APOB (Human) ELISA Kit

Transcription

1 APOB (Human) ELISA Kit Catalog Number KA assays Version: 01 Intended for research use only

2 Table of Contents Introduction... 3 Intended Use... 3 Background... 3 Principle of the Assay... 3 General Information... 4 Materials Supplied... 4 Storage Instruction... 4 Materials Required but Not Supplied... 4 Precautions for Use... 5 Assay Protocol... 6 Reagent Preparation... 6 Sample Preparation... 6 Assay Procedure... 7 Data Analysis... 8 Calculation of Results... 8 Resources... 9 Trouble Shooting... 9 References Plate Layout KA / 11

3 Introduction Intended Use The APOB (Human) ELISA Kit (Apo-B Test Kit) is an enzyme linked immunosorbent assay (ELISA) for the quantitative determination of apolipoprotein B in human plasma. The kit allows up to 42 duplicate sample determinations. The Exocell Apo-B Kit is intended for research purposes, and not for in vitro clinical diagnostic use. Background High plasma cholesterol levels have been associated with increased risk of atherosclerosis, and plasma LDL correlates better with atherosclerosis than any other lipoprotein. LDL transports 70 % of the total cholesterol and is the most important lipoprotein in cholesterol metabolism. High plasma LDL concentrations lead to development of atherosclerosis. Apolipoprotein B is the principle protein component of LDL and comprises approximately 25 % of the LDL particle. The Exocell Apo-B Test Kit provides a rapid, accurate and simple means for quantitating apo B concentrations in plasma. Principle of the Assay The Exocell Apo-B Test Kit is a competitive ELISA that depends on the ability of a monoclonal antibody, B36, to recognize and bind to a specific epitope of native apo B within the LDL complex. Values are expressed in mg/dl apo B. To complete the assay, B36 monoclonal antibody is added to the microtiter plate well onto which apo B is immobilized, and apo B (in standard or sample) is present in the liquid phase. The monoclonal antibody binds to apo B in either the solid phase or in liquid phase during the primary incubation, hence the notion of competitive binding. Subsequent washing removes reactants in the liquid phase, and the antibody bound to the solid phase is detected with an HRP-conjugated goat anti-mouse antibody. The conjugate is detected by chromogenic reaction using TMB. Because it is a competitive assay, color intensity is inversely proportional to the concentration of apo B in the liquid phase. KA / 11

4 General Information Materials Supplied List of component Component LDL Assay Plate: A multistrip ELISA plate that is precoated with a standardized preparation of apolipoprotein B. 10 X LDL Wash Buffer: A proprietary wash buffer that must be diluted 1:10 with distilled water before use. LDL Diluent: A proprietary reagent for the dilution of standard and for the preparation of samples. LDL Standard: A lyophillized preparation that is used to standardize the assay. It must be reconstituted with 1.0 ml of distilled water immediately before use. One vial is supplied (see the work sheet for the apo B concentration after reconstitution). B36 Monoclonal Antibody: Supplied in ready-to-use form. HRP-Conjugate: Goat anti-mouse HRP conjugate is supplied in ready to use form. Color Developer: TMB (3, 3, 5, 5 Tetramethylbenzidine in buffer salts) is supplied in ready to use form. Acid Color Stopper: A single bottle of 2 N sulfuric acid is included to stop color development. Amount 96 (8x12) wells 100 ml 100 ml 1 vial 6.0 ml 12 ml 12 ml 12 ml Storage Instruction Store the kit at 4 C. Materials Required but Not Supplied Micropipettors that accurately dispense 10 μl, 50 μl, 100 μl and 640 μl. A multichannel pipettor for dispensing 50 μl and 100 μl. 1.5 ml microcentrifuge tubes or similar disposible tubes sample dilution. ELISA plate reader. KA / 11

5 Precautions for Use This Product is manufactured for Abnova by Exocell. Handle all kit components and patient samples as if they were capable of transmitting infectious agents. All human body fluids used as sources for reagents in this kit were screened for hepatitis B antigen and for HIV antibody, and were found negative. However, there is no test that can guarantee that the causative agents for hepatitis and AIDS are absent. All reagents except TMB Color Developer and Color Stopper contain 0.05 % isothiazalones as a preservative. Color Stopper contains dilute sulfuric acid.. KA / 11

6 Assay Protocol The following steps are presented for the standardization of the assay and analysis of six (6) experimental samples. This requires one 12 well strip. A larger number of assays involving multiple strips may be performed, and these instructions may serve as a guide. Reagent Preparation Warm LDL Diluent, LDL Wash Buffer, LDL Standard, B36 Antibody, HRP-Conjugate, and TMB Color Developer to room temperature before beginning the assay. Reconstitute the LDL Standard: Open the tear seal. Add 1.0 ml of distilled water using a 1 ml syringe equipped with a 26 gauge needle inserted through the stopper. Mix the contents by rolling the bottle and by inversion. Avoid foaming. Allow the mixture to rest for 15 minutes, and repeat the mixing procedure. Immediately before use, vortex, remove stopper and aspirate an aliquot for dilution. Add entire contents of 10X LDL Wash Buffer to 900 ml of distilled water and mix thoroughly. Sample Preparation Sample Collection and Preparation Draw fresh blood by venipuncture using either EDTA or citrate as an anticoagulant. Heparin is not suitable. Centrifuge the specimens as soon as possible, and aspirate off the plasma fraction. The fluid should be assayed as soon as possible. The best results are obtained with fresh plasma. Prepare dilutions of LDL Standard: Label five (5) microfuge tubes 0 through 4. Add 0.4 ml of reconstituted LDL Standard to tube 0. Pipet 0.2 ml of LDL Diluent into each of the remaining microfuge tubes (tubes 1-4). Transfer 0.2 ml from tube 0 to tube 1. Mix contents by repeated pipetting (5-10 times). Transfer 0.2 ml from tube 1 to tube 2. Mix as above. Continue diluting standard in this pattern to obtain a standard series of dilutions (undilute through 1:16). Prepare dilutions of experimental samples: Place 640 μl of LDL diluent into a microcentrifuge tube. Add 10 μl of experimental sample to the diluent. Wash the inside of the tip into the mixture by repeated aspiration/discharge, and discard tip. Mix the contents by vortexing, this is a 1:65 dilution of plasma. Repeat this manipulation for each experimental sample. KA / 11

