Heterologous Radioimmunoassay for Llama and Alpaca Luteinizing Hormone with a Monoclonal Antibody,an Equine Standard and a Human Tracer

A radioimmunoassay for llama and alpaca LH was developed using a human I¹²⁵LH tracer from a commercial kit, equine LH diluted in human LH free serum as standard, and a monoclonal antibody (518B7) specific for LH but with low species specificity. A 60-min delay in the addition of the tracer and overnight incubation gave a sensitivity of 0.8 μg L⁻¹. The intra-assay coefficient of variation was 37% at 1 μg L⁻¹, declined to 15% at 4 pg L⁻¹ and was below 6% for concentrations up to 32 μg L⁻¹. The inter-assay coefficients of variation for 3 control samples were 20% (2.8 μg L⁻¹), 16% (7.1 μg L⁻¹) and 9.8% (19 μg L⁻¹). In an attempt to increase sensitivity, all tubes were preincubated for 4 h at room temperature before adding the tracer, and the sample volume was increased from 50 μL to 100 μL· (in the standard curve the increased volume was compensated for by human LH free serum). With this protocol, the assay sensitivity was 0.5 μg L⁻¹. The assay was validated clinically and demonstrated increased concentrations of LH after mating in llamas and alpacas. Furthermore, the assay was used to monitor LH responses to a single dose of GnRH in llamas (adult males and females at different ages).     

Preparation and comparison of biological properties of recombinant carp (Cyprinus carpio) growth hormone and its Cys-123 to Ala mutant

Carp growth hormone (cGH) cDNA, in which Cys-123 was mutated to Ala, was prepared, transferred to the expression vector, expressed in Escherichia coli and the mutant was purified to homogeneity. The mutation only slightly improved yield of the monomeric fraction, indicating that Cys-123 is not involved in improper refolding. As compared to cGH, the mutant (cGH-C123A) exhibited lower binding affinity toward homologous liver receptors and lower bioactivity in a 3T3-F442A preadipocyte bioassay despite the fact that both hormones exhibited almost identical cross-reactivity with anti-cGH antibodies. These results, along with those of a structural comparison to hGH, suggest that Cys-123 is located in the hydrophobic core of the hormone, and is most likely affecting the conformation of the binding site. Dimeric forms of the hormone and its mutant were less active than their respective monomers. Homologous binding experiments using a carp liver microsomal fraction revealed a single receptor population with Kd = 0.77 nM and B_max = 241 fmol/mg microsomal protein.

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Résumé Un ADN complémentaire (cDNA) de l'hormone de croissance de carpe (cGH), dans lequel l'acide aminé Cys-123 a été muté en Ala, a été préparé, inséré dans un vecteur d'expression, et exprimé dans Escherichia coli. Le mutant a ensuite été purifié jusqu'à homogénéité. La mutation améliore seulement faiblement la production de la fraction monomérique, indiquant que le Cys-123 n'est pas impliqué dans un repliement erroné. Comparé à la cGH, sa forme mutée (cGH-C123A) montre une plus faible affinité de liaison vis à vis de récepteurs hépatiques homologues, et une plus faible activité biologique dans un test réalisé sur des préadipocytes 3T3-F442A; cela en dépit du fait que les deux hormones présentent des réactions croisées presque identiques avec un anticorps anti-cGH polyclonale. Ces résultats, associés à une comparaison à la structure de l'hGH, suggèrent que le Cys-123 est localisé dans la partie hydrophobique de l'hormone, et affecte, le plus vraisemblablement, la conformation du site de liaison. Les formes dimériques de l'hormone et de sa forme mutée sont moins actives que leurs monomères respectifs. Les études de liaison homologue, réalisées avec des fractions microsomales de foie, révèlent une population unique de récepteurs de Kd = 0,77 nM et de B_max = 241 fmol/mg de proteine microsomale.

     

幼龄山羊腺垂体分泌TSH活动的季节和昼夜变化

用RIA法测定幼龄去势莎能公山羊血清中TSH含量的季节和昼夜变化,发现血清中TSH的年平均含量为3.198±1.02μIU/ml。冬季昼夜平均含量最高,为4.454±0.34μIU/ml,春季、秋季、夏季昼夜平均含量分别为3.510±0.39μIU/ml,2.748±0.41μIU/ml,2.078±0.59μIU/ml。各季度血清中TSH含量,有着极明显的季节差异。全年平均含量的昼夜变化及各季度含量的昼夜变化,均无明显的昼夜差异,但血清中TSH含量有明显的个体差异。     

Radioimmunoassay of Bovine,Ovine and Porcine Luteinizing Hormone with a Monoclonal Antibody and a Human Tracer

Forsberg, M., R. Tagle, A. Madej, J.R. Molina and M.-A. Carlsson. Radioimmunoassay of bovine, ovine and porcine luteinizing hormone with a monoclonal antibody and a human tracer. Acta vet. scand. 1993, 255-262.– A radioimmunoassay for bovine (bLH), ovine (oLH) and porcine (pLH) luteinizing hormone was developed using a human ¹²⁵ ILH tracer from a commercial kit and a monoclonal antibody (518B7) specific for LH but with low species specificity. Standard curves demonstrated similar binding kinetics when bLH, oLH and pLH were incubated with tracer and antibody for 2 h at room temperature. A 30-min delay in the addition of the tracer gave sufficient sensitivity when analysing pLH. Separation of antibody-bound LH from free hormone was achieved by using second antibody-coated micro Sepharose beads. The assay was validated and the performance compared with that of an RIA currently in use for determination of bLH and oLH (coefficient of correlation: 0.99 and 0.98). Regardless of the standards used, intra-assay coefficients of variation were <10% for LH concentrations exceeding 1 µg/L. The inter-assay coefficients of variation were <15%. The assay was used for clinical evaluation demonstrating the pre-ovulatory LH surge in two cyclic cows, LH pulsatility in an oophorectomized ewe and LH response to GnRH injection in a boar.     

