Immunoaffinity purification of baculovirus-expressed rubella virus E1 for diagnostic purposes

Abstract

Three monoclonal antibodies, termed 4E10, lE11:10, and 2D9:1, were generated against rubella virus. lmmunoblot analysis with purified authentic rubella virus or recombinant baculovirus-expressed rubella virus structural proteins El, E2, and C demonstrated that they were directed against the El envelope glycoprotein of the rubella virus particle. By using the yeast Ty virus-like particle system, it was possible to map the binding site of lE11:10 within amino acids 236 to 286 of the El protein and the binding sites of 2D9:1 and 4E10 outside this region. Immunoaffinity purification with these monoclonal antibodies made it evident that they are useful for obtaining large quantities of pure baculovirus-expressed rubella virus envelope protein El. The diagnostic potential of this immunoaffinity-purified recombinant rubella virus El protein compared with that of authentic rubella virus is demonstrated.

Soluble thymic stromal lymphopoietin receptors are absent in murine sera – detection with anti‐mTSLPR monoclonal antibodies

“…The discrepancy between the reported theoretical molecular weight of 37 kDa and the observed migration in SDS‐PAGE gels corresponding to a 50 kDa size is most likely due to glycosylation. This has been reported for a number of proteins, including mTSLPR (10–12, 26–28). In addition, both a commercial anti‐mTSLPR antiserum as well as a monoclonal anti‐histidine antibody stained identical 50 kDa bands, whereas none of the tested antibodies could identify a corresponding band in the control cell lysate prepared from recombinant FastBacHisC baculovirus‐infected insect cells (Fig.…”

Detection of rubella virus-specific immunoglobulin M antibodies with a baculovirus-expressed E1 protein

“…It has previously been shown that the baculovirus expression vector system is capable of producing significant amounts of RV-specific proteins (21,23,29) and that recombinant proteins have potential in screening for RV-specific antibodies (12,28). In addition, procaryotic and other eucaryotic systems, as well as synthetic peptide technology, have been used to produce RV-specific antigens in hopes of replacing the expensive and infectious authentic RV antigen (1,9,19,31,34).…”

“…Purification of recombinant E1. The supernatant fraction was applied to an immunoaffinity column with the monoclonal antibody, 4E10, coupled to a CNBractivated Sepharose 4B gel (Pharmacia, Uppsala, Sweden), as recently described (12). The cytoplasmic extract containing recombinant RV-E1 protein was mixed (end over end) with the affinity matrix at 4ЊC overnight.…”

Evaluation of a new enzyme immunoassay based on recombinant Rubella virus-like particles for detection of immunoglobulin M antibodies to Rubella virus

“…Concerning the rubella virus antigen, most assays were based upon whole-virus extracts. But recently, EIAs using synthetic rubella virus proteins (8,12) and recombinant E1 proteins (6) have been developed.…”