title: Rational recombinant XMRV antigen preparation and bead coupling for multiplex serology in a suspension array.
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 (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.
authors.affiliation: Section of Clinical Microbiology, Department of Medical Sciences, Uppsala University, Sweden.
authors.forename: Ali
authors.initials: A
authors.lastname: Sheikholvaezin
authors.forename: Fredrik
authors.initials: F
authors.lastname: Blomberg
authors.forename: Christina
authors.initials: C
authors.lastname: Ohrmalm
authors.forename: Anna
authors.initials: A
authors.lastname: Sjösten
authors.forename: Jonas
authors.initials: J
authors.lastname: Blomberg
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000914:Antibodies, Viral / Q000276:immunology; D000918:Antibody Specificity; D000956:Antigens, Viral / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism; D003001:Cloning, Molecular; D003593:Cytoplasm / Q000235:genetics / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism; D015686:Gene Products, env / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism*; D015683:Gene Products, gag / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism*; D005822:Genetic Vectors / Q000235:genetics / Q000378:metabolism; D006801:Humans; D007106:Immune Sera / Q000276:immunology; D002479:Inclusion Bodies / Q000378:metabolism; D008969:Molecular Sequence Data; D010957:Plasmids / Q000235:genetics / Q000378:metabolism; D011489:Protein Denaturation; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification* / Q000378:metabolism; D012698:Serologic Tests; D012995:Solubility; D013879:Thioredoxins / Q000235:genetics / Q000378:metabolism; D058008:Xenotropic murine leukemia virus-related virus / Q000276:immunology*
chemical_list: D000914:Antibodies, Viral; D000956:Antigens, Viral; D002352:Carrier Proteins; D029968:Escherichia coli Proteins; D015686:Gene Products, env; D015683:Gene Products, gag; D007106:Immune Sera; D011993:Recombinant Fusion Proteins; C517609:TrxA protein, E coli; D013879:Thioredoxins