (Calprotectin, MPO, Lactoferrin) only calprotectin was qualified for discriminating between an organic diarrhoea and a functional diarrhoea.

Sample volume Matrix Standards Incubation time Test principle Tests Cat. No.

100 mg Stool 13 - 840 ng/ml 1 h; 1 h; 10-20 min ELISA 96 Determinations K 6937

MRP

= Macrophage inhibitory factorrelated protein

MRP8/14

= Heterodimer composed of light (MRP8) and heavy (MRP 14) chains (8 and 14 kDa)

Reference Values Calprotectin Stool: Grey area:

< 15 µg/g 15 - 50 µg/g

Cut off :

50 µg/g

High stability in faeces

• Chronic diarrhoea • Morbus Crohn, Colitis ulcerosa • Polyps (not sold in the USA)

 Calprotectin= MRP 8/14 = Calgranulin A/B = S100A8/A9

Calprotectin in faeces:

Determination of faecal Calprotectin

PhiCal® Calprotectin (Stool)

PhiCal® is a German trademark of Immundiagnostik AG, Bensheim

Ideal for monitoring disease activity (e.g. of M. Crohn or after polyp resection) and early detection of relapse For the discrimination between an organic and a functional diarrhoe High sensitivity in detection of colorectal carcinoma (CC) and polyps also available as 1-pointcalibration test (Cat. No. K 6947)

NEW! PhiCal® Calprotectin (Stool) Sample volume Matrix Incubation time Test principle Tests Cat. No.

(not sold in the USA) 100 mg Stool 2 x 30 min ELISA 96 Determinations K 6927

30min nly 2 x o s e im t bation gate  Incu se conju u o t y  Read also available as 1-pointcalibration test (Cat. No. K 6967)

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2. Infl ammatory bowel diseases

PhiCal® Calprotectin (serum, plasma, urine) * Sample volume Matrix Standards Incubation time Test principle Tests Cat. No.

100 µl Serum, plasma, urine 3.9 - 250 ng/ml 3 x 1 h; 10-20 min ELISA 96 Determinations K 6935

* Not sold in the USA

S100A8/A9 in biological liquids Only for research purposes: S100A8/A9 in biological liquids Suitable for mouse/rat and other animal samples Not suitable for human samples

The heterocomplex Calprotectin consists of the two proteins, S100A8 (calgranulin A) and S100A9 (calgranulin B), also designated as MRP8 and MRP14, respectively. Expression of S100A8 and S100A9 in epithelial tissues was first described in context with squamous epithelia and with murine and human wound repair. More recently, an association of S100 protein expression with adenocarcinomas in humans has emerged.

Various conditions have shown significant correlation of S100A8/A9 (or S100A8, S100A9) levels with disease activity: • Concentrations of S100A8/A9 in serum, and particularly in synovial fluid, correlate strongly with disease activity in rheumatoid arthritis. • Plasma S100A8/A9 levels are very early, specific and sensitive prediction markers for acute rejection in kidney allograft transplantation. • Serum S100A8/A9 concentration is a prognostic marker of recurrent infection and of poor survival in alcoholic liver cirrhosis. • S100A8/A9 is useful for evaluating the extent of periodontal inflammation. • In cerebral malaria, S100A8/A9 expression correlates with microglial activation in brain. • S100A8/A9 is present in urinary stones and in dental calculus. • S100A9 in serum may serve as a useful marker for discrimination between prostate cancer and benign prostatic hyperplasia (BPH).

For Research only; not suitable for human samples Worldwide unique!

S100A8/A9 (MRP 8/14, Calprotectin, Mouse/Rat) Sample volume 100 µl Matrix Serum, plasma, urine, cell culture supernatant, tissue extract Stool (100 mg) Standards 0 - 15.6 ng/ml Incubation time 4 x 1 h, 20 min Test principle ELISA Tests 96

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2. Inflammatory bowel diseases

S100A12 (Calgranulin C, EN-RAGE) Sample volume 100 mg Matrix Stool Standards 0.22-54 ng/ml Incubation time 2x1h Test principle ELISA Tests 96 Cat. No. K 6938

References Loitsch SM et al. (2010). Gastroenterol 138:5, Suppl 1 , S-528 Barassi A et al. (2009) Abstract D-52 presented at AACC, July 21-23, Chicago; published in Clin Chem Vol 55(6), Supplement, A167 Langhorst J et al. (2009) Am J Gastroenterol 104(2):404-10 Langhorst J et al. (2008) Am J Gastroenterol 103(1):162-9 Shastri YM et al. (2008) Am J Med 121(12):1099-106 Pezzilli R et al. (2007) J Gastroenterol 42(9):754-60 Schröder O et al. (2007) Aliment Pharmacol Ther Pezzilli R et al. (2007) Dig Dis Sci Apr 28; [Epub ahead of print] Schröder O et al. (2006) J Gastroenterol (4): Abstract S1363 at AGA Meeting, May 20-25,2006, Los Angeles Shastri YM et al. (2006) J Gastroenterol (4): Abstract S1314 at AGA Meeting, May 20-25,2006, Los Angeles Shastri YM et al. (2006) Poster presented at Conference of Indian Society of Gastroenterology, November 7-12, 2006, Mumbai Yagmur E et al. (2006) Dtsch Med Wochenschr 131(36):1930-34 Hermani A et al. (2005) Clin Cancer Res 11(14):5146-52 Langhorst J et al. (2005) Inflamm Bowel Dis 11:1085-1091 Naumann M et al. (2005) Abstract (P519) Z Gastroenterol 43: 699-1016 Yagmur E et al. (2005) Abstract A202 of CCLM Oct 6-8, 2005, Jena Naumann M et al. (2004) Z Gastroenterol 42: 785-944 Schirrmacher et al. (2004) 59. Jahrestagung der DGVS, 1.-4. September 2004, Leipzig: P012, P013 Striz I, Trebichavsky IL (2004) Physiol Res. 53: 245-253 (Review) Tibble JA et al. (2001) Gut 49:402-408 Tibble JA et al. (1999) Gut 44:35 Gilbert JA et al. (1996) Scand J Gastroenterol 31:1001-1005

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2. Inflammatory bowel diseases

CRP - highly sensitive C-reactive protein (CRP) is an acute phase protein which is synthesized in the liver and released into the circulation. The CRP concentration in blood rises during inflammatory (infectious and non-infectious) diseases. CRP is considered a non-specific parameter which is useful for the evaluation of the severity of an inflammation. Elevated CRP levels always have to be clarified, even without clinical symptoms. Generally, the CRP concentration in plasma mirrors a disease activity with a lag of 1224 hours. CRP values close to the normal range (highly sensitive CRP or hsCRP) are an important risk parameter for cardiovascular diseases (Ridker et al. 1997). In addition, CRP is utilized as a marker for intestinal inflammation. The determination in serum is an additional tool in primary diagnosis to border irritable bowel syndrome. Furthermore, CRP levels are analysed as part of IBD therapy monitoring (Langhorst et al., 2008; Vermeire et al., 2006). During remission, persistent elevated CRP concentrations indicate a higher relapse risk in IBD patients. The combination of CRP with other laboratory parameters such as calprotectin or PMN-elastase strengthen the diagnostic value in the evalutation of an intestinal inflammation.

