Generation of soluble recombinant human acute phase serum amyloid A2 (A-SAA2) protein and its use in development of an A-SAA specific ELISA.
Human acute phase serum amyloid A (the A-SAA2 isoform) was expressed at high levels using the pGEX bacterial expression system. A-SAA2 protein was expressed in E. coli NM544 as part of a fusion protein facilitating rapid purification. A-SAA2 was cleaved from the fusion moiety in the presence of a non-ionic detergent (Triton X-100) to release a soluble A-SAA2. Further purification using ion exchange chromatography yielded a pure A-SAA2 (3 mg per litre of culture). Antibodies generated against recombinant A-SAA2 were specific for the acute phase SAAs, A-SAA1 and A-SAA2 and showed no cross-reactivity with the constitutively expressed SAA (C-SAA). These antibodies were used to develop a rapid enzyme-linked immunosorbent assay (ELISA) specific for the measurement of A-SAA in serum.
Department of Genetics, Trinity College, Dublin, Ireland.
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McCormack
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Hobson
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Doyle
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Jackson
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Kilty
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Whitehead
D000209:Acute-Phase Proteins / Q000096:biosynthesis*; D000293:Adolescent; D000328:Adult; D000368:Aged; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D006801:Humans; D008875:Middle Aged; D011994:Recombinant Proteins / Q000096:biosynthesis*; D012680:Sensitivity and Specificity; D000685:Serum Amyloid A Protein / Q000096:biosynthesis* / Q000276:immunology*; D012995:Solubility
D000209:Acute-Phase Proteins; D011994:Recombinant Proteins; D000685:Serum Amyloid A Protein