Cetuximab (Erbitux ) ADA ELISA
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1 1 Somru BioScience Quality & Speed. Delivered. Catalog SBA For the qualitative determination of antibodies to cetuximab in serum and plasma MANUFACTURED AND DISTRIBUTED BY: Somru BioScience Inc. 65 Watts Ave West Royalty Industrial Park Charlottetown, PE C1E 2B7 TEL: Fax: Web:
2 2 TABLE OF CONTENTS INTRODUCTION... 3 PRINCIPLE OF THE ASSAY... 3 MATERIALS & STORAGE... 4 SAFETY PRECAUTIONS... 5 PREPARATION OF REAGENTS... 5 SPECIMEN COLLECTION AND STORAGE... 6 ASSAY PROCEDURE... 7 KEY STEPS... 8 CALCULATIONS & RESULTS... 8 QUALITY CONTROLS... 9 REFERENCES... 9
3 3 INTRODUCTION Cetuximab (Erbitux ) is a chimeric IgG1 monoclonal antibody that binds the extra-cellular domain of the epidermal growth factor receptor (EGFR). It is a 152-kDa molecule composed of four polypeptide chains: two identical heavy chains and two identical light chains, consisting of 449 and 214 amino acids, respectively, bound by covalent and non-covalent bonds. The bond with EGFR is characterized by a higher affinity (Kd ¼ nm) than either endogenous ligand, as epidermal growth factor (EGF), or transforming growth factor alpha. This binding inhibits activation of the receptor tyrosine kinase and the associated downstream signaling that includes the mitogen-activated protein kinase, phosphoinositide 3-kinase/Akt and the Janus kinases/ signal transducers and activator of transcription (Stat) pathways. Furthermore cetuximab induces antibody-mediated receptor dimerization, internalization and degradation leading to receptor down-regulation. In addition, it exhibits antibody-dependent cellular cytotoxicity that could contribute to its antitumor effect. In various clinical trials cetuximab has been reported to generate an antibody response. Both binding and neutralizing antibodies against cetuximab have been reported. The formation of binding or neutralizing antibodies to a therapeutic agent may decrease the efficacy of these agents, leading to a loss of clinical response over time. In some cases, anti-drug antibodies may cause infusion reactions and serious anaphylactic reactions. Detection, measurement and characterization of anti-therapeutic antibodies are critical in understanding the safety, exposure and efficacy profile of the therapeutic agent. The Somru Cetuximab ELISA kit is designed for the qualitative determination of antibodies to cetuximab 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-cetuximab antibodies. The Somru Cetuximab ELISA kit can be used for monitoring anti- cetuximab antibodies during clinical research and offers the scientists a tool for understanding of the safety and efficacy of the cetuximab and its biosimilars. PRINCIPLE OF THE ASSAY This assay employs the double antigen sandwich enzyme immunoassay technique. The therapeutic antibody is coated onto a 96 well microplate. Quality control (QC) samples and test samples are pipetted into the appropriate wells. Anti-therapeutic antibodies present in biological matrices are bound by the immobilized therapeutic antibody. After washing away any unbound substances, biotin conjugated therapeutic antibody is added to the wells. Following a wash to remove any unbound reagent, an enzymelinked streptavidin is added to all the wells. The plate is washed to remove any unbound streptavidinenzyme reagent and a substrate solution is added to the wells for color development. The color development is proportional to the amount of anti-therapeutic antibody present in test samples. The color development is stopped and the intensity of the color is measured.
4 4 If a high level of therapeutic antibody is expected to be present in test samples, an acid dissociation step may be incorporated to minimize the impact of the therapeutic antibody interference in anti-therapeutic antibody detection. For further details, please contact our technical service team at tec@somrubioscience.com. This kit is designed for screening anti-therapeutic antibodies. The assay can be customized to include a confirmatory assay. If a confirmatory assay is required, please contact our technical service team at tec@somrubioscience.com. MATERIALS & STORAGE Store kit components at 2-8⁰C 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. The kit includes: Coated Microtiter Plate(s), 96 wells (1x8 strips) Wash Buffer (20X) Positive Control Concentrate Negative Control Concentrate Store at -20⁰C Assay Buffer Biotinylated Reagent (4,000X) Detection Reagent (20,000X) TMB TMB Stop Solution Do not mix or substitute reagents with those from other lots. Materials and instruments required but not supplied: Precision pipettes calibrated to deliver μl Multi-channel pipette calibrated to deliver μl Plate shaker Disposable tips Vortex mixer Distilled or de-ionized water Microplate reader capable of reading 450 nm with background subtraction at 650 nm Plate sealers
5 5 SAFETY PRECAUTIONS 1. The test protocol must be followed strictly. 2. 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. 3. 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. 4. Any liquid that has been brought into contact with potentially infectious material has to be discarded in a container with a disinfectant. 5. Disposal must be performed in accordance with local regulations. 6. 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. Wash Buffer (1X) Preparation: Dilute wash buffer concentrate with ultra-pure water 1/20 before use (for example add 1 ml concentrate to 19 ml ultra-pure water). Mix well. Prepare fresh on the day of use. 2. Preparation of Positive Controls: a. High Positive Control Preparation (RECOMMENDED): Dilute positive control concentrate 1/100 with assay buffer (for example: Add 5 µl of positive control to 495 µl of assay buffer). Mix well. Prepare fresh on the day of use. b. Low Positive Control Preparation (RECOMMENDED): Dilute high positive control 1/10 with assay buffer (for example: Add 25 µl of high positive control to 225 µl of assay buffer). Mix well. Prepare fresh on the day of use. 3. Biotinylated Reagent Preparation (1X) : Dilute biotinylated reagent with assay buffer 1/4,000 before use (for example use a two-step dilution process. First add 5 µl of concentrate to 495 µl of assay buffer, this will give a 1/100 dilution. Add 250 µl of the 1/100 dilution to 9750 µl assay buffer. This will give a final dilution of 1/4,000). Mix well. Prepare fresh on the day of use. 4. Detection Reagent Preparation (1X): Dilute detection reagent with assay buffer 1/20,000 before use use (for example use a two-step dilution process. First add 5 µl of concentrate to 495 µl of assay buffer, this will give a 1/100 dilution. Add 50 µl of the 1/100 dilution to 9950 µl assay buffer. This will give a final dilution of 1/20,000). Mix well. Prepare fresh on the day of use.