Indications Reference Values CRP Serum / Plasma:

< 0.068 - 8.2 mg/l

Stool:

< 56 ng/ml

• Intestinal inflammations • Differential diagnosis of chronic inflammatory bowel diseases • Therapy monitoring of chronic inflammatory bowel diseases

Umbilical cordblood: < 0.6 mg/l Urine:

< 6 ng/l CRP highly sensitive (ELISA) Sample volume 100 µl Matrix Serum, Plasma, Urine Stool (100 mg) Detection limit 1.9 ng/ml Calibrators 1.9 - 150 ng/ml Incubation time 1 h; 1 h; 15 min Tests 96 Cat. No. K 9710s

also available as 1-point-calibration test (Cat. No. K 9720s)

CRP Mouse* (ELISA) Sample volume Matrix Calibrators Incubation time Tests Cat. No.

CRP Rat* (ELISA) 15 µl Serum, Plasma 0.39 – 25 ng/ml 10 min; 10 min; 5 min 96 KA41CRPM90E

Sample volume Matrix Calibrators Incubation time Tests Cat. No.

* For Research use only

5 µl Serum, Plasma 6.25 – 100 ng/ml 10 min; 10 min; 5 min 96 KA41CRPR25E * For Research Use only

References Vermeire S et al. (2007) Gut 55:426-431 Langhorst J et al. (2008) Am J Gastroenterol 103:162–169 Henriksen M et al. (2008) Gut 336:973-979 Yaturu S et al. (2006) Cytokine 34(3-4):219-23. Epub 2006 Jul 5 Höffler D, Shah P (1997) C-reaktives Protein - die diagnostische Reichweite. Thieme Verlag Ridker P et al. (1997) New Engl J Med 336:973-979

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2. Inflammatory bowel diseases

b-Defensin 2 The b-defensins are an integral part of the congenital immune system and contribute with their antimicrobial effect to the barrier function of the intestinal epithelial cells. Defensins exert a variable degree of antimicrobial activity against bacteria, fungi, and some enveloped viruses. Vertebrate defensins are classified as a- or b-defensins, based on their pattern of disulfide bridges. Nine human defensins of epithelial origin have been found, three of them being b-defensins (HBD-1, -2 and -3). The expression of b-Defensins are induced by the pro-inflammatory cytokines and also through microorganisms (e.g. E. coli, H. pylori or P. aeruginosa). A reduced b-defensin 2 expression can, for example, be observed in the intestinal mucous of patients with Crohn’s disease. The defense system of the mucous membrane is therefore restricted and allows an increased invasion of bacteria, which could possibly lead to a typical infection in Crohn’s disease patients. Whether the reduced b-defensin-2 expression could even play a role in the development of Crohn’s disease is currently being researched, as is the possibility that it is the probiotic bacterium which produces b-defensin.

Reduced b-defensin expression with Crohn’s disease (HBD-2) Increased b-defensin expression with Colitis ulcerosa (HBD-2)

Indications • Inflammatory bowel diseases (IBD) • Research of intestinal barrier function b-Defensin 2 (ELISA) Sample volume Matrix Standards Incubation time Tests Cat. No.

Also available for your research:

100 mg Stool 0.1 – 3 ng/ml 3 x 1 h, 15 min 96 K 6500

→ b-Defensin 5 ELISA and → b-Defensin 6 ELISA

References Kapel N et al.(2009) J Pediatr Gastroenterol Nutr 48(1):117-20 Langhorst J et al. (2009) Am J Gastroenterol 104(2):404-10 Soto E et al. (2007) J Matern Fetal Neonatal Med 20(1):15-22 Langhorst J et al. (2007) Gut Apr 11, 2007 [Epub ahead of print] Langghorst J et al. (2006) Suppl 2 to J Gastroenterol (4):Abstract A205, S1340 vorgestellt bei 107th Annual Meeting of AGA, May 20-25, 2006, Los Angeles Wehkamp J et al. (2003) J Clin Pathol 56: 352-357 Schmid M et al. (2004) Z Gastro 42 : 333-338 Harder J et al. (2001) J Biol Chem 276 : 5705-5713

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2. Inflammatory bowel diseases

Ferritin Iron deficit and the resulting anaemia are common disorders which can be caused e.g. by malnutrition, malabsorption or bleedings. The determination of iron and the binding capacity of total iron is therefore clinically relevant. Molecules like hemoglobin, hemosiderin, myoglobin and cytochromes act as iron reservoirs in the body. In most tissues however, ferritin is the prevalent iron storage. Human ferritin has a molecular weight of 450 kDa and consists of a protein shell covering an iron core - each ferritin molecule can carry up to 4000 Fe2+ atoms. Almost 20% of total iron in healthy individuals is stored in this way. The function of ferritin is the oxidation of Fe2+, the transport of Fe3+ into the core and its mobilisation. High ferritin concentrations can be detected mainly in liver, spleen and bone marrow. The direct determination of iron in blood is not applicable for the detection of an iron deficit since these values are too inconsistent. Although the majority of ferritin molecules is intracellular, the ferritin concentration in serum is a meaningful parameter for the assessment of the total iron storage in the organism. The analysis of the ferritin level is meanwhile routine in laboratory diagnostics and serves as a significant parameter in the diagnosis of anaemia and hemochromatosis. High ferritin concentrations indicate an iron overload which might be caused by hemochromatosis. Furthermore, ferritin status is used to monitor the iron supply in pregnant women, blood donors and dialysis patients. Ferritin serum concentrations also serve as an additional diagnostic parameter in inflammations, chronic liver diseases and tumors.

Indications • • •

Diagnosis of iron deficit / anaemia Diagnosis and therapy monitoring of hemochromatosis Iron status in pregnant women, blood donors and dialysis patients

For Research Use only Ferritin (ELISA) Sample volume Matrix Calibrators Incubation time Tests Cat.No.

20 µl Serum 15 – 1000 ng/ml 45 min; 20 min 96 determinations KD1872

References White D et al. (1986) Am. J. Clin. Path. 72: 346 Valberg L (1980) CMAJ. 122: 1240 Forman D and Parker S (1980) Ann. Clin. Lab. Sci. 10: 345 Hazard JT et al. (1977) J. Blood. 49: 139 Smimes MA (1974) Blood. 43:581 Clinical Guide to Laboratory Tests. Ed. Nw. Tietz, 3rd ED., W.B. Saunders Company, Philadelphia, PA 19106, 1995

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2. Inflammatory bowel diseases

Lysozyme Lysozyme (muramidase) is a protein with a molecular weight of approx. 15 kDa and belongs to the group of alkaline glycosidases. Lysozyme is synthesised by granulocytes, monocytes and macrophages. The main source of faecal lysozyme is the intestinal granulocytes. Lysozyme can be detected in all cells of the inflammatory infiltrate during an acute flare of Crohn‘s disease. To some extent, lysozyme is also secreted actively by mononuclear cells into the bowel lumen.

Designed to be suitable for processing small amounts of specimen also Sandwich ELISA technology with a highly specific pair of antibodies

Indications • Diagnosis and monitoring of Crohn’s disease, Boech’s disease (in serum) • Bacterial, viral, allergenic or autoimmune related bowel inflammations of allergenic or auto immune origin

Reference values Lysozyme Stool

< 600 ng/ml

Lysozyme (ELISA) Sample volume Matrix Detection limit Calibrators Incubation time Tests Cat. No.

