Proinsulin (Intact) Chemiluminescence ELISA For the quantitative determination of proinsulin in human serum, EDTA plasma, heparin plasma, and tissue culture supernatants

For “In Vitro Diagnostic” use within the United States of America. This product is for “Research Use Only” outside of the United States of America.

Catalog Number: Size: Version:

80-PINHUI-CH01 96 Wells 3.0 – June 06, 2015

26-G Keewaydin Drive, Salem, NH 03079│P: (800) 592-5726│F: (603) 898-6854│[email protected]│www.alpco.com

INTENDED USE The Proinsulin (Intact) Chemiluminescence ELISA is designed for the quantitative determination of proinsulin in human serum, plasma (EDTA and heparin), and tissue culture supernatants. The results from this assay are to be used in conjunction with other clinical and laboratory findings to aid in the diagnosis and treatment of patients with abnormal insulin secretion, including diabetes mellitus. PRINCIPLE OF THE ASSAY The Proinsulin (Intact) Chemiluminescence ELISA is a sequential sandwich assay, which uses two mouse monoclonal antibodies: one as capture that is adsorbed to a 96-well black-well microplate and the other as a primary detector conjugated to biotin. Standards, sample, and controls are added to the plate and the capture-antigen is incubated at room temperature on a plate shaker set between 700-900 rpm. Post standard/sample incubation, the plate is washed six times and the biotinylated detection antibody is added to each well. This capture-antigen-detector sandwich is incubated at room temperature on a plate shaker set between 700-900 rpm. Post detection antibody incubation, the plate is washed six times, HRP (horseradish peroxidase)conjugated streptavidin (SA) is added to each well, and incubated at room temperature on a plate shaker set between 700-900 rpm. Post SA-HRP incubation, the plate is washed 6 times and the chemiluminescent substrate is added to each well. The microplate is analyzed on an appropriate plate reader 1 minute post substrate. The resulting relative light units (RLUs) are directly proportional to the amount of intact proinsulin present in the sample. MATERIALS SUPPLIED Component

Quantity

Preparation

12 x 8 strips

Ready to use

5 mL

Ready to use

8 x 1 mL

Ready to use

Controls 1, 2, and 3*

3 vials

Lyophilized*

Protease Inhibitor Stock

100 µL

100X

Assay Buffer

18 mL

Ready to use

Detector Antibody

80 µL

201X

SA-HRP

200 µL

66X

SA-HRP Buffer

12 mL

Ready to use

Substrate A

6 mL

Ready to use

Substrate B

6 mL

Ready to use

200 mL

21X

3

Ready to use

Proinsulin Microplate (96 wells) Zero Standard Standards (A-H) (5, 10, 25, 50, 100, 250, 1,000, 3,000 pg/mL)

Wash Buffer Concentrate Plate Sealers

*Please refer to the Certificate of Analysis enclosed with each kit for more information.

Proinsulin (Intact) Chemiluminescence ELISA

Page 2 of 12

3.0 – June 06, 2015

MATERIALS REQUIRED        

Precision pipettes for dispensing up to 100 µL (with disposable tips) Repeating or multi-channel pipette for dispensing up to 100 µL Volumetric containers and pipettes for reagent preparation Distilled/Deionized water for reagent preparation Microplate washer or wash bottle Microplate shaker capable of 700-900 rpm Microplate reader capable of reading luminescence Centrifuge (1,000 x g to 2,000 x g), vortex, and ice for sample preparation

