Mammalian expression of functional autologous red cell agglutination reagents for use in diagnostic assays

Abstract

The autologous red cell agglutination assay reagent consists of an antibody or antibody fragment of a human erythrocyte-specific monoclonal antibody (mAb) conjugated to an antigen of interest. This bi-functional reagent causes the agglutination of the patient's erythrocytes in the presence of the antigen-specific antibodies in the patient's serum. Previously, such reagents have been produced either by chemical conjugation or recombinant expression in bacteria. These protocols required laborious processes for purification and refolding. The aim of the work reported in this article was to explore the production of the agglutination assay reagent as both a single chain Fv (scFv) antibody fragment and recombinant full-length mAb, expressed in a secreted form in commonly used mammalian cell lines. The DNA encoding the anti-erythrocyte antibodies was linked to that of a diagnostic peptide from West Nile virus, which requires glycosylation for recognition by antibodies present in the sera of infected horses. The expression vectors were designed to allow the rapid directional insertion of DNA encoding other immunogenic peptides to mediate the secretion of agglutinating scFv and full-length mAb reagents from transfected mammalian cells. Stable cell lines were produced for the expression of most, but not all of the constructs. The recombinant reagents could be used directly from the cell culture media after a simple concentration step. The results indicate that further modifications to increase the yield of recombinant protein will enable the direct use of culture supernatant in diagnostic assays without further processing.

Approaches for the Development of Rapid Serological Assays for Surveillance and Diagnosis of Infections Caused by Zoonotic Flaviviruses of the Japanese Encephalitis Virus Serocomplex

“…Although functional reagents were produced, the recovery of the reagent required extensive extraction and purification procedures. We recently described the secreted expression of the human-based recombinant autologous agglutination reagent in mammalian cells, as both single chain (scFv) and intact mAbs [175]. The anti-RBC antibody was fused to a WNV diagnostic tag (WN19) and was shown to be functional in agglutination assays and suitable for diagnosis of WNV infection in horses.…”

A rapid, point of care red blood cell agglutination assay for detecting antibodies against SARS-CoV-2

“…14 Antibodies against West Nile virus have also been detected by autologous RBC agglutination assay. 15 Outside of infectious disease, was not certified by peer review) is the author/funder. All rights reserved.…”

Toward Low-Cost Affinity Reagents: Lyophilized Yeast-scFv Probes Specific for Pathogen Antigens

“…Hence, many yeast-displayed scFv perform poorly in other environments, especially when secreted. Although functional soluble scFv have been reported [2], [3], [4], [5], [6], [7], [8], in practice the great majority of scFv culled from yeast display libraries have exhibited unsatisfactory activity in standard immunoassay formats.…”