Immunoassay for Serum Glutamine Synthetase in Serum: Development, Reference Values, and Preliminary Study in Dementias

Abstract

Glutamine synthetase (GS) is a ubiquitous enzyme present at high concentrations in liver, muscle, kidney, and brain (1). GS in the brain is astrocyte specific, is involved in the detoxification of ammonia and glutamate, and is overexpressed after brain injury (2). Monomeric GS protein was found in 38 of 39 cerebrospinal fluid samples obtained from Alzheimer disease (AD) patients (3), and the concentration of GS in the lumbar cerebral spinal fluid of AD patients was increased significantly but nonspecifically (4). GS has also been reported in serum (4). Markers for AD have poor sensitivity and specificity for overt or preclinical AD (5)(6). We have developed an ELISA that is rapid, highly sensitive, and GS specific. This ELISA, together with the S100B and neuron-specific enolase (NSE) ELISAs, was used to retrospectively assess serum GS in healthy controls, AD patients, and other dementia patients. Recombinant human GS (rhGS) cDNA was purchased from ATCC (ATCC No. 409071). The full-length rhGS open reading frame was obtained by PCR and subcloning in pET28a ( Nde l/ Xho I). The construct included a polyhistidine tag at the N-terminal domain of the rhGS open reading frame and no extra sequence at the C-terminal domains. The protein was expressed in E. coli BL21 (DE3) (7). We prepared extracts and solubilized products as described (8). Affinity purification was performed by nickel-nitrilotriacetic acid chromatography. Female Balb/c mice (7–8 weeks of age) were immunized by subcutaneous injection of 50 μg of rhGS emulsified in complete Freund’s adjuvant. An intraperitoneal booster injection was given 3 weeks later (50 μg in incomplete Freund’s adjuvant) and then at 2- to 3-week intervals (three times or more) with a final injection of 50 μg of antigen in buffer via intraperitoneal route 3 days before cell fusion. Hybridoma cultures were screened by ELISA, and …