FOR INFORMATIONAL USE ONLY ▪ DO NOT USE FOR PERFORMING ASSAY ▪ REFER TO MOST CURRENT PACKAGE INSERT ACCOMPANYING TEST KIT

EPO (Erythropoietin) ELISA

Human EPO (Erythropoietin) ELISA Cat. No: KBBA01 Ver1.0

ELISA for Accurate Quantitation from Cell Culture Supernatant, Serum, Plasma, or Other Bodily Fluids

For Research Purposes Only. Not for use in diagnostic or therapeutic procedures. Purchase does not include or carry the right to resell or transfer this product either as a stand-alone product or as a component of another product. Any use of this product other than the permitted use without the express written authorization of KRISHGEN BioSystems is strictly prohibited.

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EPO (Erythropoietin) ELISA Introduction: Erythropoietin (EPO) is a heavily glycosylated protein with a molecular weight of about 30,000 - 34,000 Daltons. Human EPO is a polypeptide consisting of 165 amino acids, containing one O-linked and three Nlinked carbohydrate chains. The recombinant EPO is a good substitute for the native protein for use in an immunoassay. Intended Use: The EPO ELISA is intended for the quantitative determination of Erythropoietin (EPO) in cell culture supernatants, serum, plasma or other bodily fluids. Principle: The method employs the quantitative sandwich enzyme immunoassay technique. Samples, Controls and standards are pipetted into precoated Streptavidin Microwell. Anti-EPO antibody linked to Biotin and Anti-EPO: HRP Conjugate is added to the wells simultaneously. EPO present in the sample and standards will form a complex with antibodies and bind to the Streptavidin-Coated plate. Washing is done to remove the unbound complex. The ready to use substrate solution (TMB) is added to microwells and color develops directly proportional to the amount of EPO in the standards and sample. Color development is then stopped by addition of stop solution. Absorbance is measured at 450 nm. Materials Provided: 1. Streptavidin Coated Microtiter Plate (96 wells) – 1 no 2. Recombinant Human EPO Standard 0 mIU/ml - 4 ml 3. Recombinant Human EPO Standard (2ml/vial) - 10.1, 25.1, 51, 158 and 456 mIU/ml 4. Biotinylated EPO Antibody - 3.5 ml 5. Anti-EPO : HRP Conjugate - 3.5 ml 6. Control 1 – 2 ml 7. Control 2 – 2 ml 8. Wash Buffer (20X) – 30 ml 9. TMB Substrate – 20 ml 10. Stop Solution – 20 ml 11. Instruction Manual Materials to be provided by the End-User: 1. Microplate reader capable of reading at 450nm and 405nm. 2. Microplate washer. 3. Precision Pipettors to deliver 25, 200, 100 and 150 µL. 4. Timer. 5. Distilled or Deionized water. 6. Orbital rotator or shaker. 7. Linear graph Paper Reagent Preparation and Storage: 1. Store all kit components at 2-8 oC. 2. All reagents except the Standards, kit controls and the Wash Concentrate are ready-to-use. Store all reagents at 2-8 oC.

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EPO (Erythropoietin) ELISA 3. Use the standards and controls as soon as possible upon reconstitution. Freeze (-15oC) the remaining standards and controls as soon as possible after use. Standards and controls are stable at -15 oC for 6 weeks after reconstitution with up to 3 freeze thaw cycles when handled as recommended in "Procedural Notes" section. Health Hazard Warnings: 1. Reagents that contain preservatives may be harmful if ingested, inhaled or absorbed through the skin. Refer to the MSDS online for details. 2. To reduce the likelihood of blood-borne transmission of infectious agents, handle all serum and/or plasma in accordance with NCCLS regulations. Procedural notes 1. Samples that have values below the limit of detection (1.1 mIU/mL) should be reported as " 494 mIU/mL ". 4. Reagents from different lot numbers must not be interchanged. 5. If preferred, mix in equal volumes, in sufficient quantities for the assay, Biotinylated Antibody and Enzyme Labeled Antibody in a clean amber bottle. The combined reagent is stable for seven (7) days when stored at 4oC. Then use 50 µL of the mixed antibody into each well. This alternative method should replace Step (3) and (4), to be followed with the incubation. 6. When mixing avoid splashing of reagents from wells. This will affect assay precision and accuracy.

