Determination of Canine .BETA.2-Microgloblin in Plasma and Urine by Enzyme-linked Immunosorbent Assay.

Abstract

ABSTRACT. An enzyme-linked immunosorbent assay was developed for the determination of canine β 2 -microglobulin (β 2 -m) in plasma and urine. The detectable sensitivity for pure canine β 2 -m was 0.05 µg/l and the analytical range was 0.1 to 50 µg/l. The mean analytical recovery when pure canine β 2 -m was added to normal plasma was 101.9%. The mean analytical recovery in the urine was 102.1%. The intra-day variation coefficient was 3.1% in plasma, 4.3% in serum and 1.9% in urine. No difference was found between the concentration of β 2 -m in plasma and serum (n=17). The concentration of β 2 -m in the plasma of normal dogs was 1.82 ± 0.57 mg/l (n=31). The mean excretion in 24 hr urine collected from normal dogs was 17.6 ± 9.2 µg/l, 0.22 ± 0.12 µg/kg of body weight or 14.2 ± 9.4 µg/g of urine creatinine. The β 2 -m creatinine index of random urine samples was 23.5 ± 16.6 µg/g (n=26). There was a close correlation between the β 2 -m creatinine index of 24 hr urine samples and that of random urine samples (r= 0.872). KEY WORDS: canine, ELIS, β 2 -microglobulin.J. Vet. Med. Sci. 63(3): 343-345, 2001 β 2 -microglobulin (β 2 -m) is a low molecular weight protein that is present on the surface of all nucleated cells [12]. This protein is the light chain of the class I major histocompatibility antigens [13]. Determination of serum β 2 -m and the urine β 2 -m concentration is used to evaluate renal function in man [3,9]. The determination of β 2 -m has been carried out by radioimmunoassay [4], enzyme-linked Immunosorbent assay (ELISA) [2] and immunoturbidimetry [1], but there are no reports on the measurement of β 2 -m concentrations in dog body fluids. In this study, we developed an ELISA test for determination of β 2 -m in the plasma, serum and urine of dogs.Purified canine β 2 -m and antiserum were prepared in our laboratory. The protocol used for isolation of canine β 2 -m from the urine was reported previously [11]. For preparation of antiserum, a rabbit was immunized by the injection of 1 mg of β 2 -m with complete Freund's adjuvant (ORGA-NON TECHNIKA Co., Belgium) intracutaneously into its back. The rabbit was injected every three weeks for a total of 3 times. The IgG fraction of the antiserum was precipitated with ammonium sulfate and then purified by means of an Econo-Pac Protein A Cartridge (Bio-Rad Laboratories, U.S.A.) according to Tarditi et al. [14]. The purified IgG was dialyzed against ammonium acetate buffer (pH 8.6, 4°C), lyophilized and stored at 4°C. The IgG fraction was conjugated with biotin (biotinyl-N-hydroxysuccinimide ester, EY Laboratories, U.S.A.) by a method similar to that of Guesdon et al. [5].Blood samples were obtained from 32 normal dogs. Plasma was isolated from 3 ml of whole blood, with heparin as anticoagulant. In addition, simultaneous serum samples were taken from 17 dogs. Twenty-four hour urine samples and random urine samples were collected from 26 normal dogs, and each urine sample was stored in several PE-tubes. Five µl of Tween 20 (Kantokagaku Co., Ltd., Japan) was added to ...

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