100 µl Stool (100 mg), Serum 0.5 ng/ml 1.1 – 30 ng/ml 1 h; 1 h; 15 min 96 K 6900

also available as 1-point-calibration test (Cat. No. K 6901)

References Langhorst J et al. (2005) Inflamm Bowel Dis 11(12):1085-91 Hemrika et al. (1989) Neth J Med 34:174 Arndt et al. (1993) Crohn Clin Lab 11:867-876 Stein J. 3. (1996) Post-Graduiertenkurs der DGVS Braun OH (1969) Dtsch Med Wochenschr 28, 11 Juli 1969

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2. Inflammatory bowel diseases

Myeloperoxidase (MPO) Designed to be suitable for processing small amounts of specimen also Determination of MPO in different matrices

The granules of neutrophils (approx. 70 % of the white blood cells) contain a large number of different enzymes. Myeloperoxidase (MPO) catalyses the oxidation of substances via H2O2. The MPO H2O2 system has a toxic effect on many microorganisms such as bacteria, fungi, viruses and mycoplasma . The efficiency of the bacteriocide myeloperoxidase H2O2 system is increased by PMN-Elastase. THE MPO determination in the stool reflects the inflammatory activity of Crohn’s disease or ulcerative colitis.

Indications • Marker for inflammatory activities in the gastrointestinal tract • Renal transplant rejection Reference Values Myeloperoxidase (MPO) Stool

< 2000 ng/ml

Serum

mean value 340 ng/ml (SD 176.7)

EDTA-Plasma

mean value 98.31ng/ml (SD 62.9)

MPO (ELISA) Sample volume Matrix Calibrators Incubation time Tests Cat. No.

100 µl Stool (100mg), Urine 3.6 – 100 ng/ml 1 h; 1 h; 1 h; 10-20 min 96 K 6630

References Silberer H et al. (2005) Clin Lab 51(3-4):117-26 Tomohisa et al. (1998) Kurume Medical Journal 45:69-73 Kazunori et al. (1996) Am J Gastro, Vol 91(5)

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2. Inflammatory bowel diseases

PMN-Elastase PMN-Elastase from human polymorphonuclear granulocytes is a glycoprotein of 30 kDa and belongs to the group of serine proteases. Active PMN-Elastase is released from azurophil granula of neutrophil granulocytes after irritation or disintegration. The determination of the PMN-Elastase in stool is used to record inflammatory reactions where neutrophil granulocytes are involved. Especially in Crohn’s disease the inflammatory processes go hand in hand with an increased phagocytic activity and the biological decay of these cells and thus leads to an increased release of PMN-Elastase and other lysosomal enzymes.

Short incubation time (3 h) Designed to be suitable for processing small amounts of specimen also

Indications

• • • •

Diagnosis of Crohn’s disease Chronic arthropathy Pancreatitis Bacterial infection, sepsis

Reference Values PMN-Elastase Stool Serum

62 ng/ml 19 - 78 ng/ml

PMN-Elastase (ELISA) Sample volume Matrix Detection limit Calibrators Incubation time Tests Cat. No.

100 µl Stool, Serum, Plasma, Seminal plasma, Synovia 0.12 ng/ml 0.12 - 3.3 ng/ml 3x1 h; 10-20 min 96 K 6840

also available as 1-point-calibration test (Cat. No. K 6830)

References Eggert-Kruse W et al. (2008) Int J Androl, Jan 10. [Epub ahead of print] Langhorst J et al. (2008) Am J Gastroenterol 103(1):162-9 Langhorst J et al. (2007) Z Gastroenterol 45: P261 Langhorst J et al. (2005) Inflamm Bowel Dis 11(12):1085-91 Silberer H et al. (2005) Clin Lab 51(3-4):117-26 Heinichen et al. (1995) Clin Lab 41:539-545 Oremek et al. (1995) MTA 10:273-278

17

2. Inflammatory bowel diseases

Secretory IgA (sIgA) Reference Values sIgA (Age > 16 years) Stool Saliva

510 - 2040 µg/ml 102 - 471µg/ml

Secretory IgA consists of two IgA monomers, which are connected to each other by a J-chain and contain a secretory component. They are produced in plasma cells located in the lamina propria of the mucous membranes and are found in body secretions, such as saliva, tears, nasal mucus, tracheobronchial mucus, gastrointestinal secretions, breast milk and colostrum. The formation of secretory IgA occurs independently of the serum IgA synthesis. Therefore, a lack of serum IgA does not necessarily mean a lack of secretory IgA. Neonates and infants are supplied with sIgA through breast milk and are therefore passively immunized against gastrointestinal infections. Conclusions concerning the endogenic defence of the intestinal mucosa can be drawn from the concentration of the sIgA in stool. A deficiency of sIgA points to a diminished activity of the mucosa immune system, whereas increased sIgA values indicate increased activity and a local inflammation of the intestinal mucosa.

Indications • Proof of an imbalanced immunological barrier of the intestinal mucosa • Autoimmune diseases also available as 1-point-calibration test (Cat. No. K 8880)

sIgA (ELISA) Sample volume Matrix Detection limit Calibrators Incubation time Test principle Tests Cat. No.

100 µl Stool (100 mg), body liquids 13.4 ng/ml 22.2 – 600 ng/ml 1 h; 1 h; 10-20 min ELISA 96 K 8870

For Research only:

For Research only:

sIgA-1 (ELISA) Sample volume Matrix Calibrators Incubation time Tests Cat. No.

sIgA-2 (ELISA) 100 mg Stool 3,3-100 U 1 h; 1 h; 15 min 96 K 6863

Sample volume Matrix Calibrators Incubation time Tests Cat. No

References Mohan R et al. (2008) Pediatr Res 64(4):418-22. Michalsen A et al. (2005) BMC Complement Altern Med 5:22 Hofman LF et al.(2002) Clin Chem Vol 48(6) Suppl A169 Noel N et al. (2001) Abstract P 155 Clin Chem Vol 47 (6), Suppl A47 Schütz B (1998) Biol Med 27:31-36 Brandtzaeg P (1981) Clin Exp Immunol 44:221-231

18

100 mg Stool 3,3-100 U 1 h; 1 h; 15 min 96 K 6864

2. Inflammatory bowel diseases

TNFa Tumour Necrosis Factor alpha (TNFa) belongs to the pro-inflammatory cytokines that encourage and uphold infection reactions. Cytokines, produced by macrophages and t-cells, play a central role in both acute and chronic infections. The TNFa concentration is raised in the affected joints in many rheumatic diseases (rheumatoid arthritis, chronic poly-arthritis, ankylosing spondylitis i.e. M. Bechterew disease) and plays a significant role in the joint destruction and in the courses of other diseases. Even in Crohn’s disease, an overproduction of TNFa can be observed that obviously affects the course of the disease. In the 1990’s, pharmaceutical companies developed bio-technologically produced medications that aimed at hindering TNFa (“TNFa blockers”) and therewith produced a positive effect on the various disease symptoms (Feldmann et al. 2001). TNFa (ELISA) Sample volume Matrix Calibrators Incubation Tests Cat. No.

100 ml Serum, Stool (100 mg), Cell Culture Supernatant, Plasma 16-500 pg/ml 2h, 1h, 1h, 10-20 min 96 K 9610

References Feldmann M et al. (2001) Annu Rev Immunol 19:163-196

19

2. Inflammatory bowel diseases

TNFa-Blocker Therapy Monitoring 1. Drug-level determination With our ELISA test, anti-TNFatherapeutic antibodies can be detected

As an orientation: To ensure clinical effectiveness of the TNFa inhibitor, the serum level should be at/above 12 mg/ ml in week 4 and at/above 5 mg/ml in week 8.

