硫酸链霉素细菌内毒素定量测定法的研究

应用鲎试剂动态浊度法，将硫酸链霉素制备成4、2、1、0．5mg／mL溶液，定量添加标准内毒素进行干扰预试验，计算回收率，确定不干扰浓度为0．5—2mg／mL。从中选出1mg／mL为最佳浓度，然后进行3个批次硫酸链霉素正式干扰试验。在供试品中定量添加标准内毒素，其回收率分别为125．8％、126．1％、127．3％，均在50％-200％之间，说明此浓度的供试品对鲎试剂反应无干扰作用。因此，内毒素含量在0．015—1EU／mL范围内，将硫酸链霉素制备成浓度为1．0mg／mL的溶液，可用于细菌内毒素的定量检测。     

ELISE测定猪尿、猪肝中克伦特罗残留的研究

ELISA(酶联免疫吸附法)测定猪尿、猪肝中违禁药物克伦特罗残留量影响实验结果表明,德国拜发与北京望尔生产的两种ELISA试剂盒具有操作简单、时间短、成本低、敏感度高等特点。在室温20-22℃条件下,试验制备过程中必须做到控温度、控转速、控流速及洗板过程中应注意事项,这样可大大提高在实际操作过程中检测的准确性。     

Molecular Cloning and Expression of IL2 cDNA from the Tibet Pig

Interleukin-2 is a vital cytokine secreted by activated T lymphocytes, and plays important role in the regulation of cellular and humoral immunity of animals. In our experiment, IL2 cDNA of the Tibet Pig was first cloned by RT-PCR from ConA-stimulated lymphocytes in the blood and subcloned into pMD-18 T vector, which then was identified with endonuclease restriction. The sequencing result showed that Tibet pig IL-2 (TPIL-2) cDNA was 503 bp long (ORF was 465 bp) (Genbank accession number: AY 294018). The recombinant prokaryotic and eukaryotic expression plasmids of the cDNA were then constructed to analyse the ability to stimulate the proliferation of porcine lymphocytes in vitro. The recombinant porcine IL-2 expressed in the prokaryotic cells was found to be of 43 kDa molecular mass, which was consistent with a 17.4 kDa protein deduced from the IL-2 cDNA sequence (glutathione S-transferase molecular mass is 26 kDa); the recombinant protein in eukaryotic cells was confirmed by use of specific rabbit anti-porcine IL-2 serum in an ELISA. The bioactivity of TPIL-2 was detected through MTT colorimetry by stimulating the proliferation of pig ConA-stimulated blasts in vitro. The results indicate that the TPIL-2 significantly promoted the proliferation of ConA-stimulated blasts of pig. This confirms that IL-2 cDNA of the Tibet pig was successfully cloned and expressed in prokaryotic and eukaryotic cells, which lays the foundation for the the preparation of specific recombinant IL-2 protein and development of novel immune adjuvants to raise the immunity of pigs against various infectious pathogens and increase the immunoprotective efficacy of vaccines.     

犬瘟热病毒抗体检测方法的比较研究

用犬瘟热（ＣＤ）弱毒和自制的高免犬血清，建立了检测犬瘟热病毒（ＣＤＶ）抗体的中和（ＳＮ）试验、间接荧光抗体技术（ＩＦＡＴ）和间接免疫酶染色法。这３种方法对３８份ＣＤ弱毒疫苗免疫犬血清抗体效价的测定结果表明，当被检血清稀释度为１∶１００时，ＳＮ试验、ＩＦＡＴ和间接免疫酶染色法测定时的阳性血清数分别为２９，７和３１份，ＩＦＡＴ和间接免疫酶染色法测定结果相对于ＳＮ试验结果的符合率分别为４２．１％和９４．７％。同时发现，当ＳＮ试验测定的血清效价在１∶１００以上时，ＩＦＡＴ测定结果总是在１∶２５以上，间接免疫酶染色法测定结果总是在１∶１００以上。３种方法以各自初步确定的ＣＤＶ血清抗体效价完全保护值指标计算所测血清的完全保护率，ＳＮ试验为７６．３％，ＩＦＡＴ为７８．９％，间接免疫酶染色法为８１．６％。据此认为，ＩＦＡＴ和间接免疫酶染色法均可部分代替ＳＮ试验用于ＣＤ疫苗免疫效果的评价、免疫犬抗体水平的监测以及ＣＤ的流行病学调查     

