A Direct Immunoassay Assessment of Streptavidin- and N-Hydroxysuccinimide-Modified Biochips in Validation of Serological TNFα 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—α (TNFα) serodiagnosis of hemophagocytic lymphohistiocytosis (HLH). Interactions of TNFα antigen and TNFα 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 TNFα and TNFα 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 TNFα-receptor complexes (TNFR1 and R2) in HLH, a biochip assay strategy was developed. Plasma levels of TNFα, TNFα antibody, and TNFα-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 TNFα, 30.9% (13/42) for TNFα antibody, 28.6% (12/42) for TNFα—receptor 1 complex, and 26.1% (11/42) for TNFα—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. ( Journal of Biomolecular Screening 2008:515-526)