Catalog: DEIABL218. For the qualitative determination of antibodies to pegfilgrastim in human serum or plasma. For Research Use Only

Transcription

1 Pegfilgrastim ADA ELISA Kit Catalog: DEIABL218 For the qualitative determination of antibodies to pegfilgrastim in human serum or plasma. For Research Use Only 1

2 INTRODUCTION Filgrastim is a granulocyte colony-stimulating factor (G-CSF) analog used to stimulate the proliferation and differentiation of granulocytes[1]. It is produced by recombinant DNA technology. Pegfilgrastim is a pegylated form of the recombinant human granulocyte colonystimulating factor (GCSF) analog filgrastim. It serves to stimulate the level of white blood cells (neutrophils). Granulocyte-colony stimulating factor (G-CSF) is a growth factor and an essential cytokine belonging to the CSF family of hormone hematopoietic cell proliferation and differentiation. Filgrastim is used to treat neutropenia,[2] stimulating the bone marrow to increase production of neutrophils. The causes of neutropenia include chemotherapy and bone marrow transplantation. Pegylated filgrastim treatment can be used to stimulate bone marrow to produce more neutrophils to fight infection in patients undergoing chemotherapy. The current research effort towards the development of biosimilars and development of therapeutic agents for second-generation treatment of neutropenia requires a selective and sensitive bioanalytical method. The CD pegylated filgrastim ELISA kit is designed for the quantitative measurement of pegylated filgrastim in serum and plasma. Our ELISA-based kits combine a fast, user-friendly format with a sensitive and specific assay. The CD pegylated filgrastim ELISA kit can be used for monitoring antibodies to pegylated filgrastim during clinical research and offers scientists a tool for understanding the safety and efficacy of the pegylated filgrastim and its biosimilars. PRINCIPLE OF THE ASSAY This assay employs the sandwich enzyme immunoassay technique. The therapeutic protein is coated onto a 96 well microplate. Quality control (QC) samples and test samples are pipetted into the appropriate wells. Anti-pegfilgrastim 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 color development. The color development is proportional to the amount of anti-pegfilgrastim antibody present in test samples. The color development is stopped and the intensity of the color is measured. If a high level of pegfilgrastim is expected to be present in test samples, an acid dissociation step may be incorporated to minimize the impact of the pegfilgrastim interference in antipegfilgrastim antibody detection. 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: Coated Microtiter Plate(s), 96 wells (1x8 strips) Negative Control Concentrate Wash Buffer (20X) Reagent Diluent (1000X) Assay Buffer TMB Positive Control Concentrate 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 Adhesive plate sealer 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 legislation. 6. Only trained laboratory personnel should execute this test. 3

4 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 1/25 before use (for example add 1 ml concentrate to 19 ml ultra-pure water). Mix well. Store at 2-8 C for up to 1 week. 2. Preparation of Controls: a. High Positive Control (Recommended): Dilute positive control concentrate 1/280 with assay buffer before use (for example: add 5 µl positive control concentrate to 1395 µl assay buffer). Mix well. Do not store. b. Low Positive Control (Recommended): Dilute high positive control 1/4 with assay buffer before use (for example: add 25 µl positive control concentrate to 75 µl assay buffer). Mix well. Do not store. 3. Detection Reagent (1X) Preparation: Dilute detection reagent 1/1000 with assay buffer before use (for example add 12 µl of concentrate to 12 ml assay buffer). Mix well. Do not store. SPECIMEN COLLECTION AND PREPARATION 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 1000 x g and remove serum immediately. Assay samples immediately or aliquot and store them below -20 C. Avoid repeated freeze thaw. Plasma: To collect plasma use EDTA, heparin, or citrate as an anticoagulant. Centrifuge whole blood for 15 minutes at 1000 x 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 12 months. Avoid repeated freeze thaw samples. Avoid repeated freeze-thaw cycles. We have demonstrated samples stability up to four freeze-thaw cycles. However we strongly recommend each lab to generate their own stability data. Avoid using samples with gross hemolysis or lipemia. 4

5 ASSAY PROCEDURE Steps Step 1 Step 2 Step 3 Step 4 Step 5 Step 6 Step 7 Step 8 Description Remove coated microtiter plate(s) from 2-8 C and allow it to acclimate to room temperature for minutes. Return unused strips immediately to the foil pouch with the dessicant packs and reseal along the entire edge of zip-seal. Store until expiration date at 2-8 C Dilute prepared controls and samples 1/100 with assay buffer. For example add 5 µl control/sample to 495 µl of assay buffer. Mix well. Do not store diluted test samples Add 100µL of diluted controls and test samples to appropriate wells on the plate. Incubate for approximately 1 hours at room temperature on a plate shaker at approximately 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 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 250μL of wash buffer per well Add 100µL of TMB to each well on plate. Incubate for 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 5

6 KEY STEPS Allow plate to acclimate to room temperature Dilute Calibrators and Samles 1/100 Add Diluted Calibrators and Samples Incubate 1 hour Wash Plate Add Detection Reagent Incubate 20 minutes Wash Plate Add TMB Incubate 10 minutes Add TMB Stop Solution Read plate at 450nm against 620nm CALCULATION & RESULTS 1. The results are reported as positive or negative relative to a pre-determined cut-point. 2. The cut-point may be determined in each plate by running 3-6 negative controls ( naïve individual human samples). The cut-point is calculated by calculating the mean of the negative controls and by adding 2*Standard deviations to the calculated mean. 6

7 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. Three levels of QCs are recommended for this purpose. Each laboratory should assay these QCs and any other control materials repeatedly (at least 6 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. Ho, R.J.Y., Gibaldi, M. Biotechnology and biopharmaceuticals: transforming proteins and genes into drugs Wiley-IEEE, 2003, p. 139, Bondarenko, I, Gladkov, O, Elsaesser, R, Buchner, A and Bias, P (2013). "Efficacy and safety of lipegfilgrastim versus pegfilgrastim: a randomized, multicenter, active-control phase 3 trial in patients with breast cancer receiving doxorubicin/docetaxel chemotherapy". BMC Cancer 2013, 13: Neulasta: Patient Information Leaflet". Amgen. Retrieved 24 June "NEULASTA Patient Guide". Amgen. Retrieved 22 June 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: