A fast and simple dot-immunobinding assay for quantification of mouse immunoglobulins in hybridoma culture supernatants

Journal of Immunological Methods
T Sulimenko, P Dráber

Abstract

Mouse monoclonal antibodies of IgG subclasses and IgM class in hybridoma culture supernatants were quantified using a dot-immunobinding assay. Immunoglobulins were bound to nitrocellulose (NC) membrane and, after blocking, the membrane was incubated with anti-mouse antibody conjugated to horseradish peroxidase (HRP). Binding was revealed by incubation with a sensitive chemiluminiscence reagent. Quantitation was achieved by densitometric comparison with standard curves produced by purified monoclonal antibodies of the same subclass or purified antibodies of the same clone as the antibody to be quantified. These quantitative results were compared with those obtained using purified IgG from mouse serum or purified mouse myeloma IgM as standards. The dot-immunobinding assay requires 1 microl of hybridoma culture sample and takes about 1 h in total. Good linearity between the staining intensity and the amount of immobilized immunoglobulins was observed over the range of 0.05-5 ng/spot. The assay is simple, reproducible and can process simultaneously a large number of samples.

References

Sep 16, 1983·Journal of Immunological Methods·V Horejsí, I Hilgert
Apr 12, 1995·Journal of Immunological Methods·P DráberM Nováková
Apr 12, 1995·Journal of Immunological Methods·R K Gupta, G R Siber
Apr 15, 1994·Journal of Immunological Methods·L BakkaliC Crucière
Jan 1, 1996·Cell Motility and the Cytoskeleton·M NovákováP Dr-aber

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Citations

Dec 7, 2013·Biochemical and Biophysical Research Communications·Jamie A LinesShengxi Chen
Jun 7, 2012·Molecules : a Journal of Synthetic Chemistry and Natural Product Chemistry·Zhuo ChenBaoan Song
Oct 22, 2019·Biochemical and Biophysical Research Communications·Elizabeth Ngoc Hoa TranRenato Morona

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