Plasma Steroid Variations in Bull Calves Repeatedly Treated with Testosterone,Nortestosterone and Oestradiol Administered Alone or in Combination

The aim of this work was to investigate the secretion of dehydroepiandrosterone (DHEA), testosterone (T), dihydrotestosterone (DHT) and oestradiol (E) as biological markers in response to illegal administration of testosterone, 19-nortestosterone (N) and oestradiol, either alone or in combination. Twenty male Friesian calves (age 13-14 months) were allotted to a control group (n = 5), and five experimental groups (n = 3) each. Each experimental animal was repeatedly injected with one of the following hormonal treatments: E, T, N, T+E and N+E. Circulating DHEA, T, DHT and E were determined by radioimmunoassay. The administration of T alone did not induce any variation in plasma DHEA, T, DHT and E, which were similar to those in the control group. In contrast, DHEA, T and DHT were on average significantly lower in the T+E and N-treated groups (p < 0.01), whereas the administration of N+E resulted in the reduction of plasma T and DHT without any modification of plasma DHEA. The administration of E alone or in combination increased circulating levels of E but did not affect androgen plasma profiles. The results indicate that plasma levels of T do not permit detection of illegal treatments because plasma androgens always remained within the physiological range. Illegal E treatment could be detected in blood samples when they were collected at least every 20 days.     

广西地区人工饲养雌性食蟹猴雌二醇和睾酮含量变化的研究

探索广西地区人工饲养的雌性食蟹猴在少年、青年和中年阶段睾酮和雌二醇分泌情况。使用放射免疫方法分别测定血清中的睾酮、雌二醇含量变化。试验结果表明：广西地区人工饲养的雌性食蟹猴少年组、青年组和中年组间雌二醇分泌的量分别是38.81±21.57 pg/ml、76.09±56.23 pg/ml、64.37±39.48 pg/ml,分泌水平差异极显著（P〈0.01）,其中青年组雌二醇分泌量大于中年组,少年组的含量最少。雌性食蟹猴少年组、青年组和中年组间睾酮的分泌量分别是52.36±24.93 ng/ml,37.95±21.69 ng/ml、80.91±29.19 ng/ml,分泌水平差异极显著（P〈0.01）,其中中年组分泌水平最高,青年组其次,少年组最少。     

不孕症奶牛抗卵巢抗体的检测及临床意义

为探讨奶牛不孕症与ＡＯＡｂ的关系,用放免法检测啊１４例原发性不孕、１０例继发性不孕奶牛血清ＡＯＡｂ,并与正常妊娠组对照。实验表明,ＡＯＡｂ阳性１０例,总阳性率为４１．６７％,ＡＯＡｂ为原发性不孕相关,与继发性不孕无明显关系。结果提示,ＡＯＡｂ的存在是奶牛原发性不孕的重要病因。     

动物性食品中己烯雌酚残留量检测方法评定

用于检测动物性食品中己烯雌酚残留量的常用方法有放射免疫测定(RIA)、酶免疫测定(EIA)、气相色谱(GC)、高效液相色谱(HPLC)和薄层色谱(TLC)。比较这几种方法,认为RIA法测定的灵敏度较高,测定肝脏中DES的最低检出限可达0.3μg/kg,但对放射防护的要求较高,只能在专门实验室进行。GC法需增加衍生化步骤,检出限为2～10μg/kg。HPLC法用电化学检测器的最低检出限是0.1～0.2μg/kg。TLC法用硅胶作铺板,其最低检出限是0.1μg/kg。EIA法灵敏度高、特异性强,测定中华鳖肌肉中DES的最低检出限0.02μg/kg,是目前用于检测大量样品的首选方法。     

沼泽型水牛卵泡发育波及其生殖激素变化的试验研究

运用B超跟踪观察经同期发情处理后的沼泽型水牛的卵泡发育动态,并应用放射免疫测定法(RIA)测定其发情周期中血清促卵泡素(FSH)、促黄体素(LH)、雌二醇(E2)、孕酮(P4)等生殖激素的含量,分析了沼泽型水牛的卵泡波情况,并比较了四种生殖激素在发情周期不同时间段及不同卵泡波类型之间的差异性。发现沼泽型水牛的发情周期由2个或3个卵泡波组成,以2个卵泡波为主;2个或3个卵泡波中各个波征集、选择的卵泡数之间无显著差异;血清FSH、LH、E2、P4水平在表现为不同卵泡波类型的青年水牛无显著差异(P>0.05)。     

滤纸片E_2—17β放射免疫测定方法探讨

采用滤纸片(直径9.5mm)取定量血样,风干,通过浸泡、萃取,进行雌二醇(17β—Estradiol)放射免疫测定(RIA),经与小塑料管微量法对比,二者标准曲线基本平行.微量法Y=4.27-2.24x,纸片法Y=4.35-2.11x,平均回收率为91.3%,批内CV=7.6%～9.8%(n=10),批间CV=15.1%～19.3%(n=4).纸片法与微量法相比,具有同样准确性、特异性,还具有完整性和可行性,其灵敏度可达10~(-12)～10~(-15)g.干血点纸片法采血量少,方法简便,样品易于保存和运输,可用于畜禽及牛奶中激素含量测定.