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EPO (Erythropoietin) ELISA Specimen Collection and Handling: Serum: Collect whole blood without anticoagulant and allow blood to clot between 2-8°C for 30 mins, if possible. It has been reported that serum samples clotted at room temperature (22 oC to 28oC) caused a decrease in EPO value as assessed by radioimmunoassay of about 30% over clotting on ice. It is highly recommended that the specimen be collected between 7:30 a.m. to 12:00 noon, because diurnal variation of erythropoietin has been reported in literature. Centrifuge the serum sample preferably in a refrigerated centrifuge for 15 mins. at 700 – 800 x g. Store at -20°C if the samples are not used on the same day. Avoid repeated freezing and thawing of the same sample. Plasma: Collect blood in tubes containing anticoagulants. Centrifuge samples at 700 – 800 x g. Collect the plasma and dispense into aliquots of 400µl or more. Store at -20°C. Avoid repeated freezing and thawing of the same sample. Cell Culture Supernatant: Centrifuge samples at 700 – 800 x g. collect the supernatant and store at 2 - 8° C. If the samples are not used the same day, it is recommended to dispense the supernatant into aliquots and store at -20°C. Avoid repeated freezing and thawing of the sample. Reagent Preparation: 1. For Zero Standard (Standard A) reconstitute vial with 4 mL of distilled or deionized water and mix. For each of the non-zero Standards (Standard B through H) and kit controls 1 and 2, reconstitute each vial with 2 mL of distilled or deionized water and mix. Allow the vials to stand for 10 minutes and then mix thoroughly by gentle inversion to insure complete reconstitution. 2. Add 30 ml Wash Buffer (20X) to 570ml of distilled water to make 1X working concentration. The diluted wash solution is stable for 90 days when stored at room temperature. Assay Procedure: 1. Place sufficient Streptavidin Coated Strips in a holder to run Standards, Controls and patient samples. 2. Pipette 200 µL of Standards, controls and samples into the designated well. 3. Add or dispense 25 µL of Biotinylated EPO Antibody into each of the wells, which already contain the Standards, controls and samples. 4. Add or dispense 25 µL of Anti-EPO: HRP Conjugate into each of the same wells. Cover the microplate(s) with aluminum foil or a tray to avoid exposure to light, and place it on an orbital shaker or rotator set at 170 ± 10 rpm for 2 hours ±15 minutes at room temperature (22 -28oC). 5. Aspirate and wash plate 5 times with Wash Buffer (1X) and blot residual buffer by firmly tapping plate upside down on absorbent paper. 6. Add or dispense 150 µL of the TMB Substrate into each of the wells. 7. With appropriate cover to avoid light exposure, place the microplate(s) on an orbital shaker or rotator set at 170 + 10 rpm for 30 ± 5 minutes at room temperature (22o-28oC). 8. Add or dispense 100 µL of the Stopping Solution into each of the wells. 9. Read the absorbance of the solution in the wells within 10 minutes, using a microplate reader set to 450 nm against 250 µL of distilled or deionized water. Read the plate again with the reader set to 405 nm against distilled or deionized water.

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EPO (Erythropoietin) ELISA 10. By using the obtained final absorbance values, construct two calibration curves using 405 nm reading and 450 nm reading via cubic spline, 4 parameter logistics, or point-to-point interpolation to quantify the concentration of EPO. Calculation of Results: 1. For the 450 nm readings, construct a dose response curve using the first five Standards provided, i.e. Standards A, B, C, D and E. For the 405 nm readings, construct a second dose response curve using Standards A, D, E and F. Construct a dose response curve (calibration curve) using Standards A, B, C, D and E. 2. Assign the concentration for each Standard stated on the vial in mIU/mL. Plot the data from the calibration curve on linear graph paper with the concentration on the X-axis and the corresponding A.U. on the Y-axis. 3. Draw a straight line between 2 adjacent points. Obtain the concentration of the sample by locating the absorbance unit on the Y-axis and finding the corresponding concentration value on the X-axis. 4. Computer programs using cubic spline or 4 PL (4 Parameter Logistics) or Point-to-Point can generally give a good fit. Typical Data: This standard curve was generated at KRISHGEN for demonstration purposes only. A standard curve must be run with each assay.

Precautions: Do not mix reagents from different kits or lots. Reagents and/or antibodies from different manufacturers should not be used with this set. Quality Control: It is recommended that for each laboratory assay appropriate quality control samples in each run to be used to ensure that all reagents and procedures are correct.

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EPO (Erythropoietin) ELISA Troubleshooting Problem

Possible cause

Investigation/Actions

High Absorbances

1. Cross-contamination from other specimens

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Repeat assay taking care when washing and pipetting.

2. Insufficient or inefficient washing or reading

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Check washer efficiency

3. Wavelength of filter not correct.

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4. High assay background.

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5. Contaminated TMB

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Check that the wavelength is 450nm. If a dual wavelength spectrophotometer is available, set the reference filter between 600-650 nm. Repeat assay and include a well that contains only sample diluent or sample absorbent (i.e. a blank well). Check that TMB is colorless or faint blue.

6. Incubation time too long or incubation temperature too high.

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Check incubation time and temperature. Check incubator is at the correct temperature.

7. Incorrect dilution of serum

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Repeat assay, ensuring correct serum dilution is used.

1. Incubation time too shot or incubation temperature too low. 2. Incorrect dilution or pipetting of sera

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Ensure time and temperature of assay incubation are correct Check incubator is set at the correct temperature.

3. Incorrect filter wavelength.

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4. Contaminated Conjugate solution.

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Low Absorbances

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5. Kit has expired.

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Repeat assay ensuring correct dilutions and volumes are used Ensure controls are sufficiently mixed. Check the wavelength is set at 450nm. If a dual wavelength spectrophotometer is available, set the reference filter between 600-650nm. Dispense conjugate directly from the bottle using clean pipette tip; avoid transferring Conjugate to another container if possible. Do not return unused Conjugate to bottle. Ensure all pipettes and probes used to dispense the Conjugates are clean and free from serum, detergent and bleach. Check expiration date of kit and do not use if expired.

