Enzyme-linked immunosorbent assay for serum apolipoprotein B-100, a major triglyceride-transport protein in dairy cows

Osamu Yamamoto From the Matsumoto Livestock Hygiene Service Center, Matsumoto, Nagano 390 (Yamamoto); Morioka Livestock Hygiene Service Center, Takizawa, Iwate 020-01 (Oikawa); and Laboratory of Biochemistry, National Institute of Animal Health, 3-1-1 Kannondai, Tsukuba, Ibaraki 305 (Katoh), Japan.

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Shin Oikawa From the Matsumoto Livestock Hygiene Service Center, Matsumoto, Nagano 390 (Yamamoto); Morioka Livestock Hygiene Service Center, Takizawa, Iwate 020-01 (Oikawa); and Laboratory of Biochemistry, National Institute of Animal Health, 3-1-1 Kannondai, Tsukuba, Ibaraki 305 (Katoh), Japan.

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Norio Katoh From the Matsumoto Livestock Hygiene Service Center, Matsumoto, Nagano 390 (Yamamoto); Morioka Livestock Hygiene Service Center, Takizawa, Iwate 020-01 (Oikawa); and Laboratory of Biochemistry, National Institute of Animal Health, 3-1-1 Kannondai, Tsukuba, Ibaraki 305 (Katoh), Japan.

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SUMMARY

An elisa was developed to determine serum concentration of apolipoprotein B-100, a major triglyceride-binding protein in very low-density lipoproteins and a putative maker for hepatic lipidosis of dairy cows. Serum apolipoprotein B-100 was prepared electrophoretically, and antibodies to this protein were raised in rabbits. The antiserum prepared was further purified by affinity chromatography, using bovine serum albumin-Sepharose 4B, to remove antibodies to albumin. For the elisa, addition of 2-mercaptoethanol to the coating buffer (50 mM sodium carbonate, pH 9.6) was required to evaluate apolipoprotein B-100 concentration in serum. The elisa developed was sensitive (detection limit was 300 to 400 ng/ml of serum) and reliable (coefficients of variance were in the range of 3.3 to 7.6%). By use of the established elisa, the serum apolipoprotein B-100 concentration was found to be significantly (P < 0.01) lower during the early lactating stage than during other stages of lactation. Reduced hepatic synthesis or secretion of apolipoprotein B-100 during the early lactating stage, together with the excess uptake by the liver of serum nonesterified fatty acids, is suggested to be relevant in the accelerated accumulation of triglycerides in the liver of dairy cows during the periparturient period.

SUMMARY

An elisa was developed to determine serum concentration of apolipoprotein B-100, a major triglyceride-binding protein in very low-density lipoproteins and a putative maker for hepatic lipidosis of dairy cows. Serum apolipoprotein B-100 was prepared electrophoretically, and antibodies to this protein were raised in rabbits. The antiserum prepared was further purified by affinity chromatography, using bovine serum albumin-Sepharose 4B, to remove antibodies to albumin. For the elisa, addition of 2-mercaptoethanol to the coating buffer (50 mM sodium carbonate, pH 9.6) was required to evaluate apolipoprotein B-100 concentration in serum. The elisa developed was sensitive (detection limit was 300 to 400 ng/ml of serum) and reliable (coefficients of variance were in the range of 3.3 to 7.6%). By use of the established elisa, the serum apolipoprotein B-100 concentration was found to be significantly (P < 0.01) lower during the early lactating stage than during other stages of lactation. Reduced hepatic synthesis or secretion of apolipoprotein B-100 during the early lactating stage, together with the excess uptake by the liver of serum nonesterified fatty acids, is suggested to be relevant in the accelerated accumulation of triglycerides in the liver of dairy cows during the periparturient period.

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