ELISA/assay

Exendin-4 (Exenatide ® ) - ADA ELISA

DEIABL206

96T

Exendin-4 (Exenatide ® ) - ADA ELISA

Exendin 4

Exendin-4 (Exenatide ® ) - ADA ELISA

sELISA

Exendin 4

Human

Quantitative

Serum, plasma

96T

The kit is shipped at ambient temperature and should be stored at 2-8°C. Keep away from heat or direct sun light. The storage and stability of specimen and prepared reagents is stated in the corresponding chapters. The microtiter strips are stable up to the expiry date of the kit in the broken, but tightly closed bag when stored at 2–8°C.

Exendin-4

Human

For the quantitative determination of free antibodies to exendin-4 in human serum and plasma.

Exendin-4 is a hormone found in the saliva of the Gila monster. It displays biological properties similar to human glucagon-like peptide-1 (GLP-1), a regulator of glucose metabolism and insulin secretion. Exenatide is a synthetic version of exendin-4 approved by the FDA on April 28, 2005 for patients whose diabetes was not controlled well with oral medication. Exenatide is a 39-amino-acid peptide that enhances glucose-dependent insulin secretion by the pancreatic beta-cell, suppresses inappropriately elevated glucagon secretion, and slows gastric emptying. In clinical trials, Exenatide has been reported to generate antibody response. In some clinical trials, up to 45% of the patients formed low titer antibodies and up to 12% of the patients formed high-titer antibodies. Both binding and neutralizing antibodies against Exenatide have been reported. The formation of binding or neutralizing antibodies to therapeutic agents may decrease the efficacy of these agents leading to losses of clinical responses over time. In some cases, anti-drug antibodies may cause infusion, and serious anaphylactic reactions. Given the sequence similarities between Exenatide and GLP-1, anti-Exenatide antibodies may cross-react with GLP-1 and glucagon. This may interfere with the measurement of Exenatide in biological matrices. Accurate measurement of Exenatide is critical in understanding the safety, exposure, and efficacy of the drug. The Exendin-4 (Exenatide®) ELISA kit is designed for the qualitative determination of free antibodies to exendin-4 in serum and plasma. Our ELISA-based kits combine a fast, user-friendly format with a sensitive and specific assay. This assay is capable of detecting all isotypes of anti-exendin-4 antibodies. The kit can be used for monitoring anti-exendin-4 antibodies during research and offers the scientists a tool for understanding the safety and efficacy of exendin-4 and it's biosimilars.

This assay is a bridging type ELISA for the determination of free antibodies against the drug exendin-4 in serum and plasma samples. Standards and test samples are incubated in the presence of the biotinylated and horseradish peroxidase (HRP)-conjugated exendin-4 as capture and detection reagent, respectively. ADA, if present in sample, will make a bridge. Immune complexes are then transferred to a streptavidin-coated microtiter plate and incubated to immobilize immune complexes. Following incubation, wells are washed, and the bound enzymatic activity is detected by addition of tetramethylbenzidine (TMB) chromogen-substrate. Finally, the reaction is terminated with stop solution. The positive reaction is expected to be related to the presence of ADA in the sample.

1. Microtiter Plate . Break apart strips pre-coated with streptavidin. 12 × 8 wells 2. ADA Standards (20 μg/mL) . Used for construction of the standard curve. Contains anti-drug antibody, preservative and stabilizer. The quantity is indicated on the vial label and/or QC datasheet. 1 × 100 μL 3. TRACER (1 μg) , Lyophilized. Contains biotinylated exendin-4, preservative and stabilizer. The quantity is indicated on the vial label and/or QC datasheet. 1 × 1 Vial 4. CONJ (250×) , Concentrate (250×). Contains horseradish peroxidase (HRP) -conjugated exendin-4, preservative and stabilizer. The quantity is indicated on the vial label and/or QC datasheet. 1 × 100 μL 5. Blocking Buffer , Ready to use. Contains proteins and preservative. 1 × 20 mL 6. Dilution Buffer , Ready to use. Contains proteins and preservative. 1 × 50 mL 7. Wash Buffer , Concentrate (20×). Contains buffer, Tween-20. 1 × 50 mL 8. TMB Substrate Solution , Ready to use. Contains 3,3',5,5'-Tetramethylbenzidine (TMB). 2 × 6 mL 9. Stop Solution , Ready to use. 0.5 M Sulfuric Acid (H 2 SO 4 ). 1 × 7 mL

