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. 2022 Aug 11;14(16):3887. doi: 10.3390/cancers14163887

Figure 1.

Figure 1

GRP78 measurements using an ultrasensitive ELISA with thio-NAD cycling. (A) Schematic representation of an ultrasensitive ELISA with thio-NAD cycling. A conventional sandwich ELISA was combined with a thio-NAD cycling assay, producing signals in a quadratic-function-like response (i.e., triangle number) over time. This system comprised ALP linked with a secondary antibody against the target protein, an androsterone derivative as the first substrate, 3α-HSD as the enzyme for thio-NAD cycling, and their coenzymes (NADH and thio-NAD). Thio-NADH accumulation was measured as absorbance at 405 nm. (B) A typical linear calibration curve of GRP78 standard proteins (i.e., antigen) from the ultrasensitive thio-NAD cycling ELISA. The absorbance was obtained from a 60-min cycling reaction time. The antigen was applied in the range of 1.89–15.0 pg/mL.