Exendin-4 (Exenatide) ELISA Kit

Transcription

1 Exendin-4 (Exenatide) ELISA Kit Catalog: DEIABL227 For the quantitative determination of Exendin-4 in serum or plasma using competitive ELISA method For Research Use Only. Protocol Provided for Informational Use Only. 1

2 INTRODUCTION 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 well-controlled on oral medication [1]. 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 [2]. 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 reactions and serious anaphylactic reactions. Given the sequence similarities between exenatide and GLP-1, anti-exenatide antibodies may cross-react with GLP-1 and glucagon [3]. 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 CD Exendin-4 ELISA kit is designed to measure exenatide in biological matrices with high specificity and enhanced sensitivity. The assay is based on the principles of competitive enzyme immunoassay. An optional acid dissociation step can be incorporated to improve drug tolerance of the assay if a high amount of anti-exenatide antibodies is expected to be present in biological samples PRINCIPLE OF THE ASSAY The ELISA plate is coated with an anti-species antibody. The plate is blocked and the primary anti-exendin- 4 antibody is added to the plate. The primary antibody binds both biotinylated exenatide and the exendin- 4 in the samples. The biotinylated peptide interacts with streptavidin conjugated to horseradish peroxidase (SA-HRP). The substrate solution is added to the wells for color development. The color development is inversely proportional to the amount of exendin-4 present in test samples. The color development is stopped and the intensity of the color is measured. The concentration of exendin-4 is calculated using a standard curve of known concentrations. 2

3 MATERIALS & STORAGE Store kit components at 2-8 C unless specified otherwise. DO NOT USE past kit expiration date. Some vials contain a small amount of reagents. Spin tubes on pulse setting prior to opening. Each kit includes Microtiter Plate(s), 96wells (1x8 strips) Coating Buffer (10X) Blocking Buffer (1000X) Primary Antibody (400X) Wash Buffer (20X) Assay Buffer Calibrator (500 µg/ml) Calibrator Diluent Biotinylated Exenatide (100,000X) recommend to aliquot and store at -20 C; avoid repeated freeze/thaws Detection Reagent (10,000X) recommend to aliquot and store at -20 C; avoid repeated freeze/thaws TMB TMB Stop Solution Do not mix or substitute reagents with those from other lots. Materials and instruments required but not supplied Coating Protein Precision pipettes calibrated to deliver μL Multi-channel pipette calibrated to deliver μL Plate shaker Disposable tips Vortex-mixer Distilled or deionized water Plate sealers Microplate reader capable of reading 450nm with background subtraction at 620nm 3

4 SAFETY PRECAUTIONS The test protocol must be followed strictly. All reagents containing human material should be handled as if potentially infectious. Operators should wear gloves and protective clothing when handling any patient sera or serum based products. The kit reagents contain antimicrobial agents, acid and 3,3,5,5 -tetramethylbenzidine. Avoid contact with the skin and eyes. Rinse immediately with plenty of water if any contact occurs. Any liquid that has been brought into contact with potentially infectious material has to be discarded in a container with a disinfectant. Disposal must be performed in accordance with local legislation. Only trained laboratory personnel should execute this test. PREPARATION OF REAGENTS Prepare only the appropriate amount of required reagent on the day of use. Store all reagents as per instructions stated on the label. 1. Coating Buffer (1X) Preparation: Dilute coating buffer concentrate with ultra-pure water 1/10 before use (for example add 1.2m L concentrate to 10.8 ml ultra-pure water). Mix well. 2. Wash Buffer (1X) Preparation: Dilute wash buffer concentrate with ultra-pure water 1/20 before use (for example add 1.25mL concentrate to ml ultra-pure water). Mix well. 3. Coating Solution (1X) Preparation: Dilute coating protein concentrate with coating buffer before use (for example add 12uL coating antibody to 11988uL of coating buffer). Mix well. 4. Primary Antibody (1X) Preparation: Dilute primary antibody with assay buffer 1/400 before use (for example add 30μL concentrate to 11970µL of assay buffer). Mix well. 5. Biotinylated Exenatide (1X) Preparation: Dilute biotinylated exenatide with assay buffer 1/100,000 before use (for example use a two-step dilution: add 5µL concentrate to 495µL of assay buffer [1/100], then add 12µL diluted biotinylated exendin-4 to 11988uLof assay buffer [1/1,000]. Final concentration 1/100,000). Mix well. 6. Detection Reagent (1X) Preparation: Dilute reagent concentrate with assay buffer 1/10,000 before use (for example use a two-step dilution: add 5 μl concentrate to 495μL of assay buffer [1/100 ]; then add 12μL diluted reagent to 11988uLof assay buffer [1/1 00 ]. Final concentration 1/10,000). Mix well. 7. Preparation of Calibrators: Prepare Calibrators with desired concentrations ranging from 1000ng/mL to 0.5ng/mL using provided calibrator diluent. DO NOT SOTRE.The following is an example of calibration cure, where the stock calibrator is 150ng/mL.Mix well. 4