1. Micropipettes ( 2. Ultrapure water and calibrated glass wares. 3. Wash bottle, automated or semi-automated microtiter plate washing system. 4. Microtiter plate reader capable of reading absorbance at 450 nm and 620 nm. 5. Absorbent paper towels, standard laboratory glass or plastic tubes, a timer and a shaker.

This kit is compatible with EDTA, heparin or citrate plasma, and serum samples. Samples can be stored at or below -20°C for up to 1 year. Serum: To collect serum, use a serum separator tube and allow the whole blood to clot for 25-35 minutes. Then centrifuge blood for 15 minutes at 1500 ×g and remove serum immediately. Plasma: To collect plasma, use EDTA, heparin, or citrate as an anticoagulant. Within 30 minutes of whole blood collection, centrifuge for 15 minutes at 1500 ×g and remove plasma immediately. Sample Storage: Store assay samples immediately after collection, or aliquot and store at or below -20°C for up to 1 year. Avoid repeated freeze-thaw. We have demonstrated sample stability up to five freeze-thaw cycles. We strongly recommend each lab generate their own stability data. Avoid using samples with gross hemolysis or lipemia.

Bring all reagents to room temperature (15-30°C) prior to use. 1.To run the assay more than once, ensure that reagents are stored at conditions stated on the label. Prepare only the appropriate amount necessary for each run. The kit can be used up to 4 times within the expiry date stated on the label. 2. Preparation of the Wash Buffer (1×) : Dilute Wash Buffer to 1× with ultrapure Water before use (50 mL Wash Buffer (20×) + 950 mL ultrapure water). Mix well. The Wash Buffer is stable at 2-8°C until the expiry date stated on the label. Wash buffer (1×) can be stored in a closed flask at 2-8°C for 1 month. 3. Preparation of Tracer (80 ng/mL) : The Tracer (1 μg) is provided as a lyophilized stock. Reconstitute it with 1 mL ultrapure water as a 1 μg/ml stock and vortex gently. The reconstituted reagent should be aliquoted and stored below -20°C. After addition of ultrapure water, diluent prepared 1 μg/ml stock to 80 ng/ml with Dilution Buffer (for example, add 0.4 mL 1 μg/ml stock to 4.6 mL of Dilution Buffer for 80 ng/ml in 5 mL). Mix well. Tracer (80 ng/mL) is not stable and cannot be stored. 4. Preparation of Conjugate (1×) : Dilute CONJ (250×) to 1× with Dilution Buffer before use(20 μL CONJ (250×) + 4.98 mL Dilution Buffer). Mix well. Conjugate (1×) is not stable and cannot be stored. 5. Preparation of Working Solution : A few minutes before use, add 5 mL Conjugate (1×) to 5 mL of Tracer (80 ng/mL). Mix well. Working Solution is not stable and cannot be stored. All reagents should be balance to room temperature (20°C-25°C) before use. If crystals have formed in buffer solution, worm to room temperature until the crystals have completely dissolved.