Tumor Necrosis Factor alpha (TNFa) belongs to the pro-inflammatory cytokines that establish and sustain inflammation reactions. Cytokines, produced by macrophages and T-cells, play a central role in both acute and chronic inflammations. The TNFa concentration is elevated in the affected joints in many rheumatic diseases (rheumatoid arthritis, chronic poly-arthritis, ankylosing spondylitis i.e. M. Bechterew disease, psoriasis) and plays a significant role in joint destruction as well as in other manifestations of the diseases. Even in Crohn’s disease, an overproduction of TNFa has been observed, obviously affecting the activity of the disease. In the 1990’s, pharmaceutical companies developed biotechnologically produced drugs that are aimed to neutralize TNFa (“TNFa blockers”) and expected to have a positive effect on the various symptoms of the diseases. In 1998, the first TNFa blockers were approved for use in the therapy of rheumatoid arthritis. Since then other TNFa blockers have been marketed: • Infliximab (Remicade®), • Adalimumab (Humira®). The two TNFa blockers are approved for treatment of rheumatoid arthritis as well as ankylosing spondylitis and psoriasis. Although the TNFa blockers differ in respect to their chemical structures and mechanisms of action, the pharmacological effects are the same for both substances. They are comparable in their effectiveness in facilitating improvement in clinical symptoms of rheumatoid arthritis, but in Crohn’s disease, Infliximab has proved to be the most effective one. The therapeutic effect of the anti TNFa antibodies on chronic inflammations, i.e. Crohn’s disease or rheumatoid arthritis, depends on the serum concentration of the corresponding pharmaceutical. In many patients, the treatment is of limited value, because of a fast degradation of the pharmaceutical or generation of antibodies against it. For a better control of the therapy, drug level monitoring is necessary. Our ELISA test can be used for monitoring anti-TNFa-antibodies (e.g. Remicade®, Humira®) and provides a basis for possible preventive strategies.

Indication: • Continuous TNFa Blocker therapy monitoring

TNFa-Blocker-Monitoring (drug-level, e.g. Remicade®) Sample volume Matrix Incubation Test principle Tests Cat. No.

TNFa-Blocker-Monitoring (drug-level, e.g. Humira®)

100 ml Serum 1h, 1h, 10-20min ELISA 96 K 9655

Sample volume Matrix Incubation Test principle Tests Cat. No.

20

100 ml Serum 4h, 1h, 10-20min ELISA 96 K 9657

2. Inflammatory bowel diseases

2. ADA (anti-drug-antibodies) determination Problems in the therapy with TNFa antibodies occur when the human organism develops antibodies against the therapeutical antibody i.e. against the TNFa-blocker (among others). In a cohort study 61% of M. Crohn patients treated with Infliximab reacted with the development of antibodies against Infliximab (Baert et al. 2003). The presence of antibodies in 7-19% of patients could be associated with infusion reactions. Allergic reactions, and also therapy failure, could be attributed to the development of such antibodies. Through the parallel administration of immuno-suppressants (mostly Methotrexat) a reduction of antibody development could, at least, be achieved (Colombel et al. 2004).

Our ELISAs enable the detection of human antibody against therapeutical antibodies / TNF receptors Bender et al. (2006) found out that adalimumab is immunogenic and induces antibodies in a high rate in adalimumab-treated patients

Our ELISAs enable the detection of human antibodies against: • Adalimumab (fully humanistic antibody to TNFa, e.g. Humira®) • Infliximab (chimeric, monoclonal antibody to TNFa, e.g. Remicade®) • Etanercept (genetically produced variant of TNF receptors, e.g. Enbrel®)

Indications: Therapy monitoring for  therapy with antibodies against TNFa or  therapy with TNFa-receptors TNFa-Blocker-ADA (anti-drug-antibodies, e.g. Remicade®) Sample volume Matrix Incubation Test principle Tests Cat. No.

TNFa-Blocker-ADA (anti-drug-antibodies, e.g. Humira®)

5 ml Serum o.N., 1h, 5-10min ELISA 96 K 9650

Sample volume Matrix Incubation Test principle Tests Cat. No.

TNFa-Blocker-ADA (anti-drug-antibodies, e.g. Enbrel®) Sample volume Matrix Incubation Test principle Tests Cat. No.

5 ml Serum o.N., 1h, 5-10min ELISA 96 K 9653

References Seow et al. (2009) Gut, publ. online, doi:10.1136/gut.2009.183095 Bendtzen et al. (2009) Scand J Gastroenterol 44:774-781 Ainsworth et al. (2008) Am J Gastroenterol 103:944-948 Radstake et al. (2008) Ann Rheum Dis, publ. online, doi:10.1136/ard.2008.092833 Baert F et al. (2007) Acta Gastroenterol Belg. Apr-Jun;70(2):163-70 Bender N et al. (2006) Rheumatol Int, publ. online, doi:10.1007/s00296-006-0183-7 Baert F et al. (2003) N Engl J Med 348 : 601-608 Bender N et al. (2006) Rheumatol Int 2006 Sep 28 [Epub ahead of print] Colombel J et al. (2004) Gastroenterology 126: 19-31 Cohen R D et al. (2000) Am J Gastroenterol 95 : 3469-3477

21

5 ml Serum o.N., 1h, 5-10min ELISA 96 K 9652

2. Inflammatory bowel diseases

Zonulin ELISA for the detection of tight junctions now available for your research

Zonulin is a novel human protein analogue to the Vibrio cholerae derived Zonula occludens toxin, which participates in tight junctions between cells of the wall of the digestive tract. Zonulin binds to a specific receptor on the surface of intestinal epithelia and triggers a cascade of biochemical events which induces tight junction disassembly and a subsequent permeability increase of the intestinal epithelia, allowing some substances to pass through and activate immune reactions. A. Fasano and his co-workers found out that the zonulin-zonulin-receptor-system is more activated in celiac disease and type 1 diabetes mellitus patients. Patients with active celiac disease showed higher levels of zonulin and anti-zonulin antibodies compared to non-celiac patients and patients in remission, who were eating a gluten-free diet. In addition, it was reported that many people who suffer from celiac disease also suffer from other autoimmune disorders. It is suggested that increased levels of zonulin are a contributing factor to the development of celiac disease and other autoimmune disorders such as insulin dependent diabetes, multiple sclerosis, and rheumatoid arthritis. Zonulin*

(ELISA)

Sample volume Matrix Standards Incubation time Cat. No.

50 µl Serum, EDTA-Plasma Stool (100 mg) 0.97–1000 ng/ml 1.5 h, 1.5 h, 1.5 h, 10 min K 5600 * For Research only

References Wang W et al. (2000) J Cell Sci 113 Pt 24: 4435-40. Fasano A (2001) Gut 49: 159-62. Fasano A et al.(2000) Lancet 355(9214): 1518-19. Thomas KE et al. (2006) J Immunol 176: 2512-21. Freemark M et al. (2003) Diabetes Care 26: 1932-39. Lazzarotto F et al. (2003) Diabetes Care 26: 248-49. Watts T et al. (2005) Proc Nat Acad Sci USA 102(8): 2916-21. Sapone A et al. (2006) Diabetes 55: 1443-9. De Magistris MT (2006) 24 Suppl 2: S2-60-1 Hilbig H et al. (2008) Poster presented at the CIMT Meeting, 15.-16.05.2008, Mainz, Germany Wex T et al. (2009) Peptides 30(6):1082-7

22

3. Exocrine pancreatic function The pancreas is an important exocrine gland of the digestive system. It secretes enzymes and their precursors for the cleavage of proteins, sugars, nucleic and fatty acids. This enzymatic "cocktail" enables the decomposition of food in the small intestine. Next to this basic exocrine function, the pancreas acts as an endocrine gland by releasing hormones that regulate the blood sugar level into the circulation, e.g. insulin and glucagon. Immundiagnostik offers assays for the analysis of the exocrine pancreatic functions which can be used for diagnosis and therapy monitoring of diseases, such as exocrine pancreas insufficiency or chronic pancreatitis.

