Development of a Diagnostic IgM Antibody Capture ELISA for Detection of Anti-Cache Valley Virus Human IgM

Abstract

Cache Valley virus (CVV), a mosquito-borne orthobunyavirus, causes epizootics in ruminants characterized by congenital malformations and fetal death in North America. Only seven human infections have been identified; limited information exists on its potential as a human teratogen. Diagnosis of CVV infections relies on the plaque reduction neutralization test (PRNT), which requires live virus, is time-consuming, and cannot differentiate between recent and past infections. To improve diagnostics for CVV, we developed an IgM antibody capture ELISA (MAC-ELISA) for detection of anti-CVV human IgM in diagnostic specimens that can be performed faster than PRNT and is specific to IgM, which is essential to determine the timing of infection. Conjointly, a cell line constitutively expressing human-murine chimeric antibody with the variable regions of monoclonal antibody CVV-17 and constant regions of human IgM was developed to provide positive control material. The new cell line produced antibody with reactivity in the assay equivalent to that of a human serum sample positive for anti-CVV IgM. Five of seven archived human specimens diagnostically confirmed as CVV positive tested positive in the MAC-ELISA, whereas 44 specimens confirmed positive for another arboviral infection tested negative, showing good initial correlation of the CVV MAC-ELISA. Two of 27 previously collected serum samples from febrile patients in Yucatán, Mexico, who tested negative for a recent flaviviral or alphaviral infection were positive in both the MAC-ELISA and PRNT, indicating a possible recent infection with CVV or related orthobunyavirus. The MAC-ELISA described here will aid in making diagnostics more widely available for CVV in public health laboratories.

Figure 1.

Evaluation of Cache Valley virus (CVV) antigen produced in tissue culture in IgM antibody capture (MAC)-ELISA. Cache Valley virus antigen was made by infecting Vero, Vero E6, BHK21c.15 (B15), and LLC-MK2 (LLC) cells, inactivating viral cell culture supernatant with 0.1% beta-propiolactone, and concentrating inactivated supernatant approximately 10-fold. Antigen was titrated in the MAC-ELISA to observe optical density (OD) reactivity when tested using positive human serum (PHS) samples with high (A) and low (C) titers of CVV neutralizing antibody. P/N values (defined as the mean OD450 value of the serum sample reacted on CVV antigen divided by the mean OD450 value of normal human serum [NHS] [E] reacted on CVV antigen) of the human serum samples with high (B) and low (D) titers of CVV neutralizing antibody were calculated.

Figure 2.

Evaluation of the performance of horseradish peroxidase (HRP)-conjugated anti-CVV-14 as detector antibody in IgM antibody capture (MAC)-ELISA. (A) Anti-CVV-14 was conjugated to HRP and titrated in the MAC-ELISA to observe optical density (OD) reactivity when tested using positive human serum (PHS) samples with high (PHS-1) and low (PHS-2) titers of CVV neutralizing antibody and normal human serum (NHS). (B) P/N values (defined as the mean OD450 value of PHS reacted on CVV antigen divided by the mean OD450 value of NHS reacted on CVV antigen) were calculated. CVV = CVV Vero cell antigen; Normal = uninfected Vero cell antigen.

Figure 3.

Selection and performance of Cache Valley virus human IgM antibody (CVV-hIgM) from a recombinant cell line. (A) P/N values (mean value of optical density at 450 nm [OD450] of the CVV-hIgM reacted on CVV antigen divided by the mean OD450 value of normal human serum [NHS] reacted on CVV antigen) of supernatant from transiently transfected HEK-293 cells expressing CVV-hIgM with variable regions from murine monoclonal antibodies (MAbs) CVV-4, CVV-13, CVV-14, CVV-15, CVV-17, and CVV-18 diluted 1:10 to 1:320 in the CVV IgM antibody capture (MAC)-ELISA. (B) Nonspecific background reactivities (NBRs) (mean OD450 value of the CVV-hIgM reacted on CVV antigen divided by the mean OD450 value of CVV-hIgM reacted on Vero normal antigen) of supernatant from transiently transfected HEK-293 cells expressing CVV-hIgM with variable regions from murine MAbs as described for panel A. (C) Predicted mean values of CVVhIgM17 endpoint titer (EPT) (black line) from the cell line were plotted as log (P/N) versus log₂(dilution), with confidence bands shown as dashed black lines. The reference line in blue for determining the EPT is shown as log₂. The estimated EPT (95% CI) is indicated with vertical red lines with confidence bands as dashed red lines. (D) EPT against passage number evaluated by least absolute deviation (LAD) regression test (black line) shows stable secretion of CVVhIgM17 from the cell line tested by MAC-ELISA. (E) Purified CVVhIgM17 was titrated in the MAC-ELISA with results expressed as a P/N value.