PRECAUTIONS 1. The human blood products incorporated into this kit have been tested for the presence of HIV (Human Immunodeficiency Virus), HBV (Hepatitis B Virus), and HCV (Hepatitis C Virus). Test methods for these viruses do not guarantee the absence of a virus; therefore, all reagents should be treated as potentially infectious. Handling and disposal should be in accordance with all appropriate national and local regulations for the handling of potentially biohazardous materials. 2. All materials derived from animal sources are bovine spongiform encephalopathy (BSE) negative. However, all materials should be kept from ruminating animals. 3. Avoid direct contact with skin. 4. This product is not for internal use. 5. Avoid eating, drinking, or smoking when using this product. 6. Do not pipette any reagents by mouth. 7. Reagents from this kit are lot-specific and must not be substituted. 8. Do not use reagents beyond the expiration date. 9. Variations to the test procedure are not recommended and may influence the test results. 10. An appendix has been included with examples of instrument settings for analyzing a chemiluminescent assay. Each lab should optimize their instrument settings according to the manufacturer’s instructions. Please contact the technical services department of the manufacturer of the microplate reader for optimal instrument settings. STORAGE CONDITIONS The kit should be stored at 2-8°C. The kit is stable until the expiration date on the box label. If desired, the reconstituted controls can be stored at ≤ -20°C in aliquots for up to 6 months. The controls should not be repeatedly frozen and thawed. SAMPLE HANDLING Serum, plasma (EDTA and heparin), and tissue culture supernatant samples are appropriate for use in this assay. For serum samples, allow collected blood to clot on ice for 20 minutes and then subject the samples to 1,000 to 2,000 x g centrifugation for 20 minutes at 2-8oC, because proinsulin is not stable in blood at room temperature1. If testing will occur within 2 hours after collection, keep the serum samples on ice. If the assay is to be performed at a later time, store the serum samples at ≤ -20°C.

Proinsulin (Intact) Chemiluminescence ELISA

Page 3 of 12

3.0 – June 06, 2015

If EDTA or heparin plasma samples are to be assayed within 2 hours after collection, keep the samples on ice. If the assay is to be performed at a later time, store the samples at ≤ -20°C. Upon thawing samples, keep the samples on ice until performing the assay. Avoid repeated freeze/thaw cycles. It is recommended that all samples be centrifuged at 13,000 RPM at 2-8°C for 10 minutes before loading them on the plate. In order to reduce potential time-associated sample drift, it is recommended to thoroughly vortex each sample before use and perform each pipetting action without pausing. For samples with a greater concentration of intact proinsulin than the highest standard, dilute the sample in Zero Standard and repeat the analysis.

REAGENT PREPARATION All reagents must be equilibrated to room temperature prior to preparation and subsequent use in the assay. Prepare enough reagents for only the number of strips used. Controls (Levels 1, 2, and 3) are provided in a lyophilized form. Refer to the Certificate of Analysis provided with each kit for the appropriate volume of deionized water for reconstitution. Close the vial with the rubber stopper and cap, gently swirl the vial, and allow it to stand for 30 minutes prior to use. The contents of the vial should be in solution with no visible particulates. If desired, the controls can be stored at ≤ -20°C in aliquots for up to 6 months. The controls should not be repeatedly frozen and thawed. Protease Inhibitor Stock (100X) is to be diluted with Assay Buffer for the Working Strength Protease Inhibitor. Number of plates

Amount of Concentrate

Amount of Assay Buffer

0.5 (6 strips) 1 (12 strips)

30 µL 60 µL

2.970 mL 5.940 mL

2 5

120 µL 300 µL

11.880 mL 29.700 mL

10

600 µL

59.400 mL

Detector Antibody (201X) is to be diluted with 200 parts Assay Buffer for the Working Strength Detector Antibody. Number of plates

Amount of Concentrate

0.5 (6 strips)

30 µL

6 mL

1 (12 strips) 2

60 µL 120 µL

12 mL 24 mL

5 10

300 µL 600 µL

60 mL 120 mL

Proinsulin (Intact) Chemiluminescence ELISA

Page 4 of 12

Amount of Assay Buffer

3.0 – June 06, 2015

SA-HRP (66X) is to be diluted with SA-HRP Buffer for the Working Strength SA-HRP. Number of plates

Amount of Concentrate

Amount of SA-HRP Buffer

0.5 (6 strips) 1 (12 strips) 2 5 10

90 µL 180 µL 400 µL 800 µL 1800 µL

5.85 mL 11.70 mL 26 mL 52 mL 117 mL

Wash Buffer Concentrate (21X) is to be diluted with 20 parts distilled water for the Working Strength Wash Buffer. NOTE: All labs should account for plate washer void volume to prime the machine. The values below do not account for priming automated plate washers.