23

3. Exocrine pankcreas function

Chymotrypsin Determination of the chymotrypsin concentration: The enzyme substitution does not have to be discontinued before taking the stool sample (no false normal values in continued substitution) Long storage of stool samples (up to 10 days at bei RT)

Chymotrypsin is a serine protease, which is secreted as anexcretory enzyme from the pancreas after food intake into the duodenum. Here food proteins are hydrolytically cleaved, preferentially next to aromatic residues. A small part of the active form of the enzyme is exreted in the stool. In pancreas insufficiency secondary to a chronic pancreatitis, the secretion of the enzyme is reduced markedly. In the past an exocrine pancreatic insufficiency within the scope of a chronic pancreatitis was diagnosed e.g. by determining faecal elastase or chymotrypsin activity in stool. The colorimetric determination of chymotrypsin activity, however, is less reliable because the enzyme´s activity is influenced by enzyme inhibitors in stool. Our ELISA allows the determination of the chymotrypsin concentration in stool. Besides the determination of the chymotrypsin concentration, we offer a photometric test for the determination of chymotrypsin activity in stool.

Indications • Chronic pancreatitis • Exocrine pancreas insufficiency

Chymotrypsin Activity (photometric) Sample volume 100 mg Matrix Stool Tests 20 Cat. No. K 6990

Chymotrypsin Concentration (ELISA) Sample volume 100 mg Matrix Stool (100 mg) Standards 15.6 – 1000 mg/ml Incubation time 1 h; 1 h; 1 h; 30 min Tests 96 Cat. No. K 6910

References Goldberg et al. (1971) Comp Biochem Physiol B38: 697 Kaspar P et al. (1984) Clin Chem 30:1753 – 1757 Stein et al. (1996) Clin Chem 42:222 – 6

24

3. Exocrine pankcreas function

Pancreatic Amylase Like lipase and elastase, pancreatic amylase is synthesised in the pancreas. In the case of chronic pancreatitis, pancreatic amylase concentration is decreased. Some forms of disease (alcoholism, accidental trauma, fibrosis), are associated with a pancreatic insufficiency. In the routine laboratory pancreatic amylase has proved itself to be a genuine alternative to elastase-I in stool diagnosis with apparently higher clinical specificity and sensitivity.

Good stability of Pancreatic amylase in stool

Indications • Chronic pancreatitis

Reference Values Pancreatic Amylase Serum

26 - 325 µg/l

Stool

< 124 mg/l

Pancreatic Amylase (ELISA) Sample volume Matrix Calibrators Incubation time Tests Cat. No.

Sandwich ELISA technology with a highly specific antibody pair (no cross-reaction to salivary amylase and porcine amylase)

100 µl Serum, Stool (100 mg) 440 - 28000 mU/l 1h; 30 min; 15 min 96 K 6410

References Bishop M. et al. (1996) Pancreas 13:226-30 Katschinsky M. et al. (1997) Pancreas 15 :1991-200

25

3. Exocrine pankcreas function

Pancreatic Lipase The pancreatic lipase (triacylglycerol acylhydrolase) is a specific pancreatic enzyme which hydrolyzes primarily glycerol ester of long chain fatty acid. The activity of this enzyme is elevated in patients with acute pancreatitis. The determination of pancreatic lipase is therefore an important parameter for the detection and differentiation of pancreatic diseases, esp. in combination with the determination of the pancreatic amylase. Conventional turbidimetric methods to assess lipase activity are complex, non-specific and exhibit a low sensitivity. In contrast, our ELISA detects human pancreatic lipase in serum quantitatively and with high specificity.

Pancreatic Lipase (ELISA) Sample volume Matrix Calibrators Incubation time Tests Cat. No.

100 µl Stool (100 mg), Serum 10 - 400 U/l 30 min; 10 min 96 determinations K 6495

References Lott JA et al. (1986) Clin Chem 32:1290 Lott JA et al. (1986) Clin Enzymol: 245 Tietz NW (1986) Clin Chem 32:301-307 Tietz NW (1987) Clin Chem 33: 624 Panteghini M et al. (1989) Clin Chem 35:417 Verduin PA (1973) Clin Chi Acta 46:11-19

26

4. Food intolerances Food intolerances include enzyme defects (e.g. lactose or histamine intolerance) as well as immunological intolerances (e.g. gluten intolerance). Often, these intolerances are caused by gene defects and are therefore congenital. In addition, they can be caused by diseases or diet. Depending on the kind of food intolerance, different methods are used for diagnosis. Next to provocation tests and bowel biopsies, a number of laboratory parameters in urine, serum/plasma or stool along with DNA-analysis tools enable a reliable detection of food intolerances. Immundiagnostik offers specific assays for the detection and differentiation of various intolerances. A special focus of Immundiagnostik is the basic understanding of gluten intolerance and the supply of comprehensive laboratory analysis tools for the detection of even atypic and silent forms.

27

4. Food intolerances

EDN (Eosinophil-derived Neurotoxin) Proof of a food allergy with immediate reaction A non-invasive examination of the intestinal eosophile activity. Validated in stool, urine, serum, plasma Stable in stool for seven days

EDN (eosinophil derived neurotoxin, eosinophil protein x, EPX) measuring in stool is recommended for diagnosing a food allergy with an immediate reaction or to test the clinical efficiency of an elimination diet. EDN measurements also support an examination of the integrity of the intestinal mucous, when looking into an inflammable intestinal disease, investigating Colon Carcinoma or for the diagnosis of an intestinal parasite. The classic ways of diagnosing an allergy (determine allergy specific IgE antibodies and the prick-test) are only limitedly reliable when used to determine a food allergy. For example a normal IgE level and a negative result from the prick-test doesn’t rule out an intestinal food allergy. In this case, an EDN measurement in stool is recommended. EDN, a cationic glycoprotein, which is released by activated eosinophiles, has strong cytotoxic characteristics and plays a large part in virus prevention. It is released by the eosinophile granules in places where eosinophiles are mainly to be found, in the skin, lungs, urogenital and gastrointestinal tracts, that is, in the organs which act as an entry point for pathogen. The accumulation of EDN in the intestine is associated with tissue damage. Measuring EDN in stool can serve as an objective parameter for a current clinical or sub-clinical cronic inflammation which is noticeable in the gastrointestinal area. With Colitis ulcerosa and Crohn’s disease, the EDN measurement helps the evaluation of a disease’s activity and the prediction of it possible.

Indications • Proof of a food allergy with immediate reaction • Assessment of an elimination diet • Proof of damaged integrity of the intestinal mucous membrane caused by an invasive disease (e.g. CED, CC etc.) • Proof of intestinal parasites EDN (ELISA) Sample volume Matrix Detection limit Standards Incubation time Tests Cat. No.

