Membranes that were probed 1 were stripped with Restore Western Blot Stripping Buffer (no. to the manufacturers suggested protocol. Eltd1 Polyacrylamide Gel Electrophoresis and Western Blot Analysis Proteins were separated by electrophoresis in 4%C15% polyacrylamide gradient gels (no. 161-1122; BioRad, Hercules, CA, USA) by using Tris/glycine/sodium dodecyl sulfate (SDS) buffer (no. 161C0732; BioRad). The proteins were then either stained with Coomassie brilliant blue or transferred to a polyvinylidene difluoride membrane (no. LC2002; Invitrogen) for Western blot immunoassay. Membranes were blocked with 5% nonfat milk in phosphate-buffered saline with Tween 20 (PBS-T) for 1 h, then incubated with the primary antibody followed by peroxidase-conjugated Protein A/G (no. 32490; Pierce Biotechnology, Rockford, IL, USA). The proteins were visualized by using a SuperSignal West Pico kit (no. 34077; Thermo Scientific, Rockford, IL, USA). Membranes that were probed 1 were stripped with Restore Western Blot Stripping Buffer (no. 21059; Thermo Scientific) and reblocked with 5% nonfat milk in PBS-T between immunoassays. Antibody Production WUPyV VP1 peptide sequence (TAKPGRSPRSQPTRC) and KIPyV VP1 peptide sequence (CRPQKRLTRPRSQV) were each synthesized and injected into rabbits to produce polyclonal antibodies against WUPyV VP1 and KIPyV VP1 (service provided by GenScript, Piscataway, NJ, USA). Rabbit hyperimmune antiserum against the virus-like particles of BKV (BKVLP), JCV (JCVLP), or SV40 were kindly provided by Joakim Dillner ((at 0.6 g each, RVX-208 in solution), or in the blocking buffer alone. The ELISA was then used as described above. Cutoff Value and Statistical Analysis To calculate a cutoff value for the WU ELISA, we used 31 pediatric serum samples that gave signals below that of rabbit preimmune serum. Samples with absorbance intensity 3 SDs above the mean of these 31 samples (0.404 0.103 SD) were considered positive. A parallel set of 31 negative samples (mean 0.286 0.095 SD) were used to calculate a cutoff value for the KI ELISA. For each WU ELISA 96-well plate, the same negative control sample (serum from a 3-month-old child previously considered negative by initial ELISA experiments) and the same positive control sample (convalescent-phase serum from a patient previously found to be WU positive) were used to control for interplate variations. The cutoff value for percentage coefficient of variation of these 2 control samples was set 30%, as described by Jacobson ( em 18 /em ). All blank wells had absorbance values 0.1. Results WUPyV VP1 and KIPyV VP1 Proteins as Target Antigens in ELISAs WUPyV VP1 and KIPyV VP1 were expressed in bacteria as N-terminal, GST-tagged fusion proteins and subsequently purified by using glutathione-affinity chromatography. We used SDS polyacrylamide gel electrophoresis coupled with Coomassie blue staining to analyze the production and purification of the recombinant proteins (Figure 1, panel A). The purified GST-WUPyV VP1 or GST-KIPyV VP1 was then used as the capture antigen in ELISA to detect antibodies against WUPyV VP1 or KIPyV VP1, respectively. Open in a separate window Figure 1 ELISA using WU polyomavirus (WUPyV) viral protein 1 (VP1) or KI polyomavirus (KIPyV) VP1 as the target antigen. A) Coomassie blue staining of a sodium dodecyl sulfateCpolyacrylamide gel RVX-208 that contains bacterially expressed glutathione S-transferase (GST)CKIPyV VP1 and GSTCWUPyV VP1 before and after glutathione-affinity purification. B) ELISA using rabbit hyperimmune serum and human WU polyomavirus convalescent-phase serum preincubated with buffer alone, GST protein, or GSTCWUPyV VP1. Error bars indicate mean and SD. The results of a WU ELISA using WU-hyperimmune rabbit serum and WU-positive human convalescent-phase serum are shown in Figure 1, panel B. Both the rabbit and human serum samples gave strong signals, which were effectively inhibited by preincubation with soluble GST-WUPyV VP1. By contrast, preincubation with GST RVX-208 alone had only marginal effects on the ELISA signal intensity. An ELISA performed on a GST-KIPyV VP1 coated plate using KI-hyperimmune rabbit serum also showed similar KI-specific binding activity (data not shown). Detection of Antibodies against WUPyV VP1 and KIPyV VP1 in Human Serum Samples Of the 419 serum samples analyzed, RVX-208 a representative WU ELISA result of 29 serum samples in the 3-year age group is shown in Figure 2, panel A. A range of absorbance intensities were observed; 17 samples were above the cutoff value. In a parallel KI ELISA conducted on this same set of serum samples, using rabbit serum immunized with RVX-208 a synthetic KIPyV VP1 peptide as positive control, 15 samples were considered ELISA positive (Figure 2, panel B). Open in a separate window Figure 2 ELISA results from 3-year age group. A) 29 serum samples in the 3-year age.