Sensitive, Specific and Cost Effective
Human IL-12p40 ELISA kit is an in vitro enzyme-linked immunosorbent assay for the quantitative measurement of human IL-12p40 and ProIL-12p40 in serum, plasma, and cell culture supernatants. This assay employs an antibody specific for human IL-12p40 coated on a 96-well plate. Standards and samples are pipetted into the wells and IL-12p40 present in a sample is bound to the wells by the immobilized antibody. The wells are washed and biotinylated anti-human IL-12p40 antibody is added. After washing away unbound biotinylated antibody, HRP-conjugated streptavidin is pipetted to the wells. The wells are again washed, a TMB substrate solution is added to the wells and color develops in proportion to the amount of IL-12p40 bound. The Stop Solution changes the color from blue to yellow, and the intensity of the color is measured at 450 nm.
Suggested Application:Quantitative Protein Detection; Establishing Normal Range; Validation of Antibody Array Results.
· Pre-Coated 96-Well Strip Microplate
· Wash Buffer
· Stop Solution
· Assay Diluent(s)
· Lyophilized Standard
· Biotinylated Detection Antibody
· Streptavidin-Conjugated HRP
· TMB One-Step Substrate
Other Materials Required:
· Distilled or deionized water
· Precision pipettes to deliver 2 μl to 1 ml volumes
· Adjustable 1-25 ml pipettes for reagent preparation
· 100 ml and 1 liter graduated cylinders
· Tubes to prepare standard and sample dilutions
· Absorbent paper
· Microplate reader capable of measuring absorbance at 450nm
· Log-log graph paper or computer and software for ELISA data analysis
1.Prepare all reagents, samples and standards as instructed in the manual.
2.Add 100 μl of standard or sample to each well. Incubate 2.5 h at RT or O/N at 4°C.
3.Add 100 μl of prepared biotin antibody to each well. Incubate 1 h at RT.
4.Add 100 μl of prepared Streptavidin solution to each well. Incubate 45 min at RT.
5.Add 100 μl of TMB One-Step Substrate Reagent to each well. Incubate 30 min at RT.
6.Add 50 μl of Stop Solution to each well. Read at 450 nm immediately.
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