passages: PMC-2660343-sec-18__text
passages: sec
passages: Peptide-BSA conjugates as diagnostic antigens
  To analyse the potential of the peptides to behave as antigen mimics, their ability to react with IgM antibodies from individuals infected with EBV was assessed. In our previous study, we demonstrated that the sensitivity of detection was greatly improved when the peptides were coupled to a carrier molecule such as BSA prior to immobilisation onto a solid surface (Casey et al., 2006). This strategy was adopted to test peptides Eb1-4 and H1. A set of 40 clinical samples that were classified as EBV seropositive (n = 16), seronegative (n = 16) or potentially cross-reactive sera (n = 8) were assessed for reactivity with Eb1-4 and H1 peptides individually. The cut-off level was defined as the mean optical density of the seronegative samples plus 3 standard deviations shown as a line on the graphs in Fig. 5. Readings above this level were defined as positive and below this level negative. The same set of samples were analysed on BSA alone and these values were subtracted from the peptide-BSA conjugate readings and the corrected absorbance readings were plotted individually for our new peptides Eb1-4 and H1 in Fig. 5. There was a clear difference in the detection of seropositive antibodies by all the peptides (Fig. 5A-E) compared with the analysis of BSA alone (Fig. 5F), with the majority of absorbance readings above the cut-off level. We compared the ability of our panel of peptide mimotopes to be recognised by antibodies in the same set of seropositive samples in Fig. 6A and the sensitivity of detection is shown in Fig. 6B. We also included F1 and Gp125 mimotopes specific for two mAbs in our previous study (Casey et al., 2006). Of the peptides identified from polyclonal sera Eb1, Gp125 and F1 had the highest sensitivity (94%). Slightly lower sensitivity was observed for Eb2, 3 and 4 (88%) and H1 peptide had the lowest sensitivity (81%) as summarised in Fig. 6B. The sensitivity of F1 and Gp125 was similar to that produced by the mimotopes selected in our previous study, 95% for F1 and 92% for Gp125.
  Fig. 5
  Evaluation of peptides Eb1-4 and H1 coupled to BSA as EBV diagnostic reagents. Human serum (n = 40) previously analysed using a diagnostic test for VCA IgM was allowed to react with the peptides and the bound IgM antibodies were detected using anti-human IgM HRP. The absorbance readings for 1 (positive), 2 (negative) and putative cross-reactive sera for 3 (Parvo), 4 (HSV), 5 (CMV) and 6 (RF) are plotted for (A) Eb1, (B) Eb2, (C) Eb3, (D) Eb4, (E) H1 and (F) BSA, respectively. The cut-off value is defined as the mean of the negative population +3SD indicated by a solid horizontal line; since there were no false positives, the specificity for each mimotope was 100%.
  Fig. 6
  Comparison of the reactivities of our panel of mimotopes Eb1-4, H1, F1 and Gp125 conjugated to BSA with EBV IgM-positive sera (n = 16) absorbance values are plotted and the cut-off levels are depicted by a horizontal line in (A). (B) Summary of the false-negative results from the 5/16 serum samples seropositive for IgM EBV and the overall sensitivity for each mimotope for diagnosis of EBV IgM antibodies.
  We also considered which seropositive EBV samples contained antibodies that did not recognise the panel of peptides, i.e. false-negative readings, listed in Fig. 6B. The antibodies in serum 1 (s1) were unreactive with all of the peptides identified in this study, s2 was not reactive with Eb3, Eb4 and H1 and s3 was unreactive with H1. Gp125 and F1 that were selected in our previous study were recognised by s1, 2 and 3; however, two different serum samples (s4 and 5) did not recognise F1 or Gp125, respectively. This demonstrates that individual peptides are not recognised by all EBV antibodies and confirms that different peptides are required to represent different epitopes. Therefore, a combination of Eb1 peptide F1 and Gp125 peptides could be recognised by antibodies present in all this set of EBV clinical samples resulting in 100% sensitivity.
  For the samples defined as EBV-seronegative, there were no readings above the cut-off level and therefore no false positives, resulting in 100% specificity. In addition, there were no absorbance readings above the cut-off levels for the potentially cross-reactive serum samples, inferring that the peptides identified in this study have high specificity for EBV antibodies.
  