Procedure Notes 1. Any improper handling of samples or modification of the test procedure may influence the results. The indicated pipetting volumes, incubation times, temperatures and pre-treatment steps have to be performed strictly according to the instructions. Use calibrated pipettes and devices only. 2. Once the test has been started, all steps should be completed without interruption. Make sure that required reagents, materials and devices are prepared readily at the appropriate time. Allow all reagents and specimens to reach room temperature (20-25 °C) and gently swirl each vial of liquid reagent and sample before use. Mix reagents without foaming. 3. Kits are validated using shakers set at 200 rpm and room temperature. Performance of the assay at lower temperatures and/or mixing speeds will likely result in lower absorbance values. 4. Avoid contamination of reagents, pipettes and wells/tubes. Use new disposable plastic pipette tips for each reagent, standard or specimen. Do not interchange the caps of vials. Always cap not used vials. Do not reuse wells or reagents. 5. Use a pipetting scheme to verify an appropriate plate layout. 6. Incubation time affects results. All wells should be handled in the same order and time sequences. It is recommended to use an 8-channel Micropipette for pipetting of solutions in all wells. 7. Microplate washing is important. Improperly washed wells will give erroneous results. It is recommended to use a multichannel pipette or an automatic microplate washing system. Do not allow the wells to dry between incubations. Do not scratch coated wells during rinsing and aspiration. Rinse and fill all reagents with care. While rinsing, check that all wells are filled precisely with Wash Buffer, and that there are no residues in the wells. 8. Humidity affects the coated wells. Do not open the pouch until it reaches room temperature. Unused wells should be returned immediately to the resealed pouch including the desiccant. Procedure 1. Preparation of ADA standards and samples All the test samples and ADA standards must be diluted with Working Solution. a.Preparation of ADA Standards: Diluent ADA Standard (20 μg/ml) with Working Solution as described below. b.Preparation of test samples: Diluent test samples with Working Solution . The recommended dilution rate of the sample is 1:20 (for example, add 12.5 μL sample to 237.5 μL of Working Solution ). 2. Dispense 250 μL of diluted standards and samples into tubes (we recommend using 2 mL tubes). All the tubes are incubated overnight (16-20 h) at room temperature (RT) on a shaker (200 rpm). Avoid light. 3. Pipette 150 μL of Blocking Buffer into each of the wells to be used, cover plate with adhesive seal. Shake plate carefully. Incubate 1 hours at 37°C . 4. Discard the content of each well and wash 3 times with 350 μL wash buffer. After the final washing step, remove residual wash buffer by firmly tapping the plate on absorbent paper. 5. Add 100 μL of preincubated standards/samples (in step 2) into each well (we recommend testing in duplicate), cover plate with adhesive seal. Shake plate carefully. Incubate 30 min at RT . 6. Discard the content of each well and wash 5 times with 350 μL wash buffer. After the final washing step, remove residual wash buffer by firmly tapping the plate on absorbent paper. 7. Pipette 100 μL of Ready-to-Use TMB Substrate Solution into each well. Incubate 15 min at RT. Avoid exposure to direct sunlight. 8. Stop the substrate reaction by adding 50 μL of Stop Solution into each well. Briefly mix contents by gently shaking the plate. Color changes from blue to yellow. 9. Determine absorption immediately with an ELISA reader at 450 nm against 620 nm as a reference.

The test results are only valid if the test has been performed following the instructions. Moreover, the user must strictly adhere to the rules of GLP (Good Laboratory Practice) or other applicable standards/laws. All standards/controls must be found within the acceptable ranges as stated above and/or label. If the criteria are not met, the run is not valid and should be repeated. In case of any deviation, the following technical issues should be reviewed: Expiration dates of (prepared) reagents, storage conditions, pipettes, devices, incubation conditions and washing methods.