6. Air blank reading high

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Investigate causes of high background absorbance.

7. Incorrect storage of kit.

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10. Over washing of plate (e.g. inclusion of a long soak step).

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Ensure kit is stored at 2-8ºC,plate is sealed in foil pouch and desiccant sachet is blue/purple. Allow sufficient time for reagents to equilibrate to room temperature prior to assay. Check the reagents used match those listed on the specification sheet. Repeat assay using recommended wash procedure.

1. Poor mixing of samples.

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Mix reagents gently and equilibrate to room temperature.

2. Poor pipette precision

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3. Addition of reagents at inconstant timing intervals; reagent addition takes too long, air bubbles when adding reagents.

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Calibration may need to be checked. Check pupating technique-change pipette tip for each sample and ensure excess liquid is wiped from the outside of the tip. Use consistent timing when adding reagents. Ensure all dilutions are made before commencing addition to plate. Improve pipetting technique and skill. Tap out wash buffer after washing. Check wells are sufficiently and uniformly filled and aspirated when washing. Check reader precision Check reader manual to ascertain warm up time of instrument. Gently wipe bottom of plate. Check reader light source and detector are clean.

8. Kit reagents not equilibrated temperature 9. Incorrect reagents used.

Poor Duplicates

at

room

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4. Inefficient washing - Wash buffer left in wells, inconsistent washing, inadequate washing.

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5. Reader not calibrated or warmed up prior to plate reading.

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6. Optical pathway not clean

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7. Spillage of liquid from wells

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Cat No# KBBA01, Ver1,0

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EPO (Erythropoietin) ELISA

All wells Yellow

8. Serum samples exhibit microbial growth, haemolysis or lipaemia.

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It is not recommended to use serum samples exhibiting microbial

9. Uneven well volumes due to evaporation.

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Cover plate with a lid or plate sealer (not provided).

1. Contaminated TMB.

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Check TMB is colorless or faint blue.

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Check reagents for turbidity.

3. Incorrect dilution of serum.

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Repeat assay, ensuring correct serum dilution is used.

4. Incorrect storage of kit.

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Ensure kit is stored at 2-8ºC, plate is sealed in foil pouch and desiccant sachet is blue / purple.

5. Inefficient washing- Wash buffer left in wells, inconsistent washing, inadequate washing. 6. If conjugate reconstitution is requiredConjugate reconstituted incorrectly.

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Tap out wash buffer after washing. Check wells are sufficiently and uniformly filled an aspirated washing. Repeat assay ensuring Conjugate is reconstituted according to assay method.

1. Test not performed correctly – correct reagents not added or not added in the correct sequence

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2. Contaminated reagents Wash buffer

(e.g. Conjugate,

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All wells Negative

 2. Contaminated Conjugate solution.

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Check procedure and check for unused reagents. Ensure that Stop Solution was not added before Conjugate or TMB. Ensure that serum was diluted in correct Sample diluent; e.g. do not use Sample Absorbent for an IgG ELISA. Dispense Conjugate directly from the bottle using a clean pipette tip; avoid transferring Conjugate to another container if possible. Do not return unused Conjugate to bottle. Ensure all pipettes and probes used to dispense the Conjugate are clean and free from serum, detergent and bleach. Repeat assay using recommended wash procedure.

3. Over- washing of plate (e.g. inclusion of a long soak step).

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4. Incorrect storage of kit.

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Ensure kit is stored at 2-8ºC, plate is sealed in foil pouch and desiccant sachet is blue / purple.

5. Wash Buffer made up with Stop Solution instead of Wash Buffer Concentrate

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Ensure Wash Buffer is made up correctly.

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EPO (Erythropoietin) ELISA LIMITED WARRANTY Krishgen Biosystems does not warrant against damages or defects arising in shipping or handling, or out of accident or improper or abnormal use of the product; against defects in products or components not manufactured by Krishgen Biosystems, or against damages resulting from such non-Krishgen Biosystems made products or components. Krishgen Biosystems passes on to customer the warranty it received (if any) from the maker thereof of such non- Krishgen made products or components. This warranty also does not apply to product to which changes or modifications have been made or attempted by persons other than pursuant to written authorization by Krishgen Biosystems. THIS WARRANTY IS EXCLUSIVE. The sole and exclusive obligation of Krishgen Biosystems shall be to repair or replace the defective product in the manner and for the period provided above. Krishgen Biosystems shall not have any other obligation with respect to the products or any part thereof, whether based on contract, tort, and strict liability or otherwise. Under no circumstances, whether based on this Limited Warranty or otherwise, shall Krishgen Biosystems be liable for incidental, special, or consequential damages. This Limited Warranty states the entire obligation of Krishgen Biosystems with respect to the product. If any part of this Limited Warranty is determined to be void or illegal, the remainder shall remain in full force and effect. Krishgen Biosystems. 2014

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