7 Assay Procedure 1. Prepare the control and standard wells. Remove a 12 well strip from the plate. Drain the wells and wash five times with LDL Wash Buffer, filling the wells each time. Be sure to remove residual fluids by inverting the strip on absorbent paper and gently tapping it upon the bench top. Add 100 μl of LDL DILUENT to the left most well, A1. This is control C0, and will serve to "Blank" the plate reader. Discard the pipet tip. Pre-wet tip, and add 50 μl from LDL Standard tube 0 to well A2, discard tip. Pre-wet tip, and add 50 μl of diluted LDL Standard from tube 1 to well A3, discard tip. Pre-wet a fresh tip, and add 50 μl of diluted LDL Standard from tube 2 to well A4, discard tip. Pre-wet a fresh tip, and add 50 μl of diluted LDL Standard in tube 3 to well A5, discard tip. Pre-wet a fresh tip, and add 50 μl of diluted LDL Standard in tube 4 to well A6. Note: Wells A2-A6 contain serial two-fold dilutions of LDL STANDARD. 2. Prepare sample wells: Vortex first experimental sample tube, and transfer 50 μl well A7. Discard the tip. Repeat for each experimental sample in the remaining wells. 3. First Incubation Add 50 μl of B36 monoclonal antibody to each of the wells that contain either standard or sample except for well A1. Cover and incubate for 30 minutes at room temperature (approximately 23 C). 4. Second Incubation: Drain and wash strip 10 times as previously described. Add 100 μl of HRP-Conjugate to each well. Cover and incubate for 15 minutes at room temperature. 5. Color Development: Drain and wash strip 10 times as previously described. Blot as described above. Add 100 μl of TMB Color Developer to each well. Cover and develop for 10 minutes at room temperature. 6. Color Stopper Add 100 μl of Color Stopper to each well. Determine absorbance in a Microplate Reader at 450 nm; use well A1 as "blank." 7. Data Summary Table: Secure a copy of table 1 (see enclosure), and complete it for subsequent analysis. This table is based upon the concentration of Apo B of the standard enclosed in your kit. KA / 11

8 Data Analysis Calculation of Results Determine the Absorbance of experimental wells at 450 nm in respect to well A1 ("blank"). Calculate the average absorbances and enter the values in a photocopy of the enclosed worksheet. Prepare a semi-logarithmic plot of standard dilutions with the log [apo B] vs absorbance. The data that fall into the linear portion of the dose-response curve constitute the usable portion of the assay. Subject these data to semi-logarithmic analysis to yield a mathematical model, of the form log 10 [ apo B ] = m A b Determine the estimated [apo B] for each experimental sample. Multiply by 65 to correct for the dilution. KA / 11

9 Resources Trouble Shooting 1. No color appears after adding Color Developer: Peroxidase Conjugate was not added Kit contents were stored above 8o C or below freezing. 2. Color in wells is too light: Incubation with secondary Antibody-HRP conjugate was too short. Incubation with TMB was too short. 3. Color in wells too dark: Plate was developed too long with TMB, reduce development period appropriately or read plate at 405 nm. 4. Poor agreement between replicate wells: Reagents are above or below room temperature at the start of the assay, and unequal warming in plate during incubation has produced edge effects. Be sure to warm all reagents to room temperature before use! Poor control of sample delivery. Be sure to use calibrated pipettors, and that the tips are securely attached for the addition of samples to the wells. Further, pre-wetting of tips will improve accuracy. Washing steps were poorly completed and/or wells not blotted; wash plates thoroughly and blot completely. 5. Dark wells appear out of sequence: Well is contaminated with HRP conjugate when TMB is added; be sure to wash wells completely and blot thoroughly. Plate has been contaminated with peroxidase containing materials, i. e. airborne dust and dander; cover plate during incubations. 6. Poor correlation coefficient (rxy): Complete a graphical analysis as described (remember that the dose-response follows a semi-logarithmic relationship.) Omit any data that fall outside the linear portion of the dose-response curve. Re-analyze the data taking care that appropriate transformations (i. e. logarithms) have been made. KA / 11

10 References 1. Goldstein, J., Brown, MS. The low density lipoprotein pathway and its relation to atherosclerosis. Ann Rev Biochem 46: , Kukita, H., Hiwada, K, Kobuta, T. Serum apolipoprotein A-I, A-II, and B levels and their discriminitive value in relatives of patients with coronary artery disease. Atherosclerosis 51: , Mahley, RW, Innearity, TL, Rall, SC, Weisgraber, K. Plasma lipoproteins: apolipoprotein structure and function. J Lipid Res 25: , Naito, HK. The clinical significane of apolipoprotein B measurements. J Clin Immuno 9(1):11-19, Roscene, M, Vercaemest, R, Steinberg, K, Cooper, G. Some considerations of methodology and standardization of apolipoprotein B assays. Clin Chem 29(3): , KA / 11

11 A B C D E F G H Plate Layout KA / 11