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Published on: October 31, 2012
Cell-ELISA using beta-galactosidase conjugated antibodies
Z Liu1, T Gurlo, H von Grafenstein
1School of Pharmacy, University of Southern California, 1985 Zonal Avenue, Los Angeles, CA 90033, USA.
Journal of Immunological Methods
|February 12, 2000
Summary
Beta-galactosidase offers a superior antibody-tag for cell-enzyme-linked immunosorbent assay (cell-ELISA), reducing background noise in cell surface molecule detection. This method enables efficient quantification of cell surface markers on live cells.
Area of Science:
- Immunology
- Biochemistry
- Cell Biology
Background:
- Cell-enzyme-linked immunosorbent assay (cell-ELISA) is crucial for detecting cell surface molecules.
- Existing methods using horseradish peroxidase (HRP) or alkaline phosphatase (AP) have limitations, including higher background binding.
- The availability of beta-galactosidase conjugated antibodies is limited.
Purpose of the Study:
- To evaluate beta-galactosidase as an antibody-tag for cell-ELISA.
- To develop and optimize direct and indirect beta-galactosidase based cell-ELISA protocols.
- To establish an efficient method for conjugating beta-galactosidase to antibodies.
Main Methods:
- Evaluation of beta-galactosidase substrate (CPRG) hydrolysis by murine splenocytes.
- Comparison of background binding of beta-galactosidase, HRP, and AP antibody conjugates to murine T cells.
- Development of step-by-step protocols for direct and indirect cell-ELISA using beta-galactosidase.
- Conjugation of beta-galactosidase to antibody fragments using sulfosuccinimidyl 4-[N-maleimidomethyl]-cyclohexane-1-carboxylate (sulfo-SMCC).
Main Results:
- Murine splenocytes do not hydrolyze the beta-galactosidase substrate CPRG.
- Beta-galactosidase antibody conjugates exhibit significantly lower background binding to murine T cells compared to HRP or AP conjugates.
- Established protocols are effective for unfixed, live cells (adherent and non-adherent) and adaptable for large-scale screening.
- Efficient conjugation of beta-galactosidase to F(ab')(2) anti-B7-2 antibody fragments was achieved using sulfo-SMCC.
Conclusions:
- Beta-galactosidase is a suitable and advantageous antibody-tag for cell-ELISA, offering reduced background.
- The developed protocols facilitate accurate quantification of cell surface molecules on live cells.
- The novel conjugation method expands the utility of beta-galactosidase in cell-ELISA applications, including hybridoma screening and cell surface molecule analysis.
