Human CD276 protein, His tag

Cluster Of Differentiation 276 (CD276) also known as B7-H3 in human, is a member of the B7/CD28 superfamily of costimulatory molecules serving as an accessory modulator of T-cell response. B7 proteins are immunoglobulin (Ig) superfamily members with extracellular Ig-V-like and Ig-C-like domains and short cytoplasmic domains. Among the family members, they share about 20-40% amino acid (aa) sequence identity. CD276 may play a protective role in tumor cells by inhibiting natural-killer mediated cell lysis as well as a role of marker for detection of neuroblastoma cells. It has also been found to enhance the induction of primary cytotoxic T lymphocytes and stimulate IFN-gamma production. Besides, Sialic acid-binding Ig-like lectin 12 (SIGLEC12) has been identified as an interactor of CD276, thus a binding ELISA assay was conducted to detect the interaction of recombinant human CD276 and recombinant human SIGLEC12. Briefly, CD276 were diluted serially in PBS, with 0.01% BSA (pH 7.4). Duplicate samples of 100 μl were then transferred to SIGLEC12-coated microtiter wells and incubated for 2h at 37ºC. Wells were washed with PBST and incubated for 1h with anti-CD276 pAb, then aspirated and washed 3 times. After incubation with HRP labelled secondary antibody, wells were aspirated and washed 3 times. With the addition of substrate solution, wells were incubated 15-25 minutes at 37ºC. Finally, add 50 μl stop solution to the wells and read at 450nm immediately. The binding activity of CD276 and SIGLEC12 was in a dose dependent manner.