Plasmodium vivax aldolase-specific monoclonal antibodies and its application in clinical diagnosis of malaria infections in China

Abstract

Background: Most rapid diagnostic tests (RDTs) currently used for malaria diagnosis cannot distinguish the various Plasmodium infections. The development of a Plasmodium vivax specific RDTs with high sensitivity to sufficiently differentiate the two most common Plasmodium infections would be very crucial for disease treatment and control.

Method: Plasmodium vivax aldolase gene (PvALDO) was amplified from the extracted genomic DNA and constructed into pET30a vector. Plasmodium vivax aldolase protein was successfully expressed in Escherichia coli in soluble form and the overall purity was over 95% after one-step affinity chromatography purification. The purified products were used for the immunization of mice and rabbits. Rabbit polyclonal antibodies generated were deployed to develop a novel antibody-capture ELISA for hybridoma screening.

Results: Three PvALDO specific mAbs (14C7, 15F1 and 5H7) with high affinities were selected and used in immunochromatographic test strips. Clinical blood samples (n=190) collected from Yunnan (China) were used for evaluation and the RDT's sensitivity for P. vivax was 98.33% (95% Confidence Interval (CI): 91.03% to 99.72%) compared with microscopic examination. There was specificity of 99.23% (95% CI: 95.77% to 99.87%) for P. vivax. Only one Plasmodium falciparum sample was detected among the P. falciparum samples (n=20). All Plasmodium malariae samples (n=2) as well as healthy uninfected samples (n=108) were negative. Overall performance of this RDT was excellent with positive predictive value (PPV) and negative predictive value (NPV) of 98.33% and 99.23%, respectively, at 95% CI and a very good correlation with microscopic observations (kappa value, K=0.9757). Test strips show high sensitivity even at 6.25 ng/ml of recombinant P. vivax aldolase (rPvALDO).

Conclusion: This study further elucidates the possibility of developing aldolase-specific RDTs which can differentiate the different Plasmodium infections and improve accurate diagnosis of malaria. This RDT could adequately differentiate between P. vivax and P. falciparum infections. The novel mAb screening method developed here could find application in the screening of highly specific antibodies against other antigens.

Figure 1

Gene amplification and protein expression. (A) PCR of PvALDO gene. M: DL2000 marker, 1 and 2 represent annealing temperatures 50.4°C and 52.4°C, respectively. (B) Protein expression and purification. M: Marker, lanes 1 and 2 are the whole cell lysate and supernatant after induction, respectively. Lanes 3, 4 and 5 are pooled portions of purified products. (C) Western blot analysis of PvALDO protein. M: Marker, 1: Purified PvALDO protein (10 μg).

Figure 2

Antibody titer of cell supernatants, ascites and purified antibodies. The OD values (mean ± SD) of hybridoma cell supernatants, ascites (10^-3 dilution), and mAbs (1 μg/ml) were measured by the antibody-capture ELISA at 450 nm. Polystyrene 96-well plates were coated with GAM (2 μg/ml), antibodies were added and rPvALDO used was at a concentration of 2 μg/ml and HRP conjugated rabbit-anti-ALD IgG (1:5000) was used as secondary antibody. PBS and normal mouse serum were used as control.

Figure 3

Antibody affinity. Plates were coated with GAM (2 μg/ml) and mAbs 14C7, 15F1 and 5H7 at concentrations of 1.25, 0.63, 0.313, 0.156, 0.078, 0.039 and 0.01953 μg/ml were added for 1h. rPvALDO (0.5 μg/ml) was added and PBST was used as negative control. Rabbit-anti-ALD IgG-HRP (1:5000) was used as secondary antibody.

Figure 4

Sensitivity of assay to rPvALDO antigen. Test strips were treated with different concentrations of rPvALDO antigen and the colour development observed after 15 mins. T and C represent the test and control lines, respectively.