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J Immunol Methods. 1993 Jan 14;158(1):107-22.

Epitope mapping of human immunoglobulin-specific murine monoclonal antibodies with domain-switched, deleted and point-mutated chimeric antibodies.

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  • 1Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, MD 21224.

Abstract

27 engineered chimeric antibodies possessing human gamma, epsilon, mu or alpha constant regions and V region specificity for nitrophenyl or dansyl were used to study the isotype specificity of 29 murine monoclonal antibodies (MAbs) specific for human immunoglobulins (IgG1-4, IgE, IgM, IgA or secretory piece). The isotype-restricted immunoreactivity observed with wild-type chimeric antibodies paralleled the pattern of each MAb's reactivity with purified human myeloma proteins. 16 mutant IgG anti-dansyl chimeric antibodies with genetically engineered domain switches, deletions or point-mutations were used as antigens to further characterize the epitopes recognized by the human IgG subclass-specific MAbs. The binding of three human IgG1-specific MAbs (HP6069, HP6070 and HP6091) was mapped to similar epitopes on the CH2 domain of human IgG1. Of the two anti-human IgG2 MAbs tested, HP6002 reacted with the CH2 of IgG2 while HP6014 bound to the CH1 domain. Both anti-human IgG3 MAbs (HP6047, HP6050) reacted with different regions of the IgG3 hinge. The anti-human IgG4 MAbs (HP6023, HP6025) bound to a similar epitope on the carboxyl terminus of CH2 or the CH3 of human IgG4. The three exclusion antibodies (HP6019, HP6030 and HP6058) bound to different epitopes in the CH2 domain of three of four IgG subclasses. The domain mapping was confirmed by competitive inhibition experiments. These results were used to select a group of IgG-reactive MAbs for construction of a poly-monoclonal anti-IgG capture and detection reagent that uniformly bound all four subclasses of human IgG. This study provides support for the use of engineered chimeric human chimeric antibodies as replacements for increasingly rare, purified human paraproteins in the specificity analysis of immunochemical reagents used in clinical and research laboratories for the detection and quantitation of human antibodies. Moreover, these studies demonstrate how the MAbs can serve as effective probes for examining conformational differences among the four human IgG subclasses.

PMID:
7679128
[PubMed - indexed for MEDLINE]
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