Rational recombinant XMRV antigen preparation and bead coupling for serology

[Jemal]

Rational recombinant XMRV antigen preparation and bead coupling for multiplex serology in a suspension array

Ali Sheikholvaezina, Fredrik Blomberg, Christina hrmalm, Anna Sjsten and Jonas Blomberg,
Section of Clinical Microbiology, Department of Medical Sciences, Uppsala University, Sweden

Received 18 June 2011; revised 9 August 2011. Available online 19 August 2011.

Abstract

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 (210 mg fusion protein per 100 ml culture) was enough for 20100 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.

http://www.sciencedirect.com/science/article/pii/S1046592811002051

 

[Jemal]

I posted a couple of studies and papers I found the last few weeks.

This one is also interesting I think...

 

[eric_s]

I agree, even though i don't even try to really understand it, i need to give my brain a break. What i'm wondering is why Blomberg still seems to be quite interested in XMRV. One year ago we had high hopes for his study that he was to present at the 1'st Int'l XMRV Conference (or was it workshop?) that then turned out to be negative. I can't remember him reporting actually finding XMRV, but still he seems to be very interested in it. Would you do this work for something that you believe is a lab contaminant? I guess you wouldn't.