Fetz Kathrin, Ruaux Craig G, Steiner Jörg M, Suchodolski Jan S, Williams David A
Gastrointestinal Laboratory, Department of Small Animal Medicine and Surgery, College of Veterinary Medicine, Texas A&M University, College Station, TX 77843-4474, USA.
Biochimie. 2004 Jan;86(1):67-75. doi: 10.1016/j.biochi.2003.10.005.
Alpha(1)-proteinase inhibitor (alpha(1)-PI) of the domestic cat (Felis catus) was purified from serum and a radioimmunoassay (RIA) for the measurement of feline alpha(1)-PI concentration in serum was developed and validated. Feline alpha(1)-PI (falpha(1)-PI) was isolated using ammonium sulfate precipitation, anion-exchange, size-exclusion, ceramic hydroxyapatite, and hydrophobic interaction chromatography. The molecular weight of falpha(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 alpha(1)-PI was raised in rabbits. Tracer was produced by iodination ((125)I) of feline alpha(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 alpha(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 alpha(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 alpha(1)-PI in cats.
从家猫（Felis catus）血清中纯化出α1-蛋白酶抑制剂（α1-PI），并开发和验证了一种用于测定血清中猫α1-PI浓度的放射免疫分析（RIA）方法。使用硫酸铵沉淀、阴离子交换、尺寸排阻、陶瓷羟基磷灰石和疏水相互作用色谱法分离猫α1-PI（fα1-PI）。fα1-PI的分子量估计为57,000，相对分子质量（Mr）测定约为54.5 kDa。等电聚焦显示出四条等电点（pI）在4.3至4.5之间的条带。前19个残基的N端氨基酸序列为Glu-Gly-Leu-Gln-Gly-Ala-Ala-Val-Gln-Glu-Thr-Val-Ala-Ser-Gln-His-Asp-Gln-Glu。用兔制备了抗猫α1-PI的抗血清。采用氯胺T法对猫α1-PI进行碘化（125I）制备示踪剂。通过测定灵敏度、稀释平行性、加标回收率、批内变异和批间变异建立并验证了放射免疫分析方法。使用50只健康猫的第95百分位数中心值确定了血清猫α1-PI浓度的对照范围。该分析方法的灵敏度为0.042 mg/ml。在1/35,000、1/70,000、1/140,000和1/280,000的稀释度下，四种不同血清样品的观察值与预期值之比在105%至141.18%之间。在四种不同血清样品和五种不同加标浓度下，加标回收率的观察值与预期值之比在88.14%至152.17%之间。四种不同血清样品的批内变异系数分别为4.57%、6.45%、8.52%和4.27%，批间变异系数分别为6.88%、9.57%、7.44%和9.94%。参考范围确定为0.25 - 0.6 mg/ml。总之，采用快速有效的方法成功地从血清中纯化出猫α1-PI。本文所述的放射免疫分析方法灵敏、线性、准确、精密且可重复，将有助于进一步研究α1-PI在猫体内的生理或潜在病理作用。
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