5 Calibration Curve Solution ID Intermediate 1 Source Stock Calibrator (500 µg/ml) Source Volume (μl) Calibrator Diluent (μl) Final Volume (µl) Final Concentration ,000 5,000 ng/ml Calibrator 1 Stock Calibrator ng/ml Calibrator 2 Calibrator ng/ml Calibrator 3 Calibrator ng/ml Calibrator 4 Calibrator ng/ml Calibrator 5 Calibrator ng/ml Calibrator 6 Calibrator ng/ml Calibrator 7 Calibrator ng/ml Calibrator 8 Calibrator Diluent ng/ml SPECIMEN COLLECTION & STORAGE This kit is compatible with plasma, preferably EDTA plasma. Protease Inhibitor: The peptide must be protected from proteolysis during assay procedures and sample storage. Collect blood samples into vacutainer tubes containing anti-coagulant, preferably EDTA. Gently mix the contents immediately after collection of blood. Transfer the blood from the vacutainer tubes to the centrifuge tubes containing aprotinin (0.6 TIU /ml of blood) and gently mix several times to inhibit the activity of proteinases. Centrifuge the blood at 1,600 x g for 15 minutes at 4 C and collect the plasma. If vacutainer tubes are centrifuge-safe, the aprotinin may be added into the initialcollection step. Care must be taken when using heparin as an anticoagulant, since an excess will provide falsely high values. Use no more than 10 IU heparin per ml of blood collected. Serum: To collect serum,use a serum separator tube(sst) and allow the whole blood to clot for 25 to 30 minutes. Then centrifuge the blood for 15 minutes at 1000xg 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 1000xg and remove plasma immidiately. Sample Storage: Store assay samples immediately after collection or aliquot and store them below -20 C. Samples can be stored at or below -20 C for up to 6 months. Avoid repeated freeze-thaw samples. We have demonstrated sample stability up to four freeze-thaw cycles however, we strongly recommend the each lab generates their own stability data. Avoid using samples with gross hemolysis or lipemia. 5

6 ASSAY PROCEDURE Steps Step 1 Step 2 Step 3 Step 4 Step 5 Step 6 Step 7 Step 8 Step 9 Step 10 Step 11 Step 12 Step 13 Description Add 100µL of prepared coating antibody to wells on plate. Incubate for 1 hour at room temperature on a plate shaker at 300rpm Discard the contents of the plate and add 200µL of blocking buffer to wells on plate. Incubate for 1 hour at room temperature on a plate shaker at 300rpm Discard the content of the plate and wash the wells 3x with 250μL of wash buffer per well Add 100µL of prepared primary antibody to each well on plate. Incubate for 1 hour at room temperature on a plate shaker at 300rpm Discard the content of the plate and wash the wells 3x with 250μL of wash buffer per well Add 50µL of prepared calibrators and samples to the appropriate wells on the plate Add 50µL of prepared biotinylated exenatide to the well containing calibrator or samples on the plate. Mix by carefully pipetting up and down. Incubate for 1 hour at room temperature on a plate shaker at 300rpm Discard the content of the plate and wash the wells 3x with 250μL of wash buffer per well Add 100µL of prepared detection reagent to each well on plate. Incubate for 1 hour at room temperature on a plate shaker at 300rpm Discard the content of the plate and wash the wells 3x with 250μL of wash buffer per well Add 100µL of TMB to each well on plate. Incubate for 5-10 minutes at room temperature protected from light Add 100µL of TMB stop solution to each well on plate. Mix by gently tapping the side of the plate Determine absorbance with a microplate reader at 450nm against 620nm 6

7 KEY STEPS Add Coating Protein Add Blocking Buffer Wash Plate Add Primary Antibody Wash Plate Add Samples/Calibrators Plus Biotinylated Exenatide Wash Plate Add Detection Reagent Wash Plate Incubate Add TMB 5-10 Minutes Add TMB Stop Solution Read Plate at 450nm against 620nm CALCULATIONS & RESULTS 1. Construct a standard curve by plotting the absorbance obtained from each calibrator (disregarding the zero calibrator) against its concentration with absorbance value on the vertical (Y) axis and concentration on the horizontal (X) axis. Use a 4 or 5 parameter curve fit with appropriate weighting. 2. The concentration of the samples can be read directly from this standard curve using the absorbance value for each sample Determine the corresponding concentration of the analyte from the standard curve. 3. Any sample, undiluted or diluted, still reading greater than the highest standard should be further diluted appropriately with calibrator diluent and retested. If the samples have been diluted, the concentration determined from the standard curve must be multiplied by the dilution factor. 7

8 QUALITY CONTROLS Good Laboratory Practice (GLP) requires that quality control (QC) samples be included in each run with each standard curve to check the assay performance. Each laboratory should prepare and test at least three levels of QCs repeatedly (at least 3 times) to establish mean values and acceptable ranges. Each individual laboratory is responsible for defining their system for quality control decisions and is also responsible for making this system a written part of their laboratory procedures. It is strongly recommended to use 4-6-x criteria to accept or reject a run based on in FDA Bioanalytical guidance. REFERENCES 1. CDER Drug and Biologic Approvals for Calendar Year 2005, from the U.S. Food and Drug Administration. 2. Fineman MS, Mace KF, Diamant M, Darsow T, Cirincione BB, Booker Porter TK, Kinninger LA, Trautmann ME. (August 28, 2008). Clinical relevance of anti-exenatide antibodies: safety, efficacy and cross-reactivity with long-term treatment. Diabetes Obes Metab Jun;14(6): E. Petrella, G. Heintz (2004). Validation of an ELISA Used to Determine Anti-Exenatide Antibody Cross-reactivity to GLP-1 and Glucagon in Human Plasma Samples. AAPS Poster session. 4. US FDA, Guidance for Industry: Bioanalytical Method Validation. US Department of Health and Human Services, US FDA, Center for Drug Evaluation and Research, Rockville, MD USA (2001). 8