Purification and partial characterization of feline α_1-proteinase inhibitor (fα_1-PI) and the development and validation of a radioimmunoassay for the measurement of fα_1-PI in serum

摘要

Alpha_1-proteinase inhibitor (α_1-PI) of the domestic cat (Felis catus) was purified from serum and a radioimmunoassay (RIA) for the measurement of feline α_1-PI concentration in serum was developed and validated. Feline α_1-PI (fα_1-PI) was isolated using ammonium sulfate precipitation, anion-exchange, size-exclusion, ceramic hydroxyapatite, and hydrophobic interaction chromatography. The molecular weight of fα_1-PI was estimated at 57,000 and the relative molecular mass (M_r) was determined to be approximately 54.5 kDa. Isoelectric focusing revealed four bands with isoelectric points (pI) between 4.3 and 4.5. The N-terminal amino acid sequence of the first 19 residues was Glu-Gly-Leu-Gln-Gly-Ala-Ala-Val-Gln-Glu-Thr-Val-Ala-Ser-Gln-His-Asp-Gln-Glu. Antiserum against feline α_1-PI was raised in rabbits. Tracer was produced by iodination (~(125)I) of feline α_1-PI using the chloramine T method. A radioimmunoassay was established and validated by determination of sensitivity, dilutional parallelism, spiking recovery, intra-assay variability, and inter-assay variability. A control range for serum feline α_1-PI concentration was established from 50 healthy cats using the central 95th percentile. The sensitivity of the assay was 0.042 mg/ml. Observed to expected ratios for serial dilutions ranged from 105% to 141.18% for four different serum samples at dilutions of 1 in 35,000, 1 in 70,000, 1 in 140,000 and 1 in 280,000. Observed to expected ratios for spiking recovery ranged from 88.14% to 152.17% for four different serum samples and five different spiking concentrations. Coefficients of variation for four different serum samples were 4.57%, 6.45%, 8.52%, and 4.27% for intra-assay variability and 6.88%, 9.57%, 7.44%, and 9.94% for inter-assay variability. The reference range was established as 0.25-0.6 mg/ml. In summary, feline α_1-PI was successfully purified from serum using a rapid and efficient method. The radioimmunoassay described here is sensitive, linear, accurate, precise, and reproducible and will facilitate further studies of the physiological or potential pathological role of α_1-PI in cats.