Klassevirus 3C ELISA for matched serum samples from klassevirus PCR-positive children from India. Endpoint OD₄₅₀ values for matched preinfection serum samples (1:100 dilution; white) and postinfection serum samples (1:100 dilution; shaded) are grouped together, along with polyclonal rabbit antisera generated against klassevirus 3C and blocking buffer (A). The dotted line indicates an OD₄₅₀ cutoff value of 0.30 that separates pre- and postinfection OD₄₅₀ values. Asterisks indicate significant increases in OD₄₅₀ (P < 0.005). Remaining serum was available from two matched serum samples (2246 and 3516) for Western confirmation, which showed a 4-fold and 8-fold increase in signal between pre- and post-klassevirus infection serum samples at a dilution of 1:200, respectively (B).

Purification of klassevirus 3C protease. N-terminal His-tagged klassevirus 3C protease was expressed in E. coli and purified by Ni-NTA chromatography (A and B). Ni-NTA eluates were further purified by gel filtration chromatography using a Superdex 200 column (C). The 91-ml fraction was tested via silver stain to confirm purity of klassevirus 3C protease (D). Lane M, molecular mass markers.

Klassevirus 3C ELISA for 353 age-banded samples from St. Louis Children's Hospital and Barnes-Jewish Hospital. Approximately 30 samples for each age band were included. A specific OD₄₅₀ cutoff value of 0.30 based on the matched Indian serum in Fig. 2 suggested 24 (6.8%) of the serum samples were seropositive for klassevirus.