The Human IgG Easy Quantification ELISA Kit provides a rapid and easy method (one antibody step ELISA) for the quantitative determination of human IgG in cell culture supernatant and serum. The kit includes ready-to-use reagents necessary to analyze up to 89 samples in 30 min. Buffer solutions are color coded in order to simplify pipetting steps.
Principle of the Assay:
The method employs the quantitative sandwich enzyme immunoassay technique. A polyclonal antibody specific to the gamma heavy chain of human IgG is pre-coated onto the microwells. Samples and standards are pipetted into microwells, and human IgG present in the sample is bound by the capture antibody. Next, a HRP (horseradish peroxidase) conjugated anti-human IgG (H+L) antibody is pipetted and incubated simultaneously with samples. After washing microwells in order to remove any non-specific binding, the ready to use substrate solution (TMB) is added to microwells. Color develops proportionally to the amount of human IgG in the sample. Color development is then stopped by addition of stop solution. Absorbance is measured at 450 nm.
The method enables the detection of natural human IgG as well as recombinant humanized or fully human monoclonal antibodies. Chimeric (human-mouse) recombinant antibodies are detected with the method, but need a specific standard curve. Cross reactions (determined by ELISA) are < 1% for the following IgG: Mouse, Cow, Horse, Sheep and Swine and are < 2 % for Rabbit IgG. No cross reaction was observed with pure mouse or rat serum, indicating the ability of the method to detect human IgG in in vivo studies.
Matrix effect: Culture medium generally does not interfere in the assay. It is nevertheless recommended to evaluate the matrix effect in case of the use of “homemade” culture medium.
The detection range is from 16 ng/ml to 1000 ng/ml.
The detection threshold is 4 ng/ml.