100 µl Serum, Urine, Plasma, Stool (100 mg) 0.125 ng/ml 0.6 – 40 ng/ml 1 h, 1 h, 10 min 96 K6811

References Magnusson J et al. (2003) Clin Exp Allergy 33 : 1052-1059 Bengtsson U et al. (1997) J Allergy Clin Immunol :100 : 216-221 Bischoff SC et al. (1997) Dig Dis Sci 42(2) 394-403 Petersen CGB et al. (2002) Am J Gastroenterol 97(7) 1755-1762

28

4. Food intolerances (Gluten sensitivity)

Gluten sensitivity Gliadin / Transglutaminase Antibodies Celiac disease/ gluten intolerance is a chronic gastrointestinal disease with a prevalence of about 1:250 in Europe. The definition of gluten intolerance has to be revised due to new data regarding its prevalence. The classic diagnosis of fully developed celiac disease by biopsy-proven villous atrophy only represents the tip of the iceberg. The iceberg model of Logan clearly indicates that the majority of patients with gluten intolerance do not present with fully developed celiac disease. The early diagnosis and the subsequent initiation of a gluten-free diet is instrumental in preventing a total atrophy of the intestinal mucous layer. In patients presenting with long-term, unspecific abdominal complaints secondary to gluten intolerance, the quality of life can be improved considerably after an early diagnosis and a timely initiation of a gluten-free diet. Gluten intolerance is, next to its classic symptoms, associated with other autoimmune diseases (rheumatoid arthritis, dermatitis herpetiformis Duhring or diabetes mellitus) or with a risk of miscarriage. We offer a unique product line for the purpose of evaluating unspecific abdominal complaints, especially when gluten intolerance is suspected. Beside the assays for the determination of anti-transglutaminase IgA antibodies from serum, we offer tests for the determination of anti-transglutaminase sIgA/IgA from faeces. The tissue transglutaminase and also the epidermal transglutaminase have been proven to be associated with gluten intolerance. The epidermal transglutaminase especially is associated with extraintestinal manifestations like Dermatitis herpetiformis Duhring. Gluten intolerance is caused by gliadins or analogous proteins in cereals. It is well known that this (auto-)immunological disease strikes individuals with a special genetic profile: there is a strong association with specific risk alleles encoding the heterodimeric HLA-DQ2 molecule (>90 % of the patients) or respectively the HLA-DQ8 molecule. The MutaGEL HLA-DQ 2+8 test allows the individual HLA-DQ genotyping by amplification of the encoding allels (for 24 determinations). For this purpose, only a small sample of EDTA-blood is necessary for the preliminary extraction of DNA (reagents not included). The subsequent processing with molecular biological methods leads to specific amplification products detectable by gel-electrophoresis.

Classic celiac diseases: only the tip of the iceberg (Logan's iceberg model)

Indications Reference Values Anti-Transglutaminase

• Poor appetite and failure to gain weight • Iron-deficiency anaemia • Neurological disturbances (e.g., depression, lethargy) • Infertility • Recurrent abortions (Untreated pregnant women are at risk of miscarriage and at risk of having a baby with a congenital malformation) • Dermatitits herpetiformis Duhring • Rheumatoid arthritis symptoms

Serum Stool

≥ 7 AU positive > 100 U/l positives

Reference Values Anti-Gliadin Serum Stool

29

≥ 18 AU positive > 100 U/l positives

4. Food intolerances (Gluten sensitivity)

* Anti-gliadin-assay (particularly the IgG subclass) can yield falsepositive results in gastrointestinal conditions other than CD, including cow´s milk protein intolerance and parasite infections (Fasano and Catassi,2001)

Affected organ

Serum diagnostics

Stool diagnostics

Gastrointestinal tract

Anti-tTG-IgA

Anti-Gliadin-sIgA* Anti-tTG-sIgA

Joints

Anti-tTG-IgA

Anti-tTG-sIgA

Skin

Anti-TGe-IgA

Sample volume

Matrix

Cali- brators

Incubation time

Test  prinicple

Tests

Cat.No

Anti-human epidermal Trans- glutaminase IgA [anti-TGe-IgA]

100 µl

Serum, Plasma

Cut off

1h; 1h; 30min

ELISA

96

K 9396

Anti-human tissue Transglutaminase IgA [anti-tTG-IgA]

100 µl

Serum, Plasma

Cut off

1h; 1h; 30min

ELISA

96

K 9399

Anti-human tissue Transglutaminase IgG [anti-tTG-IgG]

50 µl

Serum, Plasma

Cut off

2h

ELISA

96

K 9398

Anti-human tissue Transglutaminase sIgA [anti-htTG-sIgA]

100 mg

Stool

Cut off

2h; 1h; 15-25min

ELISA

96

K 9393

anti-Gliadin IgA

100 µl

Serum

Cut off

1h; 1h; 15min

ELISA

96

K 9310

anti-Gliadin IgG

50 µl

Serum, Plasma

Cut off

2h

ELISA

96

K 9300

anti-Gliadin sIgA

100 mg

Stool

Cut off

2h; 1h; 15-25min

ELISA

96

K 9311

200 µl

DNA (e.g. whole blood, cheek swab)

2h

PCR (allel specific)

24

KE09020

Product

MutaGEL® HLA-DQ 2+8

References Rose C et al. (2010) J Dtsch Dermatol Ges (JDDG) 8(4):265-70. Rose C et al. (2009) J Am Acad Dermatol 2009 Apr 1. [Epub ahead of print] Marietta EV et al. (2008) J Invest Dermatol 128(2):332-5. Donaldson MR et al. (2007) J Invest Dermatol 127(5):1268-71. Epub 2007 Jan 4. Luft LM et al. (2003) J Rheumatol 30: 2613-2619 Sardy et al. (2002) J Exp Med 195 : 747-757 Fassano and Catassi (2001) Gastroenterology 120: 636-651 Martinelli P et al. (2000) Gut 46 : 332-335 Sardy et al (1999) Clin Chem 45:2142-21 Schütz et al. (1998) Glutenunverträglichkeit EHK 11:807-810 Stern et al. (1998) Der Kinderarzt 2:159164 Dietrich et al. (1997) Nat Med 3: 797-801 Aeschlimann D et al. (1994) Thrombosis and Hemostasis 71: 402-415 Logan RFA (1992) Dyn Nutr Res 2 : 14-24

30

4. Food intolerances (Gluten sensitivity)

Gliadorphin (Gliadomorphin) Gliadorphin is a 7 amino acids peptide which is formed during digestion of the gliadin component of the gluten protein. Gluten-derived peptides bind to opioid receptors in the brain and exhibit morphine-like effects, for example like heroin. These compounds have been shown to react with areas of the brain which are involved in speech and auditory integration. Urine samples from people with autism, schizophrenia, and celiac disease contain high amounts of gliadorphin. It is suspected that this peptide may also be elevated in other disorders such as chronic fatigue, fibromyalgia, and depression. Symptom remission has been observed after exclusion of wheat and dairy products from the diet.

Indications • Autism • Schizophrenia • Celiac disease Gliadorphin (Gliadomorphin) (ELISA) Sample volume Matrix Standards Incubation time Tests Cat.No.

25 µl Urine 3-300 ng/ml o.n.; 1 h; 12-25 min 96 determinations K 7011

31

4. Food intolerances (Lactose intolerance)

Lactose intolerance MutaGEL® Laktase Patients with lactose intolerance are not able to digest milk sugar (lactose) taken in with food. Due to this fact, these persons subsequently suffer under malabsorption problems like nausea, flatulence, diarrhoea or stomach pain. The most important reason for lactose intolerance is founded in a genetical lack of the enzyme lactase which is responsible for the degradation of milk sugar in the organism. This common gene defect is very easy to detect by analysing the T/C base replacement at position -13910 from the regulatory region of the lactase gene. If this point mutation is homozygous, a lactase deficiency and subsequent lactose intolerance is predetermined. The manifestation of the disease occurs with about 20 years of age and the prevalence of the homozygous mutation in Germany is more than 15 %. The kit “MutaGEL® Laktase” allows the detection of the common T13910C polymorphism in the lactase gene LCT.

