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 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 employs the double antigen sandwich enzyme immunoassay technique. Exendin-4 is coated onto a 96 well microplate. Quality control (QC) samples and test samples are pipetted into the appropriate wells. Anti- exendin-4 antibodies present in biological matrices are bound by the immobilized exenatide. After washing away any unbound substances, a horseradish peroxidase-linked reagent is added to all the wells. The plate is washed to remove any unbound reagent and a substrate solution is added to the wells for colour development. The colour development is proportional to the amount of anti-exendin-4 antibodies present in test samples. The colour development is stopped and its intensity is measured.

107-188 ng/mL

Store kit components at 2 to 8°C unless specified otherwise. DO NOT USE past kit expiration date. Some vials contain a small amount of reagent. Spin tubes on pulse setting prior to opening. Do not mix or substitute reagents with those from other lots. Each kit includes: §Microtiter Plates (1x8 strips) § Coating Buffer (10X) §Coating Protein (200X)* §Blocking Buffer §Positive Control (100 ug/mL) §Wash Buffer (20X) §Control Diluent* §Assay Buffer §Detection Reagent (1000X)* §TMB Substrate §TMB Stop Solution * Store at -15 to -30?C upon receipt. Avoid repeated freeze-thaw.

§Microplate reader (450-620 nm) §Plate shaker § Distilled or de-ionized water §Disposable tips §Precision pipettes (5-1000 μL) §Vortex mixer § Multi-channel pipette (50-200 μL) §Adhesive plate sealers

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): Dilute Coating Buffer to 1X with Distilled or De- Ionized Water (for example, add 1.2 mL Buffer (10X) to 10.8 mL of Water for 1X in 12 mL). Mix well. Do not store. 2. Coating Solution (1X): Dilute Coating Protein to 1X with prepared Coating Buffer (1X) (for example, add 60 μL Protein (200X) to 11.94 mL of Buffer (1X) for 1X in 12 mL). Mix well. Do not store. 3. Wash Buffer (1X): Dilute Wash Buffer to 1X with Distilled or De- Ionized Water before use (for example, add 1.25 mL Buffer (20X) to 23.75 mL of Water for 1X in 25 mL). Mix well. Store at 2 to 8°C for up to 1 week. 4. Preparation of Positive Controls: a. High Positive Control (HPC) (500 ng/mL) (RECOMMENDED): Dilute Positive Control to 500 ng/mL with Control Diluent (for example, add 5 μL of Control (100 μg/mL) to 995 μL of Diluent for 500 ng/mL in 1 mL). Mix well. Do not store. b. Low Positive Control (LPC) (100 ng/mL) (RECOMMENDED): Dilute prepared HPC to 100 ng/mL with Control Diluent (for example, add 100 μL of HPC (500 ng/mL) to 400 μL of Diluent for 100 ng/mL in 500 μL). Mix well. Do not store. c. Negative Control (NC) (0 ng/mL): This assay uses Control Diluent as the negative control. 5. Detection Reagent (1X): Dilute Detection Reagent to 1X with Assay Buffer (for example, add 12 μL of Reagent (1000X) to 11.988 mL of Buffer for 1X in 12 mL). Mix well. Do not store.

When stored at 2°C to 8°C unopened reagents will be stable until expiration date. Do not use reagents beyond this date. Opened reagents must be stored at 2°-8°C. After first opening the reagents are stable for 30 days if used and stored properly. Microtiter wells must be stored at 2°C to 8°C. Take care that the foil bag is sealed tightly. Protect TMB-Substrate Solution from light.

ASSAY PROCEDURE Step 1 Add 100 μL of prepared Coating Solution (1X) to the plate. Incubate for approx. 1 hour at RT, shaking at approx. 300 rpm. Step 2 Discard plate contents and tap dry on paper towel. Add 200 μL Blocking Buffer to the plate. Incubate for approx. 1 hour at RT, shaking at approx. 300 rpm. Step 3 Discard plate contents and wash 3x with 300 μL of prepared Wash Buffer (1X) per well. Tap dry on paper towel. Step 4 Dilute prepared Controls and Test Samples 1/50 with Assay Buffer (e.g. 10 μL Control/Sample in 490 μL Buffer, total volume 500 μL). Mix well. Do not store. Step 5 Add 100 μL of diluted Controls/Samples to the plate. Incubate for approx. 2 hours at RT, shaking at approx. 300 rpm. Step 6 Discard plate contents and wash 3x with 300 μL of prepared Wash Buffer (1X) per well. Tap dry on paper towel. Step 7 Add 100 μL of prepared Detection Reagent (1X) to the plate. Incubate for approx. 1 hour at RT, shaking at approx. 300 rpm. Step 8 Discard plate contents and wash 3x with 300 μL of prepared Wash Buffer (1X) per well. Tap dry on paper towel. Step 9 Add 100 μL of TMB Substrate to the plate. Incubate for 10-15 minutes at RT protected from light. Step 10 Add 100 μL of TMB Stop Solution to the plate. Mix by gently tapping the side of the plate. Step 11 Determine absorbance with microplate reader at 450 nm with background correction at 620 nm. KEY STEPS Step 1 Add Coating Solution (1X) ◆ Incubate 1 hour ◆ Tap plate dry Step 2 Add Blocking Buffer ◆ Incubate 1 hour ◆ Wash plate Step 3 Dilute Controls and Test Samples 1/50 Step 4 Add diluted Controls and Samples ◆ Incubate 2 hours ◆ Wash plate Step 5 Add Detection Reagent (1X) ◆ Incubate 1 hour ◆ Wash plate Step 6 Add TMB Substrate ◆ Incubate 10-15 minutes Step 7 Add TMB Stop Solution ◆ Read plate at 450 nm with background correction at 620 nm CALCULATIONS & RESULTS 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 (naive individual human samples) and calculating their mean, then adding 2* standard deviation to this calculated mean. 3. An alternative cut-point may be calculated to fit the purpose of the study as described in G. Shankar, et al. (2008).

Product Datasheet Protocol