Development of an enzyme-linked immunosorbent assay using recombinant protein antigen for the diagnosis of Chikungunya virus

We describe here the development of an in-house enzyme linked immunosorbent assay (ELISA) for the diagnostic of Chikungunya virus (CHIKV) infections using a recombinant protein from CHIKV. The recombinant protein gene was designed based on 154 sequences and we used computational methods to predict its structure and antigenic potential. To confirm predictions, the gene coding for the recombinant CHIKV protein (rCHIKVp) was synthetized and expressed in prokaryotic system. Subsequently, the protein was purified by affinity chromatography and used as antigen in an indirect ELISA. We present data regarding the optimization of the recombinant antigen production and preparation of the ELISA to detect IgG against CHIKV in human sera.


Data
We design a synthetic gene coding for a recombinant Chikungunya virus protein (rCHIKp) to be used as an antigen in serologic assays. In silico analysis revealed that rCHIKp secondary structure has predominant coils and identified B lymphocyte epitopes in regions of structural disorder, which are advantageous for antigenic recognition. The overall antigenic prediction score was 0.53 (which suggests a probable efficient antigen). The gene was expressed in E. coli cells and purified by affinity chromatography. Coomassie Brilliant Blue staining showed the presence of rCHIKp with the predicted molecular mass (~42 kDa). Next, we developed an In House Enzyme-Linked Immunosorbent Assay (ELISA) for the detection of immunoglobulin G (IgG) anti-CHIKV using the rCHIKp as the ELISA's antigenic solid phase. The rCHIKVp-based ELISA showed a sensitivity value of 95% and specificity of 96% (Table 1). No cross-reactivity was found against sera from Zika-(ZIKV) and Dengue-(DENV) positive patients (Fig. 1). The developed ELISA was used in the assessment of human patients suspected of arboviral infections in Minas Gerais State, Brazil [1].

Design of the antigen gene
CHIKV genome sequences deposited at GenBank were aligned using MEGA7 software [2]. The selection criteria were: complete annotation of the genome and absence of indefinite nucleotides in the sequence. Brazilian (n ¼ 42) and foreign samples belonging to the genotypes circulating in Brazil were used. Sequences belonged to the Asian genotype (n ¼ 112) and East-Central South African (ECSA) Specifications Analyzed data are shown Experimental factors Samples are crude human sera that were simply heated to eliminate complement factors. The ELISA's recombinant antigen was obtained by affinity chromatography and maintained in 6 M urea up to use.

Experimental features
The ELISA's recombinant antigen -the E2 protein from Chikungunya virus -was expressed in E. coli transformed cells and purified by affinity chromatography. The purified protein was used as the antigenic solid phase of the ELISA and was tested against a bank of characterized human sera, composed of Chikungunya-positive sera, Dengue-positive sera or Zika-positive sera. Sera that are negative for all viruses were also used. Data source location Minas Gerais State, Brazil.

Data accessibility
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Value of the Data
This data provides details about the design and production of a recombinant Chikungunya virus protein (rCHIKp). We describe the standardization of an in house ELISA using the rCHIKp as an antigen. The developed ELISA presented the ability to distinguish chikungunya-positive human sera from virus-negative sera.
The ELISA was also able to efficiently distinguish patients that were seropositive for chikungunya from those that were seropositive for dengue or zika.
genotype (n ¼ 274). A consensus sequence was generated for each group (Group 1: Brazilian samples, Group 2: Asian genotype samples and Group 3: ECSA genotype samples). These consensual sequences were, then, compared to each other to generate a unique nucleotide sequence. The gene sequence was codon-optimized for expression in E. coli by OptimumGene ™ -Codon Optimization software (Genescript). Transmembrane and hydrophobicity analyzes were performed using TMHMM (http://www.cbs.dtu.dk/services/TMHMM/) [3] and Protscale -Expasy (http://web. expasy.org/Protscale/) [4]. From these predictions, a cut-off point for the generation of the protein without its transmembrane domain was determined. The nucleotide sequence of the truncated protein-coding gene was commercially synthesized and subcloned into the pET-21 expression vector, which included a histidine tag to the construction.

In silico analysis
To evaluate the potential of our rCHIKVp as a diagnostic tool, the protein's predicted amino acid sequence was submitted to BepiPred (http://www.cbs.dtu.dk/services/BepiPred/) for the prediction of linear B cell epitopes [5] and IUPred (http://iupred.enzim.hu/), for the prediction of intrinsic structural disorder, indicating absence of secondary structure and, consequently, regions of possible interaction with antibodies [6]. The antigenicity of the designed protein was evaluated by the VexiJen V2.0 online server (http://www.ddphpharmfac.net/vaxijen/VaxiJen/VaxiJen.html) [7]. Table 1 Relative sensitivity and specificity of the rCHIKVp-based in house-Anti-CHIKV ELISA in comparison to a commercial kit (Chikungunya IgG ELISA Euroimmun, Germany).

Recombinant protein production
The pET-21 vector containing the gene of interest was used to transform E. coli BL21(DE3) strain by heat shock. Plasmid-positive clones were induced by IPTG and expression of the recombinant protein was optimized and analyzed by SDS-PAGE. The antigen was purified by affinity chromatography using nickel columns in an € AKTAprime plus system (GE Healthcare, USA).

In house Anti-Chikungunya virus ELISA
The seroreactivity of the rCHIKVp was evaluated using a panel of sera samples from human patients, CHIKV seropositive or not, by an in-house indirect IgG ELISA. The optimal concentration of the recombinant rCHIKVp per plate well was determined based on a clear distinction of anti-CHIKV antibodies using positive and negative samples. We tested a range of 25e800 ng/well of rCHIKVp and sera dilutions ranging from 1:25 to 1:3200 (Fig. 2). The cut-off value was determined by ROC curve analysis, and an index (I) of each absorbance value of the patient sample over the value of cut-off was calculated, according to the equation: I ¼ a/c, where a is the absorbance of the patient sample and c is the cut-off value (0,279). The data is classified as follows: I < 0.9: negative 0.9 I < 1.1: borderline I 1.1: positive