Use of VHH antibodies for the development of antigen detection test for visceral leishmaniasis

Abstract

We have recently developed a sensitive and specific urine-based antigen detection ELISA for the diagnosis of visceral leishmaniasis (VL). This assay used rabbit IgG and chicken IgY polyclonal antibodies specific for the Leishmania infantum proteins iron superoxide dismutase 1 (Li-isd1), tryparedoxin1 (Li-txn1) and nuclear transport factor 2 (Li-ntf2). However, polyclonal antibodies have limitations for upscaling and continuous supply. To circumvent these hurdles, we began to develop immortalized monoclonal antibodies. We opted for recombinant camelid VHHs because the technology for their production is well established and they do not have Fc, hence providing less ELISA background noise. We report here an assay development using VHHs specific for Li-isd1 and Li-ntf2. This new assay was specific and had analytical sensitivity of 15-45 pg/mL of urine. The clinical sensitivity was comparable to that obtained with the ELISA assembled with conventional rabbit and chicken antibodies to detect these two antigens. Therefore, similar to our former studies with conventional antibodies, the future inclusion of VHH specific for Li-txn1 and/or other antigens should further increase the sensitivity of the assay. These results confirm that immortalized VHHs can replace conventional antibodies for the development of an accurate and reproducible antigen detection diagnostic test for VL.

Figure 1

Reactivity of selected VHHs with recombinant proteins Li‐isd1 or Li‐ntf2. Microtitre plates were coated with Li‐isd1 (A) or Li‐ntf2 (B), and reactivity of the selected VHHs was tested by conventional ELISA

Figure 2

Sensitivity of capture ELISAs assembled with VHH for detection of the proteins Li‐isd1 and Li‐ntf2 spiked in buffer as well as in urine samples of normal healthy subjects. Capture VHH JRD‐C1/JRD‐C1‐C1/myc (anti‐Li‐isd1) or JRO‐H8/JRO‐H8/myc (anti‐Li‐ntf2) at 2 μg/mL was used to coat the ELISA plates. Wells were then incubated with various concentrations of Li‐isd1 or Li‐ntf2 diluted either in buffer plus 1% BSA or in urine from normal healthy subjects followed by incubation with E‐tag‐labelled developing VHH JRD‐E5/JRD‐E9 (anti‐Li‐isd1) or JRF‐E3/JRF‐E3 (anti‐Li‐ntf2). Reactions were developed after addition of peroxidase‐labelled anti‐E‐tag plus the substrate H₂O₂ and the chromophore TMB. Results are expressed as OD read at 450 nm. Same results were obtained using ELISA assembled with conventional rabbit IgG and chicken IgY antibodies (not shown)

Figure 3

Antigen detection capture ELISA assembled with VHHs for the identification of Li‐isd1 in urine of VL patients and controls. ELISA plates were coated with either VHH JRD‐C1/JRD‐C1‐C1/myc (2 μg/mL) or antigen affinity‐purified IgY anti‐Li‐isd1 antibody (1 μg/mL). After overnight incubation with patients or control, urine samples plates were washed and wells were incubated with E‐tag‐labelled developing VHH JRD‐E5/JRD‐E9 or biotin‐labelled rabbit IgG anti‐Li‐isd1 antibody. Wells corresponding to reactions with VHHs were next incubated with peroxidase‐labelled rabbit anti‐E‐tag antibody. Wells incubated with biotinylated IgG were next incubated with streptavidin‐peroxidase. The substrate H₂O₂ and the chromophore TMB were then added followed by OD reading at 450 nm. Samples from VL patients and from controls were from Belo Horizonte, MG, Brazil. Dashed lines represent the cut‐off values calculated as described in the text. These are representative results of at least three experiments performed at different times with the same urine samples and same capture ELISA. VL, visceral leishmaniasis

Figure 4

Antigen detection capture ELISA assembled with VHHs for the identification of Li‐ntf2 in urine of VL patients and controls. ELISA plates were coated with either JRO‐H8/JRO‐H8/myc (2 μg/mL) or purified IgY anti‐Li‐ntf2 antibody (1 μg/mL). After overnight incubation with patients or control urine samples, plates were washed and wells were incubated with E‐tag‐labelled developing JRF‐E3/JRF‐E3 or biotin‐labelled rabbit IgG anti‐Li‐isd1 antibody. Wells corresponding to reactions with VHHs were next incubated with peroxidase‐labelled rabbit anti‐E‐tag antibody. Wells incubated with biotinylated IgG were next incubated with streptavidin‐peroxidase. The substrate H₂O₂ and the chromophore TMB were then added followed by OD reading at 450 nm. Samples from VL patients and from controls were from Belo Horizonte, MG, Brazil. Dashed lines represent the cut‐off values calculated as described in the text. These are representative results of at least three experiments performed at different times with the same urine samples and same capture ELISA. VL, visceral leishmaniasis

Figure 5

Compiled results of capture ELISA assembled with VHHs or conventional Abs for the identification of the proteins Li‐isd1 and Li‐ntf2 in urine of VL patients and controls. Note that several samples were positive only with either one of the VHH (A) or conventional Abs (B) assays, hence supporting the development of a multiplexed assay to increase the clinical sensitivity of the test. Also, the overall sensitivity of the compiled assays was highly comparable between VHH and conventional Abs assays (54.2% and 58.3% for VHH and conventional Abs, respectively). VL, visceral leishmaniasis

Figure 6

Clinical sensitivity and specificity of a capture duplexed ELISA assembled with both Li‐isd1 and Li‐ntf2 VHHs. ELISA plate wells were coated with a pool of VHH JRD‐C1/JRD‐C1‐C1/myc (anti‐Li‐isd1) and JRO‐H8/JRO‐H8/myc (anti‐Li‐ntf2) VHHs following by blocking and overnight incubation with patients or control urine samples. Plates were washed, and wells were incubated with a pool of the E‐tag‐labelled developing VHHs JRD‐E5/JRD‐E9 (anti‐Li‐isd1) plus JRF‐E3/JRF‐E3 (anti‐Li‐ntf2). Wells were next incubated with peroxidase‐labelled rabbit anti‐E‐tag antibody. The substrate H₂O₂ and the chromophore TMB were then added followed by OD reading at 450 nm. Samples from VL patients, n = 24; CL (cutaneous leishmaniasis) n = 6; CD (Chagas disease), n = 6; Sch (schistosomiasis), n = 6; and TB (tuberculosis), n = 12 were from Belo Horizonte, MG, Brazil. Healthy controls, n = 6 were from local donors. Dashed line represents the cut‐off values calculated as described in the text. These are representative results of at least three experiments performed at different times with the same urine samples. VL, visceral leishmaniasis