Qualitative 1. The results are reported as positive or negative relative to a pre-determined cut-point. 2. The cut-point may be determined on each plate by running 3-6 negative controls (Drug-naïve individual human samples) and calculating their mean, then adding 2* standard deviation to this calculated mean. 3. If the "1:20 diluted sample OD450 nm" is less ( 4. If the "1:20 diluted sample OD450 nm" is equal and higher (≥) than the "cut-point value", the sample is regarded as POSITIVE for Anti-Drug-Antibody (ADA) specific for the drug in concern. Quantitative 1. For the calculation of the positive sample concentration, polynomial regression is recommended. Standard curve is constructed by plotting the units of the 8 standard points (ng/mL) along the abscissa (X axis) and the corresponding OD450 nm values along the ordinate (Y axis). 2. The Anti-Drug Antibody (ADA) concentration of positive samples can be reported based on the principles indicated below. 3. The ADA concentration of pre-diluted (at the ratio of 1:20) positive samples can be directly read on the standard curve. In order to report the ADA concentration for the corresponding undiluted positive sample, the value must be multiplied by the factor of 20. Example: If a 1:20 pre-diluted positive sample results in an ADA concentration of 100 ng/mL on the standard curve, then its undiluted sample value corresponds to 2000 ng/mL (100 ng/mL x 20 = 2000 ng/mL). 4. If any diluted positive sample is reading OD450 nm higher than that of highest standard supplied, it should be further diluted (in such a case 1:100 dilution of the sample is proposed) appropriately with Working Solution and then retested. Also this second dilution has to be used for calculation of the final result. It is recommended that each laboratory determines the best dilution ratio for their samples in order to minimize retesting.

For anti-exendin-4 antibodies in serum and plasma, the method has been demonstrated to be highly (>0.99) linear from 0 to 1000 ng/mL.

The Detection Threshold for the assay is 1.74 ng/mL. The assay sensitivity for undiluted clinical samples corresponds to 34.89 ng/mL, Because the serum or plasma samples are instructed to be diluted at 20-fold (1:20) before starting the assay.

Recovery rate was found to be 80-120% using native serum and plasma samples spiked with exogenous Anti-Drug Antibody (ADA) positive samples.

1. Before starting the assay, read the instructions completely and carefully. Use the valid version of the package insert provided with the kit. Be sure that everything is understood. 2. Obey lot number and expiry date. Do not mix reagents of different lots. Do not use expired reagents. 3. All reagents should be handled as if potentially infectious. Operators should wear gloves and protective clothing when handling any patient sera or serum-based products. 4. Some reagents within the kit may contain antimicrobial agents, and acid. Avoid contact with the skin and eyes. Rinse immediately with plenty of water if any contact occurs. 5. Any liquid brought into contact with potentially infectious material needs to be discarded in a container with a disinfectant. Disposal must be performed in accordance with local regulations. 6. Only trained laboratory personnel should execute this test.

For the quantitative determination of free antibodies to exendin-4 in human serum and plasma.

No. Components Size Storage Conditions 1. Microtiter Plate 1×12×8 2-8°C 2. ADA Standards (20 μg/mL) 1 × 100 μL 2-8°C 3. TRACER (1 μg), lyophilized 1 × 1 Vial 2-8°C 4. CONJ (250×), concentrate (250×) 1 × 100 μL 2-8°C 5. Blocking Buffer, ready to use 1 × 20 mL 2-8°C 6. Dilution Buffer, ready to use 1 × 50 mL 2-8°C 7. Wash Buffer, concentrate (20×) 1 × 50 mL 2-8°C 8. TMB Substrate Solution, ready to use 2 × 6 mL 2-8°C 9. Stop Solution, ready to use 1 × 7 mL 2-8°C

See individual product datasheet

0.5M

See individual product datasheet

See individual product datasheet

Exenatide is a 39-amino-acid peptide that enhances glucose-dependent insulin secretion by the pancreatic beta-cell, suppresses inappropriately elevated glucagon secretion, and slows gastric emptying. The Exendin-4 (Exenatide®) ELISA kit is designed for the qualitative determination of free antibodies to exendin-4 in serum and plasma. Our ELISA-based kits combine a fast, user-friendly format with a sensitive and specific assay. This assay is capable of detecting all isotypes of anti-exendin-4 antibodies. The kit can be used for monitoring anti-exendin-4 antibodies during research and offers the scientists a tool for understanding the safety and efficacy of exendin-4 and it's biosimilars.