Indications • Nausea, cramps, bloating, gas, and diarrhea, which begin about 30 minutes to 2 hours after drinking or eating fluids, foods containing lactose

MutaGEL® Laktase (AS) Sample volume Matrix Test principle Tests Cat. No.

200 µl DNA (e.g. from EDTAwhole blood, cheek swab) PCR (allel specific) 96 Determinations KE 09009

References Tag CG et al. (2007) Clin Chem 53(1):146-48 Buning C et al. (2003): Scand J Gastroenterol 38: 538-542. Enattah NS et al. (2002): Nat Genet 30: 233-237.

32

4. Food intolerances (Fructose intolerance))

Fructose intolerance MutaGEL® Aldolase B The liver isoenzyme Aldolase B is critical for sugar metabolism, and a catalytic deficiency due to mutations in its gene may result in hereditary fructose intolerance (HFI) syndrome, with hypoglycaemia and severe abdominal symptoms. The autosomal recessive disorder HFI is a potentially lethal inborn error in metabolism and the disease poses diagnostic problems because of in part very unspecific clinical manifestations. The present Aldolase B PCR test is useful for the detection of the three most common muatations critical for gluconeogenesis and fructose metabolism: A149P (60%), A174D (11%) and N334K (8%). These mutation may account together for more than 80% of all known mutations causing HFI and their screening will be helpful for suited therapy of afflicted patients. MutaGEL® Aldolase B Sample volume Matrix Test principle Tests Cat. No.

200 µl DNA (e.g. whole blood, cheek swab) PCR (allel specific) 24 KE 09013

33

4. Food intolerances (Histamin intolerance))

Histamin intolerance Diamine Oxidase (DAO)

N

NH2

N H

Histamin

Histamin

Diamine oxidase (DAO) is a histamine-metabolizing enzyme. Although DAO is found practically in the whole body, the most important site of its action is the intestine. The enzymatic activity of DAO determines the histamine degradation speed. In the case of DAO deficiency or inhibition, incorporated or endogenous histamine cannot be degraded quickly enough, and the symptoms of histamine intolerance are presented. Millions of people suffer from gastrointestinal problems, migraine, irritations of nasal mucosa and other allergy-like symptoms after consumption of certain nutrients. Too much histamine in the body can be the reason for this wide range of symptoms. Another possibility for reduced DAO function could be the intake of activity-inhibiting substances, such as alcohol or medication. Histamine induced food intolerance is not IgE-mediated. Determination of the DAO activity in serum or plasma is a suitable marker for diagnosis of histamine intolerance and the associated symptoms. With our easy-to-use, reliable and standardised test kit it is possible to quantify the biological activity of DAO in the circulation. Only 50 µl of serum are needed for the test, results are available within 3 hours. Indications:  Detection of histamine intolerance  Monitoring of a histamine-free diet

DAO 3H (REA) Sample volume Matrix Calibrators Incubation time Tests Cat. No.

DAO (ELISA) Sample volume Matrix Incubation time Tests Cat. No.

100 µl Serum 2.1 - 80 U/ml 2,5 h 96 K 8220

References Infvesson G et al. (1969) Scand J Clin Lab Invest 24: 163-168 Sessa A et al. (1994) Agents and Actions 43: 69-77 Wantke F et al. (1993) Clin Exp Allergy 23: 982-985 Wantke F et al. (1999) Inflammation Research 48: 169-170

34

25 µl Serum, Plasma 2 h, 1 h, 1 h, 10-20 min 96 K 8500

4. Food intolerances (Histamin intolerance))

Histamin Histamin* (ELISA)

New for your research: Our ELISA for the quantitative determination of HIstamin in Stool

Sample volume 100 mg Matrix Stool Calibrators 10–2560 ng/ml Incubation time 15 min; 30 min; 10 min; 15 min Tests 96 Cat. No. K 6861 * For Research Use only

35

5. Infectious diseases



Infections of the intestinal tract with bacteria, viruses or parasites can cause life threa-

tening acute diarrhea. In addition, frequent or chronic infections play a role in the pathogenesis of a number of intestinal diseases. For routine screening, Immundiagnostik offers a comprehensive, automatable assay portfolio (ELISA, PCR) for pathogen detection.

Microscopic picture of the yeast Candida albicans

36

5. Infectious diseases

Helicobacter pylori Antigen Helicobacter pylori (H. pylori) is a spiral-shaped bacterium that can be found in the human stomach and duodenum. In order to be able to survive in the extremely acid environment of the stomach, H. pylori bacteria produce urease which in turn metabolises urea into bicarbonate and ammonia. Particularly, the highly corrosive ammonia affects the gastric mucosa adversely and might cause severe damage. Besides a possible gastritis, an H. pylori infection could eventually lead to a duodenal ulcer or a gastric tumour, resulting from the persisting immune response to the infection. Traditional diagnosis of an H. pylori infection requires invasive measures such as gastroscopy and biopsy, which for most patients are rather burdensome. Alternatively, measuring H. pylori antibodies in serum or the 13C-urease breath test provides information on a possible H. pylori infection. Our Helicobacter pylori Antigen ELISA offers a tool for the detection of the H. pylori antigen in faeces. The advantages of a faecal antigen test are obvious: • non-invasive sample collection • cost efficient alternative to the golden standard of gastroscopy • equivalent sensitivity and specifity to the 13C-urease breath test • for follow-up of treatment more useful than serological test since a com parison of pro- and post-treatment is necessary

Indications • Initial diagnosis of a H. pylori infection • Follow-up of treatment Helicobacter pylori antigen Sample volume Matrix Calibrators Incubation time Tests Cat. No.

(ELISA) 100 mg Stool Cut off 1 h; 1 h; 1h, 15 min 96 K 6920

References Gisbert JP et Pajares JM (2004) Helicobacter 4: 347-368 Uemura N et al. (2001) N Engl J Med 345: 784-89

Also available at Immundiagnostik for serum und plasma samples: •

Helicobacter pylori IgA ELISA (KBE-HLAK09)

•

Helicobacter pylori IgA CLIA (KBC-HLAK09)

•

Helicobacter pylori IgG ELISA (KBE-HLGK08)

•

Helicobacter pylori IgG CLIA (KBC-HLGK08)

37

Optimal patient screening: Combination of Helicobacter pylori antigen detection and determination of antibiotic resistance

5. Infectious diseases

Norovirus Gastrointestinal infections can cause life threatening diseases ultimately leading to death. It was recently shown, that the genetic heterogeneous group of Noroviruses (formally known as Norwalk-like viruses) are the major cause of non-bacterial gastroenteritis worldwide. Human Noroviruses are small, non-enveloped viruses with a ssRNA (single stranded) genome. Noroviruses belong to the family of Caliciviridae and are divided into genotype I and II. These viruses are resistant against higher temperatures (60°C), acid (pH 3) and chlorit (10mg/L). The viruses are transmitted via contaminated food and water but also from person-to-person and are highly contagious. MutaPLATE® Norovirus Sample volume Matrix Test principle Tests Cat. No.

New:

MutaPLEX® Norovirus Sample volume Matrix Test principle

200 µl RNA (Stool) real time RT-PCR (for open system)



(+ extraction control)

Tests Cat. No.

32 KG190132

MutaREAL® Norovirus Sample volume Matrix Test principle Tests Cat. No.

New:

200 µl RNA (Stool) real time RT-PCR (for open system) 96 KG1934196

MutaREX® Norovirus Sample volume Matrix Test principle Tests Cat. No.

