Insulin Aspart ELISA Kit
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1 Insulin Aspart ELISA Kit Catalogue DEIABL215 For the qualitative determination of antibodies to insulin aspart in human serum and plasma FOR RESEARCH USE ONLY Creative Diagnostics. All rights reserved Ramsey Road Shirley, NY 11967, USA Tel: Fax:
2 INTRODUCTION Insulin aspart (Novolog / Novorapid ) is a fast-acting insulin analog with an earlier onset of action than regular human insulin. It is produced through recombinant DNA technology so that the amino acid, B28, which is normally proline, is substituted with an aspartic acid residue. This analog has increased charge repulsion, which prevents the formation of hexamers, to create fasteracting insulin. The sequence is inserted into the yeast genome, and the yeast expressed the insulin analog, which is then harvested from a bioreactor. Insulin aspart is indicated to improve glycemic control in adults and children with diabetes mellitus. Due to the fast acting nature it, the dosage of insulin aspart must be individualized. When it is given by subcutaneous injection, it should generally be used in regimens with an intermediate or long-acting insulin Because of NovoLog s comparatively rapid onset and short duration of glucose lowering activity, some patients may require more basal insulin and more total insulin to prevent pre-meal hyperglycemia when using insulin aspart than when using human regular insulin. In various clinical trials, insulin aspart has been reported to generate an antibody response. Increases in anti-insulin antibody titers that react with both human insulin and insulin aspart have been observed in patients treated with NovoLog. Increases in anti-insulin antibodies are observed more frequently with NovoLog than with regular human insulin. Data from a 12- month controlled trial in patients with type 1 diabetes suggest that the increase in these antibodies is transient. The formation of 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 CD Insulin aspart ADA ELISA kit is designed for the quantitative determination of antibodies to Insulin aspart in serum and plasma. Our ELISA-based kits combine a fast, user-friendly format with a sensitive and specific assay. The assay is designed to detect human IgG, IgM and IgA against Insulin aspart. 2
3 PRINCIPLE OF THE ASSAY This assay employs the 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 protein. After washing away any unbound substances, enzyme conjugated detection antibody is added to the wells. The plate is washed to remove any unbound enzyme conjugated reagent and a substrate solution is added to the wells for colour development. The colour development is proportional to the amount of anti-therapeutic antibody present in test samples. The colour development is stopped and its intensity is measured. 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 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. 5. Only trained laboratory personnel should execute this test. 3
4 MATERIALS & STORAGE 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: Coated Microtiter Plate(s) (1x8 strips) Coating Protein (1500X) Positive Control Concentrate Control Diluent* Detection Reagent (1000X)* Coating Buffer (10X) Blocking Buffer Wash Buffer (20X) Assay Buffer TMB TMB Stop Solution * Store at -15 to -30⁰C upon receipt. Avoid repeated freeze-thaw. Materials and instruments required but not supplied: Microplate reader ( nm) Plate shaker Distilled or de-ionized water Disposable tips Precision pipettes ( μl) Vortex mixer Multi-channel pipette ( μl) Adhesive plate sealers 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 1.2 ml concentrate to 10.8 ml of ultra-pure water for a final volume of 12 ml). Mix well. Do not store excess solution. 4
5 2. Coating Solution (1X) Preparation: Dilute coating protein concentrate with prepared coating buffer 1/1500 before use (for example, add 8 µl concentrate to ml of prepared coating buffer for a final volume of 12 ml). Mix well. Do not store excess solution. 3. Wash Buffer (1X) Preparation: Dilute wash buffer concentrate with ultra-pure water 1/20 before use (for example, add 1.25 ml concentrate to ml of ultra-pure water for a final volume of 25 ml). Mix well. Store at 2 to 8 C for up to 1 week. 4. Preparation of Positive Controls: a. High Positive Control Preparation (RECOMMENDED): Dilute positive control concentrate with control diluent 1/2 (for example, add 50 µl of concentrate to 50 µl of control diluent for a final volume of 100 µl). Mix well. Prepare fresh on the day of use. b. Low Positive Control Preparation (RECOMMENDED): Dilute positive control concentrate with control diluent 1/20 (for example, add 5 µl of concentrate to 95 µl of control diluent for a final volume of 100 µl). Mix well. Prepare fresh on the day of use. 5. Detection Reagent (1X) Preparation: Dilute detection reagent concentrate with assay buffer 1/1000 before use (for example, add 12 µl of concentrate to ml of assay buffer for a final volume of 12 ml). Mix well. Prepare fresh on day of use. 5
6 SPECIMEN COLLECTION & STORAGE 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 minutes. Then centrifuge blood for 15 minutes at 1000 x 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 1000 x g and remove plasma immediately. Sample Storage: Store assay samples immediately after collection or aliquot and store them at or below -20 C for up to 1 year. Avoid repeated freezethaw of samples. We have demonstrated sample stability up to five freezethaw cycles; we strongly recommend that each lab generate their own stability data. Avoid using samples with gross hemolysis or lipemia. ASSAY PROCEDURE Step 1 Add 100 µl of prepared coating solution 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 Step 4 Step 5 Step 6 Discard plate contents and wash 3x with 300 μl of wash buffer per well. Tap dry on paper towel. Dilute prepared controls and test samples 1/100 with assay buffer (e.g. 5 μl control/sample to 495 μl buffer, total volume 500 μl). Mix well. Do not store diluted controls or test samples. Add 100 µl of diluted controls/samples to the plate. Incubate for approx. 1 hour at RT, shaking at approx. 300 rpm. Discard plate contents and wash 3x with 300 μl of wash buffer per well. Tap dry on paper towel. 6
7 Step 7 Step 8 Step 9 Add 100 µl of prepared detection reagent to the plate. Incubate for approx. 1 hour at RT, shaking at approx. 300 rpm. Discard plate contents and wash 3x with 300 μl of wash buffer per well. Tap dry on paper towel. Add 100 µl of TMB to the plate. Incubate for approx 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 Add coating solution Incubate 1 hour Tap plate dry Add blocking buffer Incubate 1 hour Wash plate Dilute controls and test samples 1/100 Add controls and samples Incubate 1 hour Wash plate Add detection reagent Incubate 1 hour Wash plate Add TMB Incubate 5-10 minutes Add TMB stop solution Read plate at 450 nm with background correction at 620 nm 7
8 CALCULATIONS & RESULTS 1. The results are reported as positive or negative relative to a predetermined cut-point. 2. The cut-point may be determined on each plate by running 3-6 negative controls (naïve 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). QUALITY CONTROLS Good Laboratory Practice (GLP) requires both 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 FDA Bioanalytical Guidance. 8
9 REFERENCES 1. Bolli, G. et. al. (1999). "Insulin analogues and their potential in the management of diabetes mellitus.". Diabetologia 42 (10): Kaplan, W.; et al. (2004). "Effects of Mixing Insulin aspart and Short- Acting Insulin Analogs on Glucose Control". Diabetes Care 27 (11): doi: /diacare PMID EPAR Lantus, German summary of admission report of EMEA (PDF) 4. L. Raymond Reynolds, MD, FACP, FACE, ECNU (January 2010). "Comparing Insulins Detemir and Insulin aspart in Type 2 Diabetes: More Similarities than Differences". Postgraduate Medicine 122 (1): European Medicines Agency update on safety of insulin Insulin aspart, July 23, 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: Creative Diagnostics. All rights reserved Ramsey Road Shirley, NY 11967, USA Tel: Fax: info@creative-diagnostics.com 9
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