Hepatitis B virus Pres1 Antigen ELISA Kit Catalog Number KA2118 96 assays Version: 01 Intended for research use only www.abnova.com
Introduction and Background A. Test Principle This kit was based on standard sandwich enzyme-linked immune-sorbent assay technology. Anti-Pres1 monoclonal antibodies were pre-coated onto 96-well plates. And the HRP conjugated anti-hbs polyclonal antibodies were used as detection antibodies. The test samples and HRP conjugated detection antibodies were added to the wells subsequently and then formed pre-coated antibody-pres1 antigen-hrp conjugated detection antibody complex. TMB substrate was used to visualize HRP enzymatic reaction. TMB was catalyzed by HRP to produce a blue color product that changed into yellow after adding acidic stop solution. The density of yellow is proportional to the PreS1 antigen amount of sample captured in plate. Read the O.D. absorbance at 450nm/630nm in a microplate reader. B. Application For qualitative detection of Hepatitis B Virus Pres1 Antigen in human serum or plasma. sales@abnova.com www.abnova.com 1
Material and Method A. List of Component 1. One 96-well plate pre-coated with anti-pres1 monoclonal antibody. 2. PreS1 antigen positive Control: 1ml 3. PreS1 antigen negative Control: 1ml 4. HRP conjugated anti-hbs polyclonal antibody (RTU): 6ml 5. Chromogenic reagent A (Carbamide peroxide): 6ml 6. Chromogenic reagent B (TMB): 6ml 7. Stop solution: 6ml 8. Wash buffer (20x) (Tris buffer including Tween 20): 50ml. Dilution: 1:20 9. 2 Sealers B. Additional Required Materials But Not Provided 1. Microplate reader (Dual wavelength: 450nm/630 nm) 2. Microplate oscillator 3. Automated plate washer 4. 37 C incubator 5. Double-distilled or deionized water. C. Sample Preparation and Storage Handle test samples (human serum and plasma) properly and analyze immediately, or store them at 2-8 C if analyze within 5 days, or store at -20 C. D. Storage and Expiration Store at 2-8 C in dark for one year. E. Protocol 1. Equilibrate kit components and test samples for 30 min at room temperature. 2. Dilute concentrated wash buffer 20-fold (1:20) with double-distilled or deionized water (i.e. add 50ml of wash buffer into 950ml of double-distilled or deionized water). 3. Set 1 zero well, 2 negative control and 2 positive control wells. Add 50µl of PreS1 negative and positive control into negative control and positive control wells respectively. 4. Add 50µl of test sample into each well of the assay plate. Cover plate with provided sealer and incubate at 37 C for 30 min. 5. Remove the sealer, discard the solution in the plate without touching the side walls, and wash plate. (Plate Washing Method: Fill each well with diluted wash buffer and soak for 15 seconds. Blot the plate onto paper towels or other absorbent material. Repeat this process four additional times for a total of FIVE washes. For automated washing, aspirate all wells and wash FIVE times with wash buffer, sales@abnova.com www.abnova.com 2
overfilling wells with diluted wash buffer. Blot the plate onto paper towels or other absorbent material.) 6. Except the zero well, add 50µl of HRP conjugated anti-hbs polyclonal antibody into each well, cover the plate with provided sealer and incubate at 37 C for 30 min. 7. Repeat Step 5. 8. Add 50µl of chromogenic reagent A into each well (including the zero well), subsequently, add 50µl of chromogenic reagent B into each well (including the zero well) and mix thoroughly. Cover and incubate the plate at 37 C for 10 min. The shades of blue can be seen in the wells. 9. Add 50µl of stop solution into each well and mix thoroughly. The color changes into yellow immediately. 10. Read the O.D. absorbance at 450nm in a microplate reader (O.D. absorbance at 630nm is for reference) within 10 min after adding the stop solution. sales@abnova.com www.abnova.com 3
Result A. Calculation and Results analysis 1. For calculation, (the relative O.D. 450 of PC) = (the O.D. 450 of positive control) - (the O.D. 450 of zero well); (the relative O.D. 450 of NC) = (the O.D. 450 of negative control) - (the O.D. 450 of zero well); (the relative O.D. 450 of test sample) = (the O.D. 450 of test sample) - (the O.D. 450 of zero well). 2. If O.D. 450 of negative control is greater than 0.15 (O.D. 450 of NC>0.15) or O.D. 450 of positive control is less than 0.8 (O.D. 450 of PC<0.8), the results are invalid. 3. CUTOFF value=2.1x mean O.D. 450 of negative control (mean O.D. 450 of NC 0.05, take 0.05 to calculate; mean O.D. 450 of NC>0.05, take the actual value to calculate). 4. If the relative O.D. 450 of test sample CUTOFF value, the results are positive; conversely, the relative O.D. 450 of test sample < CUTOFF value, the results are negative. Precautions 1. This kit is suitable for serum and plasma samples and is for research use only. 2. The endogenous (e.g. RF), exogenous (e.g. germ contaminated samples) interference factors or inappropriate operations (e.g. heterogeneous liquid adding, improper plate washing) would lead to false positive and false negative results. 3. Sealers cannot be used repeatedly, and use separate sealers for different lots. 4. NaN 3 cannot be used as test sample preservative. And dilute test sample with fetal bovine serum (FBS) if necessary. 5. Handle this kit as potentially infectious. sales@abnova.com www.abnova.com 4