0.5 (6 strips)

Amount of Concentrate (manual wash) 25 mL

Amount of dH2O (manual wash) 500 mL

1 (12 strips) 2

40 mL 80 mL

800 mL 1600 mL

5 10

200 mL 380 mL

4000 mL 7600 mL

Number of plates

Chemiluminescent Substrates A & B are provided individually and should be combined in equal parts to create the Working Strength Chemiluminescent Substrate prior to use. The Working Strength Chemiluminescent Substrate is stable for 1 hour. Number of plates

Amount of Substrate A

Amount of Substrate B

0.5 (6 strips) 1 (12 strips)

3 mL 6 mL

3 mL 6 mL

2 5

12 mL 25 mL

12 mL 25 mL

10

50 mL

50 mL

QUALITY CONTROL It is recommended that the controls provided with the Proinsulin (Intact) Chemiluminescence ELISA be included in every assay. The concentration ranges of the controls are provided on the certificate of analysis provided with each kit; however, it is recommended that each laboratory establishes its own acceptable ranges. ASSAY PROCEDURE All reagents and microplate strips (while sealed in foil pouch) should be equilibrated to room temperature prior to use. Gently mix all reagents before use. A standard curve must be performed with each assay run and with each microplate if more than one is used at a time. All standards, controls, and samples should be run in duplicate. 1. The microplate should be equilibrated to room temperature prior to opening the foil pouch. It is recommended to designate enough microplate strips for duplicate determinations of the standards, controls, and samples. Any remaining microplate strips should be stored at 2-8°C in the tightly sealed foil pouch containing the desiccant.

Proinsulin (Intact) Chemiluminescence ELISA

Page 5 of 12

3.0 – June 06, 2015

2. Pipette 50 µL of each standard, control, and sample into their respective wells. See Reagent Preparation for control reconstitution instructions. A suggested plate layout is included. 3. Pipette 50 µL of Working Strength Protease Inhibitor (see Reagent Preparation) into each well. 4. Cover microplate with a plate sealer and incubate for 1 hour at room temperature, shaking at 700-900 rpm on a microplate shaker. 5. Decant the contents of the wells and wash the microplate 6 times with 350 µL of Working Strength Wash Buffer per well (see Reagent Preparation) using an automated microplate washer. a. Manual Wash: Alternatively, fill the wells with Working Strength Wash Buffer using a wash bottle equipped with a wash nozzle or manual washer. (It is not recommended to use a multichannel pipette. Wash buffer must be dispensed with adequate and equal force in order to properly wash the wells.) Soak the wells for 1 minute. Invert the microplate to discard the liquid and firmly tap the inverted microplate on absorbent paper towels. Repeat the wash and soak procedure 2 more times, for a total of 3 washes. After the final wash, (automated or manual), remove any residual wash buffer and bubbles from the wells by inverting and firmly tapping the microplate on absorbent paper towels. 6. Pipette 100 µL of Working Strength Detector Antibody (see Reagent Preparation) into each well. 7. Cover microplate with a plate sealer and incubate for 1 hour at room temperature, shaking at 700-900 rpm on a microplate shaker. 8. Decant the contents of the wells and wash the microplate 6 times as in Step 5 above. 9. Pipette 100 µL of Working Strength SA-HRP (see Reagent Preparation) into each well. 10. Cover microplate with a plate sealer and incubate for 30 minutes at room temperature, shaking at 700-900 rpm on a microplate shaker. 11. Decant the contents of the wells and wash the microplate 6 times as in Step 5 above. 12. Pipette 100 µL of Working Strength Chemiluminescent Substrate (see Reagent Preparation) into each well. 13. Place the microplate in a microplate reader capable of analyzing the luminosity of the wells. The microplate should be analyzed 1 minute after the addition of the chemiluminescent substrate, and no later than 25 minutes after substrate addition. Analyze the plate using a 1 second integration time.

Proinsulin (Intact) Chemiluminescence ELISA

Page 6 of 12

3.0 – June 06, 2015

CALCULATION OF RESULTS Construct a standard curve from the standards. It is recommended to use a software program to calculate the standard curve and to determine the concentration of the samples. A 5 pl curve fit with 1/y2 weighting is recommended for data analysis. The Proinsulin (Intact) Chemiluminescence ELISA is a ligand binding assay, with responses exhibiting a sigmoidal relationship to the analyte concentration. Currently accepted reference models for such curves use a 4 or 5 parameter logistic (PL) fit, as these models optimize the accuracy and precision across a greater range. In the example below, a 5-PL curve fit with 1/y2 weighting was used to maximize the accuracy and precision of samples with low concentrations. However, the accuracy and precision of all models are limited at the lowest and highest ends of the detectable range due to the influence of individual laboratory conditions. As a result, caution should always be used when interpreting results where the analyte response becomes non-linear.2 TYPICAL STANDARD CURVE The following results are provided for demonstration purposes only and cannot be used in place of data obtained with the assay. A standard curve must be performed with each assay run and plate tested. In this example, a 5-parameter curve fit with 1/y2 weighting is used for data analysis. Note: below is a table for converting pg/mL to pM. 9.4 pg/mL = 1 pM Standard Standard (pg/mL) A B C D E F G H Zero

