Sheikholvaezin A, Blomberg F, Ohrmalm C, Sjösten A, Blomberg J
Section of Clinical Microbiology, Department of Medical Sciences, Uppsala University, Sweden
Diagnosis of infectious diseases often requires demonstration of antibodies to the microbe (serology). A large set of antigens, covering viruses, bacteria, fungi and parasites may be needed. Recombinant proteins have a prime role in serological tests. Suspension arrays offer high throughput for simultaneous measurement of many different antibodies.
We here describe a rational process for preparation, purification and coupling to beads of recombinant proteins prepared in Escherichia coli derivate Origami B, to be used in a serological Luminex suspension array. All six Gag and Env proteins (p10, p12, p15, p30, gp70 and p15E), from the xenotropic murine leukemia virus-related virus (XMRV), were prepared, allowing the creation of a multiepitope XMRV antibody assay.
The procedure is generic and allows production of protein antigens ready for serological testing in a few working days. Instability and aggregation problems were circumvented by expression of viral proteins fused to a carrier protein (thioredoxin A; TrxA), purification via inclusion body formation, urea solubilization, His tag affinity chromatography and direct covalent coupling to microspheres without removal of the elution buffer.
The yield of one preparation (2-10mg fusion protein per 100ml culture) was enough for 20-100 coupling reactions, sufficing for tests of many tens of thousands of sera. False serological positivity due to antibodies binding to TrxA and to traces of E. coli proteins remaining in the preparation could be reduced by preabsorption of sera with free TrxA and E. coli extract.
The recombinant antigens were evaluated using anti-XMRV antibodies. Although hybrid proteins expressed in E. coli in this way will not have the entire tertiary structure and posttranslational modifications of the native proteins, they contain a large subset of the epitopes associated with them. The described strategy is simple, quick, efficient and cheap. It should be applicable for suspension array serology in general.
We present a system for efficient production of recombinant protein antigens for use in a quick multiplex serology system. Suspension arrays need shorter incubation times and are more precise than ELISAs. A novel protocol for coupling proteins to color-coded beads enhances the construction of large serological panels. It should be useful for diagnosis of infectious diseases, and should have many applications.
This work was supported by Replico Medical AB, Uppsala, Sweden, by ME Research UK (Charity number SC 036942), and by the Irish ME Trust.
Comment by ME Research UK
Professor Blomberg, head of the Research Group of Clinical Virology at the University of Uppsala, received grant funding from ME Research UK and the Irish ME Trust to test for XMRV in Swedish patients and controls. The results of this investigation were subsequently published – for more on this topic, see our essay “XMRV and ME/CFS — A tumultuous journey for scientists and patients”.
The research group in Uppsala has since received another grant from ME Research UK to begin the search for the microbiological biomarkers of ME/CFS, and this report outlines the methodological strategy that is being employed.