200 µl RNA (Stool) real time RT-PCR 96 KG2934196

200 µl RNA (Stool) real time RT-PCR (+ extraction control)

96 KG290196

Literature Lopman BA et al. (2003) Viral gastroenteritis outbreaks in Europe, 1995-2000. Emerg Infect Dis Jan 9(1):90-6 Marshall JA et al. (2006) Laboratory Diagnosis of Norovirus. Clin Lab 52:571-81

38

5. Infectious diseases

Enterovirus Human Enteroviruses are small RNA viruses which belong to the family of picornaviridae. The ubiquitous pathogen is responsible for about 500 million infections per year worldwide. The 64 serotypes are divided in the following groups: * Polioviruses Typ 1-3 * Coxsackieviruses with subgroups A and B * Echoviruses * Enteroviruses 68-71

Highest quality standard of our PCR kits: 2010 evaluated by the international panel "Quality Control for Molecular Diagnostics" with best result (100%)

In moderate climates there is a seasonal accumulation of enterovirus infections in late summer and fall. Transmission of enteroviruses occur via the faecal-oral route. Airborne infections are also common. The viruses are transmitted between humans through stool or through saliva-contaminated objects and are excreted during the acute disease phase. In addition, virus particles can be detected up to several weeks after the symptoms have subsided. The median incubation time for most enterovirus infections is 3-5 days. Diseases caused by enteroviruses are very complex and can be life threatening, especially for children. Among them are febrile illness, conjunctivitis, herpangina, hand foot and mouth disease, gastroenteritis, generalized skin rashes, meningitis, pneumonia, myocarditis, pericarditis, hepatitis, encephalitis, paralyses, fetal damage up to severe neonatal diseases with pneumonia, myocarditis and meningoencephalitis. Enteroviruses are excreted mainly in stool and, depending on the virus type, through the pharyngeal route. In acute disease, the diagnosis should prefererrably be based on PCR pathogen detection. Our PCR kits are screening tests for the qualitative determination of human enterovirus RNA (Polio-, Coxsackie A-, Coxsackie B- and echoviruses) in clinical samples (stool, whole blood, plasma, respirational tract samples, cerebrospinal fluid).

The CE-marked kits are optimized for enterovirus detection in stool

New: MutaPLEX® Enterovirus Sample volume 200 µl Matrix RNA (biol. liquids) Test principle real time RT-PCR for open systems + extraction control Tests 96 Cat. No. KG190296

MutaPLATE® Enterovirus Sample volume 200 µl Matrix RNA (biol. liquids) Test principle real time RT-PCR for open systems Tests 96 Cat. No. KG1900296

New: MutaREAL® Enterovirus Sample volume Matrix Test principle Tests Cat. No.

MutaREX® Enterovirus Sample volume 200 µl Matrix RNA (biol. liquids) Test principle real time RT-PCR + extraction control Tests 96 Cat. No. KG290296

200 µl RNA (biol. liquids) real time RT-PCR 96 KG2900296

39

5. Infectious diseases

Salmonella sp. Salmonella are the main cause for food intoxications: In more than 65% of all cases, egg products, meat and poultry are the common foods which transmit salmonella. Therefore, the European regulations specify that none of the roughly 2000 known salmonella subtypes must be detected in a 25 g food sample. Until now, the necessary analysis has been performed with bacteriological cultures which take up to 5 days. The PCR offers a far more sensitive and time saving alternative (one day) to the conventional microbiological analysis of Salmonella sp. in various food samples. In addition, the PCR allows the pathogen detection in stool samples and is thereofore ideal for a clinical setting.

MutaREAL® Salmonella Sample volume 200 µl Matrix DNA (Food, Stool) Test principle real time RT-PCR Tests 96 Cat. No. KV2900196

40

5. Infectious diseases

D-Arabinitol The yeast Candida albicans is present in most human mucous membranes of the mouth and pharynx, the genital area and the intestinal tract. If the immune system is weakened or the physiological bacterial intestinal flora disrupted, the yeast multiplies and leads to a generalized candidiasis. Typical symptoms of a candidiasis include general weakness, recurring vaginal infections, fungal skin infections, fatigue, memory impairments, irritability, headaches, poor concentration, joint aches and flatulence. A systemic candidiasis is often diagnosed late. As a consequence of the multiplication of the yeast in the circulation, multiple organs can be affected. Complications include endocarditis, endophthalmitis or osteomyelitis. During a systemic candidiasis, pathogen detection occurs in blood, liquor, joint fluid and endotracheal samples and bronchial lavages. An additional possibility to diagnose invasive yeast infections is the determination of D-Arabinitol in serum or urine. D-Arabinitol is a characteristic yeast metabolite. While Candida ssp. produce exclusively D-Arabinitol, the human metabolism generates L-Arabinitol. D-Arabinitol serum levels rise during an invasive candidiasis, when the yeast multiplies in the organism. Apart from the gas chromatographic determination easy-to-use tests have been missing so far. Our Arabinitol test is a straight-forward colorimetric enzyme assay for the manual and automatic analysis of D-Arabinitol in serum or urine.

Indications • Detection of a Candida infection • Determination of the severity of a Candida infection • Therapy monitoring • Choice of the appropriate medication

D-Arabinitol Sample volume 10 µl Matrix Serum, Urine Test principle colorimetric Calibrators 20-100 µmol/l Tests 125 Cat. No. KMD-ARAB

41

Colorimetric test: • Simple handling • Automatable

5. Infectious diseases

More tests for the detection of viral and bacterial infections (determination in stool) on request:

Serazym Adenovirus

HW/E-017 HW/E-017-A2

96 determinations 2 x 96 determinations

HW/E-045 HW/E-045-A2

96 determinations 2 x 96 determinations

HW/E-020 HW/E-020-A2

96 determinations 2 x 96 determinations

HW/E-038

96 determinations

HW/E-018

96 determinations

HW/E-040 HW/E-040-A2

96 determinations 2 x 96 determinations

Serazym Verotoxin 1+2

HW/E-030 HW/E-030-A2

96 determinations 2 x 96 determinations

Serazym Campylobacter

HW/E-093 HW/E-093-A2

96 determinations. 2 x 96 determinations

Kit for automate

Serazym Astrovirus Kit for automate

Serazym Rotavirus Kit for automate

Serazym Giardia lamblia Kit for automate

Serazym Entamoeba histolytica Kit for automate

Serazym Clostridium difficile  Toxin A/B Kit for automate Kit for automate

Kit for automate

42

Appendix: Stool sample preparation systems We can spare you the cumbersome and unpleasant stool weighing procedure: Our special, cost-effective sample tube enables the preparation of a defined sample solution with minimal stool contact. For sample collection, the dip stick with screw cap is used to collect the sample (15 mg), excessive stool is stripped off at the cone-shaped insert (s. Fig.). The stool sample is now ready for storage or dilution with buffer. The resulting sample suspension can be used with our ELISAs for the determination of a respective parameter in stool. The sample tubes can be ordered empty or filled with buffer for the preparation of a defined stool sample suspension. Fig.: Practical preparation of stool samples

Dipstick

• • • •

time saving hygienic minimal stool contact suited for direct use on automates

Stool sample

Stool sample tube

Stool sample preparation systems unfilled

Cat. No. K 6998SAS

with buffer (dilution 1:50)

Cat. No. K ....SASP1

with buffer (dilution 1:100)

Cat. No. K ...SASP2

]

43

.... = Cat. Nr. of the respective ELISA test for the determination of a parameter in stool.

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Immundiagnostik AG Stubenwald-Allee 8a D-64625 Bensheim Tel.: +49 (0) 62 51/70 19 00 Fax: +49 (0) 62 51/84 94 30 [email protected] www.immundiagnostik.com