A Sample Instruction Manual
EC10196 (96T x 5)
ELISA Kit DIY Materials
For Anti-Treponema pallidum (Syphilis) human IgG detection
Instruction manual
For research use only, not for use in diagnostic procedures
1st Edition
[ INTENDED USE ]
This ‘Do it Yourself (DIY)’ assay kit contains materials for developing your own immunoassays. This may include the development of indirect ELISA to measure anti-Treponema pallidum (Syphilis)human IgG in serum, plasma, cell culture supernatants, and other biological fluids in vitro. This kit contains sufficient materials for preparation of at least five 96-well plates. Reagent Preparation and Assay Protocol are provided as suggestions only. Researchers should optimize the use of kit materials and protocols for their own model system and determine if the kit is suitable for their samples and immunoassays.
[ REAGENTS AND MATERIALS PROVIDED ]
| Components | Quantity | Form | Storage |
|---|---|---|---|
| Treponema pallidum (Syphilis) p17/p47 fusion protein (RA10196) for plate coating | 60 µg | Liquid, one vial, contains 0.1% ProClin 300 | ≤–20°C, avoid multiple freeze/thaw cycles |
| HRP labeled mouse anti-human IgG (EC20010) monoclonal antibody for detection | 60 µl | Liquid, one vial, contains 0.1% ProClin 300 and 50% Glycerol | ≤–20°C |
[ RECOMMENDED BUFFERS AND SOLUTIONS ]
DIY ELISA support pack (EC20012), which includes 96-well strip plate, coating buffer, blocking buffer, reagent diluent buffer, washing buffer, TMB substrate, stop solution.
Notes: The recommended diluents and buffers contained in EC20012 are validated in the lab, other reagents selected for use can alter the performance of an immunoassay.
| Components | Quantity | Form |
|---|---|---|
| 96-well strip plate | 5 plates | 96 well (8 strips x 12) |
| ELISA plate coating buffer (5x) | 12 ml | One bottle |
| ELISA plate blocking buffer | 110 ml | One bottle |
| ELISA diluent buffer | 120 ml | One bottle |
| ELISA plate washing buffer (30x) | 65 ml | One bottle |
| HRP TMB substrate | 30 ml A + 30 ml B | Two bottles |
| Stop solution | 30 ml | One bottle |
[ STORAGE ]
Plate coating antigen and HRP labeled antibody should be stored at -20°C. TMB should be stored at 4°C. 96-well Plates and buffers/solutions could be stored at room temperature. The unopened reagents are valid for 6 months; they are stable for one month after opening when stored at 4°C. Please make all solutions fresh before the experiment.
[ REAGENT PREPARATION ]
Bring all components to room temperature (18-25°C) before use. Working solutions should be prepared and used immediately.
Plate coating antigen: Briefly spin or centrifuge the stock plate coating antigen before use. Aspirate appropriate volume of plate coating antigen, dilute to final concentration of 1 mg/ml with ELISA plate coating buffer for plate coating.
HPR labeled mouse anti-human IgG: Briefly spin or centrifuge the stock HRP labeled antibody before use. Aspirate appropriate volume of the reagent, 1: 1000 dilution in ELISA diluent buffer.
[ ASSAY PROTOCOL ]
Plate Preparation:
1. Dilute the plate coating antigen to working concentration in Coating Buffer. Immediately coat the 96-well strip plates with 100μL per well of the diluted plate coating antigen. Cover the plate and incubate overnight at 4°C.
2. Aspirate the solution and wash with 350μL of ELISA Plate Washing Buffer to each well using a squirt bottle, multi-channel pipette, manifold dispenser or auto-washer, and let it sit for 1~2 minutes. Remove the remaining liquid from all wells completely by snapping the plate onto absorbent paper.
3. Block plates by adding 200μL of ELISA Plate Blocking Buffer to each well. Incubate at 37°C for 1.5 hours.
4. Repeat the aspiration/wash process as in step 2. The plates are now ready for sample detection.
Commonly Used Assay Procedure:
1. Add 100μL of different concentrations of controls (not provided) and samples into the appropriate wells. Cover the wells. Incubate for 1 hour at 37°C.
2. Repeat the aspiration/wash process for 3 times as in Step 2 of plate preparation.
3. Add 100μL of working solution of HRP labeled antibody to each well, cover the wells, and incubate for 30 minutes at 37°C.
4. Repeat the aspiration/wash process for total 5 times as in Step 2.
5. Add 90μL of TMB Substrate (45 μLA + 45 μLB) to each well. Cover the wells, and incubate for 10 - 20 minutes at 37°C. Protect from light.
6. Add 50μL of Stop Solution (1mol/L HCl) to each well.
7. Run the microplate reader and conduct measurement at 450nm immediately.
