Development of antigen capture ELISA for the quantification of EIAV p26 protein

Abstract

An antigen capture enzyme-linked immunosorbent assay (AC-ELISA) was established based on two monoclonal antibodies (mAbs) for the quantification of equine infectious anemia virus (EIAV). Two p26-specific monoclonal antibodies were developed in mice. The mAb 9H8 was coated in microtiter plates as the capture antibody; the other mAb, 1G11, was coupled to horseradish peroxidase (HRP) and used as the detection antibody. The limit of detection for the EIAV p26 protein was 0.98 ng/ml, and the linearity range was 3.9-62.5 ng/ml. The sensitivity of p26 AC-ELISA for the detection of the virus (EIAV infectious clone, FDDVcmv3-8) was the same as that for the purified p26 protein. No cross-reaction with other equine viruses was observed by this method. The intra- and inter-assay coefficients of variation were below 8.3 and 10.3 % for testing p26 and FDDVcmv3-8, respectively. The AC-ELISA was also compared to Western blotting (WB) and reverse transcriptase (RT) assays, validating the sensitivity, accuracy, and reliability of this method. Both the AC-ELISA and RT assay showed good agreement, with a correlation coefficient of R (2) =0.9946. Sample analysis showed that this AC-ELISA is a useful tool for quantifying EIAV p26 in cell lysates and culture medium.

Identification of three PPV1 VP2 protein-specific B cell linear epitopes using monoclonal antibodies against baculovirus-expressed recombinant VP2 protein

“…Here, we focused on the immunodominant protein of PPV1, the VP2 protein, as a potential candidate for development of diagnostic and research reagents. Analysis of the PPV1 VP2 epitopes should extend our understanding of PPV1-specific immunity and accelerate the development of epitope-based diagnostic tools and synthesized peptide vaccine for PPV1 (Hu et al 2014;Kamstrup et al 1998). The report of Kamstrup et al (1998) firstly described a total of nine linear epitopes of both PPV1 VP1 and VP2 proteins and their immunogenicity was subsequently investigated.…”

“…The optimal conditions for the VP2 epitope-based iELISA were investigated by varying the conditions at each step (except in the colorimetric reaction and reaction termination), while maintaining the conditions at all other steps constant (Huang et al, 2011a(Huang et al, , 2011b. The mean (M) and standard deviation (SD) of the OD 405 values for 24 negative controls were used to estimate the cutoff level (Hu et al 2014;Huang et al 2011aHuang et al , 2011b. Based on this criterion, a VP2 epitope-based iELISA was established to scan 135 guinea pig sera.…”

“…Because there have been no updates on the antigenic structures of PPV1 VP2 for the last 18 years, it is crucial to determine how these epitopes have changed. The analysis of antigenic sites might also facilitate the development of effective diagnostic tools for clinical practice (Hu et al 2014). …”

Development of an antigen-capture ELISA for the detection of the p27-CA protein of HERV-K(HML-2)

“…Combined with an immune serum obtained by immunizing rabbits with recombinant capsid protein, the assay was shown to be reasonably sensitive, with a detection limit of 0.130 ng/ml, similar to that reported for other non-commercial antigen-capture ELISAs, e.g. 0.100 ng/ml for HIV-1 capsid protein (Sanders-Beer et al, 2012), 1.25 ng/ml for the p27 antigen of avian leucosis virus (Yun et al, 2013) or 0.98 ng/ml for the p26 antigen of equine infectious anemia virus (Hu et al, 2014). The sensitivity of highly optimized commercial HIV-1 p24 ELISA kits is, of course, considerably higher.…”

Infection with equine infectious anemia virus vaccine strain EIAVDLV121 causes no visible histopathological lesions in target organs in association with restricted viral replication and unique cytokine response

“…The capsid protein (p26), which contains most antigenic epitopes for T cell-mediated immune responses, is the most abundant protein of EIAV (Alvarez et al, 2007;Hu et al, 2014). Immunohistology for p26 revealed that in addition to being detected in the alveolar walls in the lungs ( Fig.…”