A direct immunoassay assessment of streptavidin- and N-hydroxysuccinimide-modified biochips in validation of serological TNFalpha responses in hemophagocytic lymphohistiocytosis.

The authors report 2 biochip platforms on gold manufactured by either nanoscale biotinylated self-assembled architectures to streptavidin surface or proteins containing free NH(2) groups to N-hydroxysuccinimide (NHS)-activated surfaces and investigated the potential application of tumor necrosis factor-alpha (TNFalpha) serodiagnosis of hemophagocytic lymphohistiocytosis (HLH). Interactions of TNFalpha antigen and TNFalpha antibody on the biochips were optimized using an indirect immunofluorescence method. Variation coefficients were 1.87% to 4.56% on the streptavidin biochip and 5.03% to 8.64% on the NHS biochip. The correlation coefficients (r) in TNFalpha and TNFalpha antibody assays in HLH patients between the 2 biochip formats were 0.9623 and 0.9386 and the concordance frequencies were 92.2% and 96.1%, respectively. To detect plasma TNFalpha-receptor complexes (TNFR1 and R2) in HLH, a biochip assay strategy was developed. Plasma levels of TNFalpha, TNFalpha antibody, and TNFalpha-receptor complexes (TNFR1 and R2) were detected in plasmas from 42 HLH cases using streptavidin biochips. Frequencies of the biomarkers in the plasmas were 40.5% (17/42) for TNFalpha, 30.9% (13/42) for TNFalpha antibody, 28.6% (12/42) for TNFalpha-receptor 1 complex, and 26.1% (11/42) for TNFalpha-receptor 2 complex, respectively. The streptavidin biochip format was more sensitive than the NHS surface and was demonstrated to be a valuable tool to identify individual biomarker molecules and molecular complexes in sera and cell lysates and to track therapeutic progress of patients.