Standard (pM)

5 10 25 50 100 250 1,000 3,000 0

0.53 1.06 2.66 5.32 10.6 26.6 106 319 0

Standard

Standard (pg/mL)

Mean RLU

RLU CV (%)

A B C D E F G H Zero

5 10 25 50 100 250 1,000 3,000 0

678 1,469 3,378 7,201 13,499 36,548 139,329 369,116 87

3.5 11.6 2.1 6.5 5.9 4.3 1.2 2.5 11.8

Proinsulin (Intact) Chemiluminescence ELISA

Page 7 of 12

S:N

Mean Conc. (pg/mL)

Conc. CV (%)

% Recovery

7.8 16.9 39.0 83.1 156 422 1,608 4,259

4.8 10.8 24.8 51.9 95.8 253.9 989.7 3,015

3.8 11.8 2.1 6.3 5.8 4.2 1.3 3.2

95 108 99 104 96 102 99 100

3.0 – June 06, 2015

PERFORMANCE CHARACTERISTICS (Refer to the kit specific Certificate of Analysis for the most relevant performance data.) Sensitivity The Limit of Blank (LoB), Limit of Detection (LoD), and the Limit of Quantitation (LoQ), were determined in accordance with the CLSI Guideline EP17-A. The LoB and LoD of the assay are less than or equal to 5 pg/mL. The limit of quantitation (LoQ) is defined as the lowest concentration which has calculated concentration CV ≤20%, an accuracy of 80-120%. The LoQ of the assay is less than or equal to 10 pg/mL.

Precision: Within run (intra-assay) variation The within run precision is expressed as the percentage coefficient of variation (CV %). This was determined based on the mean and standard deviation of 16 replicates of a sample run in a single assay. The table below shows the results of 3 samples that span the range of the assay. Sample 1

Sample 2

Sample 3

Mean

35.2 pg/mL (3.7 pM)

244.9 pg/mL (26.1 pM)

1,835.7 pg/mL (195.3 pM)

CV (%)

5.7

6.6

8.1

16

16

n 16 Precision: Between run (inter-assay) variation

The between run precision is expressed as the percentage coefficient of variation (CV %). This was determined based on the mean and standard deviation across 2 assays of 32 measurements of a single sample. The table below shows the results of 3 samples that span the range of the assay.

Proinsulin (Intact) Chemiluminescence ELISA

Page 8 of 12

3.0 – June 06, 2015

Sample 1

Sample 2

Sample 3

Mean

33.4 pg/mL (3.6 pM)

244.9 pg/mL (26.1 pM)

1,881.3 pg/mL (200.1 pM)

CV (%)

7.5

6.5

6.8

n

32

32

32

Linearity The linearity of the assay was determined by preparing dilutions of samples with high proinsulin concentrations in the Zero Standard from 1:2 to 1:128. The expected values were compared to the observed values to determine a percent recovery.

Serum Heparin Plasma EDTA Plasma Tissue Culture Supernatants

Mean recovery (%)

Min (%)

Max (%)

r2

103 99 98 91

90 88 92 78

114 107 116 107

0.9981 0.9991 0.9987 0.9980

Spike and Recovery The spike and recovery of the assay was determined by adding various known amounts of proinsulin to samples. Serum, heparin plasma, EDTA plasma, and tissue culture media were evaluated. The expected values were compared to the observed values to determine a percent recovery. The table below shows the recovery for the samples.

Serum

Heparin plasma

EDTA plasma

Tissue culture media

low mid high low mid high low mid high low mid high

Mean recovery (%)

Min (%)

Max (%)

88 85 81 91 88 85 90 84 87 96 96 92

83 80 73 80 76 74 85 79 79 89 90 86

95 93 90 95 94 95 93 93 96 102 101 101

Specificity The table below indicates the analyte and the percent cross-reactivity observed in the assay. Analyte

Proinsulin (Intact) Chemiluminescence ELISA

% Cross-reactivity

Page 9 of 12

3.0 – June 06, 2015

Human Intact Proinsulin

100

Human Des (31,32) Proinsulin

0.73

Human Des (64,65) Proinsulin

70.2*

Human C-peptide

Not Detected

Human Insulin 0.17 *Physiologically, Des (64,65) comprises of only ~6% of circulating total proinsulin3, 4 and would not be detected in human samples. Hook Effect No high dose hook effect was observed with analyte concentrations up to 300,000 pg/mL.

