TIM-1 / KIM-1 ELISA Assay Kit

Additional Information

Assay Background

TIM-1 / KIM-1 is a membrane receptor for both human hepatitis A virus (HHAV) and TIMD4. The encoded protein may be involved in the moderation of asthma and allergic diseases. The reference genome represents an allele that retains a MTTVP amino acid segment that confers protection against atopy in HHAV seropositive individuals. Alternative splicing of this gene results in multiple transcript variants. Related pseudogenes have been identified on chromosomes 4, 12 and 19. TIM-1 / KIM-1 may play a role in T-helper cell development and the regulation of asthma and allergic diseases. Receptor for TIMD4 (By similarity). In case of human hepatitis A virus (HHAV) infection, functions as a cell-surface receptor for the virus. May play a role in kidney injury and repair.

Assay Principle

This assay employs the quantitative sandwich enzyme immunoassay technique. An antibody specific for TIM-1 / KIM-1 has been pre-coated onto a microtiter plate. Standards or samples are pipetted into the wells and any TIM-1 / KIM-1 present is bound by the immobilized antibody. After washing away any unbound substances, a biotin-conjugated antibody specific for TIM-1 / KIM-1 is added to each well and incubate. Following a washing to remove unbound substances, streptavidin conjugated to Horseradish Peroxidase (HRP) is added to each microplate well and incubated. After washing away any unbound antibody-enzyme reagent, a substrate solution (TMB) is added to the wells and color develops in proportion to the amount of TIM-1 / KIM-1 bound in the initial step. The color development is stopped by the addition of acid and the intensity of the color is measured at a wavelength of 450nm ±2nm.The concentration of TIM-1 / KIM-1 in the sample is then determined by comparing the O.D of samples to the standard curve.

Assay Procedure

1. Remove excess microplate strips from the plate frame, return them to the foil pouch containing the desiccant pack, and reseal it.
2. Add 100 μl of standards, samples and zero controls (Standard/Sample diluent) into wells. Incubate for 1.5 h at 37 °C.
3. Aspirate each well and wash, repeating the process three times for a total four washes. Wash by filling each well with 1× Wash Buffer (350 μl) using a squirt bottle, manifold dispenser, or autowasher. Complete removal of liquid at each is essential to good performance. After the last wash, remove any remaining Wash Buffer by aspirating, decanting or blotting against clean paper towels.
4. Add 100 μl 1X Antibody conjugate into each well. Cover wells and incubate for 1 hour at 37 °C.
5. Aspirate each well and wash as step 3.
6. Add 100 μl of 1X HRP-Streptavidin solution to each well. Cover wells and incubate for 30 minutes at 37 °C.
7. Aspirate each well and wash as step 3.
8. Add 100 μl of TMB Reagent to each well. Incubate for 10-20 minutes at 37°C in dark.
9. Add 100 μl of Stop Solution to each well. The color of the solution should change from blue to yellow. Gently tap the plate to ensure thorough mixing.
10. Read the OD with a microplate reader at 450nm immediately. (optional: read at 610-650 nm as the reference wave length)