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Introduction
This in-vitro ELISA kit employs an indirect ELISA method. Standard 96-well plates are coated with the pathogen antigen, which combines with the corresponding antibody present in a sample and positive control, which is used as a calibration curve for interpretation purposes. When a biotinylated secondary anti-human IgA/G/M antibody is added, a complex forms between the biotinylated secondary antibody and human antibody bound to the protein-coated plate. A TMB substrate is added, and a blue color is generated. The depth of color is relative to the amount of the human anti-pathogen antigen antibody present. The stop solution changes the color from blue to yellow, and the intensity of the color is measured at 450 nm.
The Positive Control consists of an anti-pathogen antigen antibody. A biotinylated anti-positive control antibody is added to the positive control wells and treated identically to the samples. The intensity of the resulting color (measured at 450 nm) is used as a calibration curve for interpretation purposes in the different assays.
Kit Components
| Component | Size / Description |
|---|---|
| Pathogen antigen coated 96-well Microplate | 96 wells (12 strips x 8 wells) coated with pathogen antigen |
| Wash Buffer Concentrate (20X) | 25 ml of 20X concentrated solution |
| Positive Control | 1 vial of Positive Control sample containing anti-pathogen antigen antibody |
| Biotinylated Anti-Human Ig(A/G/M) | 1 vial of solution |
| Biotinylated Anti-Positive Control | 1 vial of solution |
| HRP-Streptavidin concentrate | 1 vial of solution |
| TMB One-Step Substrate Reagent | 12 ml of 3,3,5,5'-tetramethylbenzidine (TMB buffer solution) |
| Stop Solution | 8 ml of 0.2 M sulfuric acid |
| Assay Diluent B | 15 ml of 5X concentrated buffer |
| 5X Sample Diluent | 25 ml of 5X diluent buffer, 0.5% proclin 300 as preservative |
Additional Materials Required
- Microplate reader capable of measuring absorbance at 450 nm
- Shaker
- Pipettes capable of accurately delivering 2 ul to 1 ml volumes
- Adjustable 1-25 ml pipettes for reagent preparation
- Graduated cylinders: 100 ml and 1 liter
- Distilled or deionized water
- Absorbent paper
Assay Procedure Summary
- Prepare all reagents, samples and standards as instructed.
- Add 100 µl positive control, or sample to each well. Incubate 1 hour at room temperature.
- Add 100 µl prepared Biotinylated Anti-Positive Control and Biotinylated Anti-Human Ig(A/G/M) Antibody into respective wells. Incubate 1 hour at room temperature.
- Add 100 µl prepared HRP-Streptavidin solution to each well. Incubate 30 minutes at room temperature.
- Add 100 µl TMB One-Step Substrate Reagent to each well. Incubate 30 minutes at room temperature.
- Add 50 µl Stop Solution to each well. Read at 450 nm immediately.
Storage / Stability
The entire kit may be stored at -20°C for up to 1 year from the date of shipment. Avoid repeated freeze-thaw cycles. The kit may be stored at 4°C for up to 6 months. For extended storage, it is recommended to store at -80°C. For prepared reagent storage, see table below.
