Rapid and Sensitive Immunoassay for the Measurement of Serum S100B Using Isoform-specific Monoclonal Antibody

Abstract

S100 is an acidic calcium-binding protein with a molecular weight of 21 000, originally discovered by Moore (1) in the bovine brain. Today, >14 different S100 members are known (2), of which S100A1 and S100B are the most studied (3)(4)(5)(6). Because a high concentration of S100B is present in the brain, S100 has been studied for use as a biochemical marker for central nervous system pathology. Numerous studies in the literature have suggested the clinical usefulness of measuring this protein in cerebrospinal fluid (7)(8)(9)(10)(11), and in recent years, serum S100B has been reported as a useful marker for early detection of metastases of melanoma and cerebral complications from head injury, cardiac surgery, and acute stroke (12)(13)(14)(15)(16)(17)(18)(19)(20)(21). With the advent of therapeutic treatments for stroke that either dissolve the clot or protect the brain, early diagnosis of stroke and the identification of appropriate patients for intervention are increasingly important. Existing assays lack sensitivity and ease of use and usually require ≥3 h to perform. We have, therefore, developed an ELISA that is rapid, highly sensitive, and S100B specific. This ELISA was used to retrospectively assess S100B concentrations in the serum of stroke patients. Human S100B cDNA (Genome System), amplified by PCR, was cloned into vector pET28a (Novagen) and expressed in Escherichia coli BL-21 (DE3)pLysS (Novagen). Recombinant human S100B (rS100B) was isolated from host cells that were induced with 1 mmol/L isopropylthio-β-galactoside for 2 h at 37 °C and were lysed with a native buffer system. S100B was purified with an Ni-NTA affinity column. The ELISA buffers were as follows: plate-coating buffer (100 mmol/L carbonate buffer, pH 9.6); phosphate-buffered …