The production of a ?universal developer? for the immunological detection of human IgG and its application in immunodiagnostics

Abstract

This study describes the development of a biospecific monoclonal antibody capable of the simultaneous recognition of horseradish peroxidase (HRP) and human IgG. This antibody, coded McC2, has been applied in a novel manner as a universal developing reagent for the detection of human IgG. McC2 cross-reacts with all human IgG subtypes and was found to recognise an epitope on the Fc portion of human IgG. McC2 does not cross-react with human IgM or IgA. This bi-specific antibody belongs to the mouse IgG1 subclass. McC2 was used for the detection of human IgG in a simple one step enzyme-linked immunosorbent assay (ELISA). Use of this bi-specific antibody in this assay resulted in an excellent signal to noise ratio with background in negative control wells virtually nonexistent. McC2 was also applied in a clinical diagnostic test for the detection of auto anti-nuclear antibodies in patient sera. McC2 was substituted, in a blind study, for a HRP-conjugated second antibody supplied with the test kit. All sera were tested both with the kit's second antibody and McC2. When using McC2, we obtained no false positive results whereas five false positives were obtained when using the kit's second antibody. However, one false negative result was obtained with the use of McC2 as a developing reagent while none were noted with the use of the kit's second antibody.(ABSTRACT TRUNCATED AT 250 WORDS)

Production of a bi-specific monoclonal antibody recognizing mouse kappa light chains and horseradish peroxidase

“…The enhanced specificity of a direct epitope-specific bi-specific developing reagent like McC10 has been previously documented. Bi-specific anti-human IgG anti-HRP monoclonal antibodies were successfully used in clinical diagnoses of systemic lupus erythematosus and found to be more reliable for the accurate detection of circulating anti-nuclear antibodies in patient sera (Semenenko et al 1988). In addition, anti anti-rabbit kappa light chain anti-HRP bi-specific monoclonal antibodies proved to be superior to conventional PAP when employed in tissue prone to display high background staining and were used successfully in a sensitive quantitative ELISA for the detection of a biologically active peptide (Kenigsberg et al 1990).…”

“…More recently, with the development of two new techniques [internal radiolabelling of monoclonal antibodies (Cuello and Milstein 1981 ;Cuello et al 1982) and the production of bi-specific monoclonal antibodies (Milstein and Cuello 1983;], hybridoma technology has renewed interest in direct immunostaining procedures. Bi-specific monoclonal antibodies which possess binding sites for the antigen under investigation and an immunohistochemical marker like horseradish peroxidase (HRP) on the same Ig molecule have been produced and used successfully in a variety of studies (Suresh et al 1986;Semenenko et al 1988;Kenigsberg et al 1990). Bi-specific monoclonal antibodies used in one-or two-step immunocytochemistry (ICC) have been found to be remarkably sensitive and highly specific, dramatically enhancing the signal-to-noise ratio.…”