REFERENCES 1. Pfützner, A. et al. Clinical and Laboratory evaluation of a new specific ELISA for intact proinsulin. Clin Lab. 2005; 51(5-6): 243-249. 2. Finlay JWA, Dillard RF. Appropriate Calibration Curve Fitting in Ligand Binding Assays. AAPS Journal. 2007; 9(2): E260-E267. 3. Sizonenko, S. et al. Kinetics of Proinsulin Conversion in Human Islets. Diabetes. 2005; 42:933-936. 4. Yu, P. et al. Relationship between proinsulin and beta cell function in different states of glucose tolerance. International Journal of Diabetes in Developing Countries. 2012; 32(4): 219-223.

Proinsulin (Intact) Chemiluminescence ELISA

Page 10 of 12

3.0 – June 06, 2015

SHORT ASSAY PROTOCOL Add 50 µL standards, controls, and samples Add 50 µL Working Strength Protease Inhibitor Incubate for 1 hour at RT, shake at 700-900 rpm Wash 6 times Add 100 µL Working Strength Detector Antibody Incubate for 1 hour at RT, shake at 700-900 rpm Wash 6 times Add 100 µL Working Strength SA-HRP Incubate for 30 min at RT, shake at 700-900 rpm Wash 6 times Add 100 µL Working Strength Chemi Substrate Incubate for 1 minute at RT Read plate Total Time = 2 hours 31 minutes

SUGGESTED PLATE LAYOUT Below is a suggested plate layout for running standards, controls, and up to 36 samples in duplicate. 1 2 3 A Std A Std A Zero B Std B Std B Ctrl 1 C Std C Std C Ctrl 2 D Std D Std D Ctrl 3 1 E Std E Std E 2 F Std F Std F Std G Std G 3 G 4 H Std H Std H Std= Standard Ctrl = Control Numbered wells = Samples

4

5

6

7

8

9

10

11

12

Zero Ctrl 1 Ctrl 2 Ctrl 3 1 2 3 4

5 6 7 8 9 10 11 12

5 6 7 8 9 10 11 12

13 14 15 16 17 18 19 20

13 14 15 16 17 18 19 20

21 22 23 24 25 26 27 28

21 22 23 24 25 26 27 28

29 30 31 32 33 34 35 36

29 30 31 32 33 34 35 36

Proinsulin (Intact) Chemiluminescence ELISA

Page 11 of 12

3.0 – June 06, 2015

APPENDIX Instrument settings: Please contact the microplate reader manufacturer’s technical services department for additional assistance. These instrument settings are to be used as a guideline. It is optional to shake the plates before reading for ≤ 3 seconds. Molecular Devices Spectramax L Read Mode: Luminescence Integration Time: 1 second (1000 ms) Sensitivity: PMT: MaxRange Target Calibration Wavelength: 470 nm Automix: Classic: 30 mm/s Automix before read: Off Plate Type: 96 well standard Injection and Delay: Off Injection wells: None Dark Adapt: Off AutoRead: Off

Molecular Devices Spectramax M5 Read Mode: Luminescence (LUM) Read Type: Endpoint Wavelength: All Plate Type: 96 well standard opaque Read Area: Variable based on experiment PMT and Optics: Integration Time 1000 ms Shake: Off More Settings: Calibrate (on); Carriage Speed (Normal); Read Order (Column); Setting Time (off)

Biotek Synergy2 Detection Method: Luminescence Read Type: Endpoint Integration: 0:01.00 (MM:SS.ss) (1 second) Emission: Hole Optics Position: Top Sensitivity: 150 Top Probe Vertical Offset: 1.00mm

Tecan Infinite 200 Plate: Corning 96 Flat Bottom Black Polystyrol Mode: Luminescence Attenuation: NONE Integration Time: 1000 ms Settle Time: 0 ms

Proinsulin (Intact) Chemiluminescence ELISA

Page 12 of 12

3.0 – June 06, 2015