A recombinant monoclonal-based Taenia antigen assay that reflects disease activity in extra-parenchymal neurocysticercosis

Abstract

Background: Antigen tests for diagnosis and disease monitoring in some types of neurocysticercosis (NCC) are useful but access to testing has been limited by availability of proprietary reagents and/or kits.

Methods/principal findings: Three previously identified IgM-secreting hybridomas whose IgM products demonstrated specificity to Taenia solium underwent variable heavy and light chain sequencing and isotype conversion to mouse IgG. Screening of these recombinantly expressed IgG anti-Ts hybridomas, identified one (TsG10) with the highest affinity to crude Taenia antigen. TsG10 was then used as a capture antibody in a sandwich antigen detection immunoassay in combination with either a high titer polyclonal anti-Ts antibody or with biotinylated TsG10 (termed TsG10*bt). Using serum, plasma, and CSF samples from patients with active NCC and those from NCC-uninfected patients, ROC curve analyses demonstrated that the TsG10-TsG10-*bt assay achieved a 98% sensitivity and 100% specificity in detecting samples known to be antigen positive and outperformed the polyclonal based assay (sensitivity of 93% with 100% specificity). By comparing levels of Ts antigen (Ag) in paired CSF (n = 10) or plasma/serum (n = 19) samples from well-characterized patients with extra-parenchymal NCC early in infection and at the time of definitive cure, all but 2 (1 from CSF and 1 from plasma) became undetectable. There was a high degree of correlation (r = 0.98) between the Ag levels detected by this new assay and levels found by a commercial assay. Pilot studies indicate that this antigen can be detected in the urine of patients with active NCC.

Conclusions/significance: A newly developed recombinant monoclonal antibody-based Ts Ag detection immunoassay is extremely sensitive in the detection of extra-parenchymal NCC and can be used to monitor the success of treatment in the CSF, serum/plasma and urine. The ability to produce recombinant TsG10 at scale should enable use of this antigen detection immunoassay wherever NCC is endemic.

Clinical trial registration: ClinicalTrials.gov Identifiers: NCT00001205 - & NCT00001645.

Fig 1. Antibody selection and adaptation.

A) Comparison of various potential IgG and IgM antibodies for the detection of Taenia antigens. Direct ELISA coated with two-fold serial dilutions of purified T. crassiceps antigen (Tc Ag). Various IgG and IgM antibodies were used for detection and the S/N ratio is shown. TsG10 is the related IgG of TsW5, while 11H12 is the related IgG of TsW11. B) Complete heavy and light chain variable region amino acid sequences used for construction of the TsG10 IgG antibody.

Fig 2. TsG10-TsG10*bt assay determination of the assay cutoff.

A) Receiver operating characteristic (ROC) curve of the S/N results testing 90 uninfected control serum/plasma/CSF samples and 56 serum/plasma/CSF samples from patients with known active SANCC and a positive antigen result by a comparator assay (see Materials and Methods). B) Using the Wilson/Brown method, while maximizing sensitivity and specificity, the S/N threshold for positivity was chosen to be >2.29 (line shown) plotted alongside the sample S/N results used for ROC curve construction shown in (A). Also shown are the S/N from serum samples from patients with active E. granulosus infection.

Fig 3. Tc Ag detection of standards using the TsG10 and comparator assays over a dilution series, shown as S/N ratios.

This standard curve was used so that Ts Ag concentrations in patient samples could be interpolated.

Fig 4

A) Serum, plasma, and CSF samples from patients with known SANCC, but at various stages of treatment (including cured) were tested by the TsG10-TsG10*bt and the comparator assay, ApDia. Both assays were run with the same standard curve and quantitative results interpolated from the standard curve. Data are antigen concentrations +1 (ng/ml). B) Spearman Correlation analysis using the sample results from (A).

Fig 5

Paired plasma/serum and CSF sample Ts Ag results in active and cured extra-parenchymal NCC (A) Twenty paired plasma/serum and (B) 10 paired CSF samples from patients with active extra-parenchymal NCC during active disease (early in treatment) and at the time of cure. Data are antigen concentrations +1 (ng/ml).