6 6 SPECIMEN COLLECTION AND STORAGE This kit is compatible with EDTA, heparin or citrate plasma and serum samples. Serum: To collect serum use a serum separator tube (SST) and allow the whole blood to clot for 25 to 35 minutes. Then centrifuge blood for 15 minutes at 1000x g and remove serum immediately. Plasma: To collect plasma use EDTA, heparin, or citrate as an anticoagulant. Centrifuge whole blood for 15 minutes at 1000x g within 30 minutes of collection and remove plasma immediately. Sample storage: 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 9 months. Avoid the repeated freezethaw of samples. We have demonstrated sample stability up to four freeze-thaw cycles however, we strongly recommend each lab to generate their own stability data. Sample Pre-treatment (optional): If a high level of rheumatoid factor (RF) is expected to be present in the test samples, a sample pre-treatment step may be incorporated to minimize the impact of RF in the detection of anti-therapeutic antibody. Please contact our technical service team at tec@somrubioscience.com for details. Avoid using samples with gross hemolysis or lipemia.
7 7 ASSAY PROCEDURE Steps Step 1 Step 2 Step 3 Step 4 Step 5 Step 6 Step 7 Step 8 Step 9 Step 10 Description Remove coated microtiter plate(s) from 2-8⁰C and allow it to acclimate to room temperature for minutes. Return unused strips immediately to the foil pouch with the desiccant packs and reseal along the entire edge of zip-seal. Store until expiration date at 2-8 C Dilute negative control and test samples 1/10 with assay buffer. For example add 25 μl control or test sample to 225 μl of assay buffer. Mix well. Do not store diluted controls or test samples. Add 100 μl of diluted controls and samples to appropriate wells on the plate. Incubate for approximately 1 hour at room temperature on a plate shaker at approximately 300 rpm. Discard the content of the plate and wash the wells 3x with 250 μl of wash buffer per well. Add 100 μl of prepared biotinylated reagent to appropriate wells on the plate. Incubate for approximately 1 hour at room temperature on a plate shaker at approximately 300 rpm. Discard the content of the plate and wash the wells 3x with 250 μl of wash buffer per well. Add 100 μl of prepared detection reagent to appropriate wells on the plate. Incubate for approximately 1 hour at room temperature on a plate shaker at approximately 300 rpm. Discard the content of the plate and wash the wells 3x with 250 μl of wash buffer per well. Add 100 μl of TMB to each well on plate. Incubate for approximately 5 minutes at room temperature protected from light. Add 100 μl of TMB stop solution to each well on plate. Mix by gently tapping the side of the plate. Step 11 Determine absorbance with a microplate reader at 450 nm against 620 nm.
8 8 KEY STEPS Allow plate to acclimate to room temperature minutes Dilute Controls and Test Samples 1/10 Add Controls and Samples Incubate 1 hour Wash plate Incubate 1 hour Add Biotinylated reagent Wash plate Incubate 1 hour Add Detection reagent Wash plate Add TMB Incubate 5 minutes Add TMB Stop Solution Read plate at 450 nm against 620 nm CALCULATIONS & RESULTS 1. The results are reported as positive or negative relative to a pre-determined cutpoint. 2. The cutpoint may be determined in each plate by running 3-6 negative controls (naïve individual human samples). The cutpoint is calculated by calculating the mean of the negative controls and by adding 2*Standard deviations to the calculated mean. 3. An alternative cutpoint may be calculated to fit the purpose of the study as described in G. Shankar, et al. (2008).
9 9 QUALITY CONTROLS Good Laboratory Practice (GLP) requires that positive and negative quality control (QC) samples be included in each run to check the assay performance. Each laboratory should prepare and assay these 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 the FDA Bioanalytical guidance. REFERENCES 1. "Remicade Becomes First Anti-TNF Biologic Therapy to Treat One Million Patients Worldwide" (Press release). Johnson & Johnson. November 6, "Cetuximab Product Approval Information - Licensing Action". Drugs@FDA. U.S. Food and Drug Administration (FDA). 3. G. Shankar, et al. (2008). Recommendations for the Validation of Immunoassays Used for Detection of Host Antibodies Against Biotechnology Products. J. Pharmaceutical and Biomedical Analysis 48:
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