Immunoinformatics-guided recombinant polypeptide-based enzyme-linked immunosorbent assay for seromonitoring of laboratory animals for minute virus of mice and Kilham rat virus

Abstract

Subclinical infection of laboratory animals with one or more of several pathogens affects the results of experiments on animals. Monitoring the health of laboratory animals encompasses routine surveillance for pathogens, including several viruses. This study aimed to explore the development of an alternative assay to the existing ones for detecting infection of mice and rats with the parvoviruses minute virus of mice (MVM) and Kilham rat virus (KRV), respectively. Full-length VP2 and NS1 proteins of these parvoviruses, besides fragments containing multiple predicted epitopes stitched together, were studied for serological detection. The optimal dilution of full-length proteins and antigenic regions containing predicted epitopes for coating, test sera, and conjugate was determined using a checkerboard titration at each step. The assays were evaluated vis-à-vis commercially available ELISA kits. The results showed that an engineered fusion of fragments containing multiple predicted MVM VP2 and NS1 epitopes was better than either of the full-length proteins for detecting antibodies in 90% of the tested sera samples. For KRV ELISA, full-length VP2 was better compared to other individual recombinant protein fragments or combinations thereof for the detection of antibodies in sera. This report is the first description of an ELISA for KRV and an improved assay for MVM. Importantly, our assays could be exploited with small volumes of sera. The results also demonstrate the utility of immunoinformatics-driven polypeptide engineering in the development of diagnostic assays and the potential to develop better tests for monitoring the health status of laboratory animals.

Fig 1. Illustrative diagram of the constructs generated in this study.

All polypeptides were designed to contain a 6-His tag at the N-terminus. The linker between the fused fragments is five amino acids long.

Fig 2. Cloning, expression, and purification of the CD polypeptide.

(a) Cloning of CD. Fragments of C and D were amplified separately and then joined by SOE-PCR. M, DNA marker; 1, PCR amplified fragment C (528 bp); 2, PCR amplified fragment D (573 bp); 3, joined CD fragment (1.1 kb). (b) SDS-PAGE analysis of CD protein. Transformed BL21 cells induced with IPTG were separated into soluble and insoluble fractions and subjected to SDS-PAGE. M, molecular weight marker (kDa); 1, total cell before induction; 2, total cell after induction; 3, total cell after sonication; 4, supernatant; 5, pellet solubilized in 8 M urea. (c) Chromatogram showing the elution profile of CD. The denatured insoluble fraction was subjected to protein purification using a Ni-Sepharose High performance (NiHP) affinity column on an AKTA pure^™ chromatography system. Fraction numbers 8–10 were pooled. (d) SDS-PAGE analysis of CD protein during purification. The proteins were visualized by Coomassie staining. M, molecular weight marker (kDa); 1, sample before loading on the column; 2, flow-through; 3–7, elution fractions after NiHP chromatography. (e) SDS-PAGE analysis of the expressed polypeptides. BL21 cells transformed with plasmids carrying the respective constructs were induced with IPTG, and the extracts were subjected to SDS-PAGE under reducing conditions, followed by Coomassie staining. 1, Polypeptide A; M, molecular weight marker (kDa); 2, Polypeptide CD; 3, Polypeptide E; 4, Polypeptide FG; 5, MVM VP2 protein; 6, MVM NS1 protein; 7, KRV VP2 protein, 8, KRV NS1 protein. (f) Western blotting. Proteins transferred onto PVDF membrane were probed using HRP-conjugated anti-His MAb. 1, Polypeptide A; M, molecular weight marker (kDa); 2, Polypeptide CD; 3, Polypeptide E; 4, Polypeptide FG; 5, MVM VP2 protein; 6, MVM NS1 protein; 7, KRV VP2 protein; 8, KRV NS1 protein. The position of three molecular weight markers (kDa) is shown on the left.

Fig 3. Comparison of in-house ELISA with Xpress Bio kit.

Wells were coated with a predetermined quantity of the polypeptides, blocked, and reacted with serum samples before detecting with HRP-conjugated anti-mouse or anti-rat antibodies. BSA was used as the control antigen to deduct the background OD. Representative data for three mouse (a) and three rat (b) serum samples are shown respectively for MVM and KRV.

Fig 4. Scatter plots showing the reactivity or sera against various polypeptides.

OD values for the positive (PS) and negative (NS) serum samples were plotted against each of the polypeptides used in the study. Data for mouse sera against MVM (a) and rat sera against KRV (b) are shown separately. PS and NS denote positive and negative sera, respectively, as determined using the commercial kit.