金免疫层析法检测囊虫循环抗原试验研究

应用胶体金和免疫层析技术进行了检测金免疫层析法(GICA)囊虫循环抗原(CA)试验研究。5株抗囊虫McAb和一株多抗用于最佳配对的筛选,一株用于标记胶体金,另一株包被NC膜。以双抗体夹心ELISA为对照,GICA用于CA检测,观察其敏感性、特异性和稳定性。结果显示,以McAb 1A5和1B6配对的结果最佳,1A5最适标记量为10μg／mL,186最佳包被浓度为1 g／L;用GICA和ELISA两种方法检测囊虫CA,痈猪血清和囊液的符合率达100％,病人血清和脑脊液的符合率达80％;CA最低检测量为10 ng,检测正常猪、人及其他疾病血清,均呈阴性;检测时间5-15 min,整个实验仅一步操作;GICA试纸条分别在4℃和室温下保存105 d以及在37℃保存45 d,检测结果均未发现改变。以上结果表明,快速检测囊虫循环抗原的金免疫层析法具有简便、快速、敏感、特异和稳定的特点,适合基层使用。     

Development of an Immunobinding Assay with Monoclonal Antibodies to Diagnose Mycoplasma bovis in Semen

Veterinary Research Communications -     

返滴定法测定蜂胶中游离酸含量

建立测定蜂胶品中游离酸含量的方法.采用弱碱溶解蜂胶再酸化萃取的方法纯化样品,用返滴法测定,以水杨酸作为对照品检测样品中游离酸含量.结果测定了5个不同产地的蜂胶样品,游离酸含量在1.89～6.54%范围内,回收率为99.30%.本方法操作简单,重现性好,有助于蜂胶质量的鉴别.     

Comparison of antibody values in sera of pigs vaccinated with a subunit or an attenuated vaccine against classical swine fever

Ten pigs, aged 85 days, were vaccinated with a subunit vaccine containing 32 g of classical swine fever virus glycoprotein E2 (gp E2) (group 1), and a further 10 pigs were vaccinated with a C strain vaccine (10^4±0.15 TCID₅₀/ml), produced by amplification in minipig kidney (MPK) cell culture (group 2). Nine non-vaccinated pigs served as a control group (group 3). Serum samples were collected before (day 0) and at 4, 10, 21 and 28 days after vaccination and were analysed by two commercially available enzyme immunoassays and by a neutralizing peroxidase-linked assay (NPLA). At the same times, peripheral blood was taken for determining the total leukocyte count and the body temperature was taken daily. Antibodies were not detected in serum samples collected before vaccination (day 0), and no side-effects that could be connected with vaccination were observed during the trial. Ten days after vaccination 6/10 pigs vaccinated with the subunit vaccine were seropositive. On days 21 and 28, the ratios of serologically positive to vaccinated pigs were 9/10 and 10/10, respectively. Four of the ten pigs that were vaccinated with the C strain vaccine were positive on day 21 and 9/10 on day 28. However, the results of the NPLA showed that only 4/10 pigs had an antibody titre >1:32 at the end of the trial in both the vaccinated groups, even though the subunit vaccine initiated an earlier and higher level of neutralizing antibodies than the vaccine produced from the C strain. Challenge was performed 28 days after vaccination on four randomly selected pigs from both vaccinated groups. The pigs survived the challenge without showing any clinical signs of classical swine fever (CSF), while two nonvaccinated control pigs died on the 10th and 12th days after infection.     

快速凝集试验与补体结合试验检测牛边缘无浆体感染的效果对比

从138份血清样品的比较试验结果显示，快速凝集试验（RCA）比补体结合试验（CF）检测边缘无浆体感染的敏感性高（88．9％：81．5％），假阴性率低（11．1％：18．5％），两者都具有良好的特异性和预测性，检测阳性符合率高，快速凝集试验对一次感染牛的持续检出阳性时间更长久（303天：92天）     

Effect of blood sample handling post-collection on Erysipelothrix rhusiopathiae antibody titres

A study was conducted to determine the effect of blood sample mishandling on the performance of an enzyme-linked immunosorbent assay for the detection of antibodies against Erysipelothrix rhusiopathiae. Eleven sample maltreatments (storage at −10 °C, storage at 4 °C, heat treatment of clotted blood, haemolysis, repetitive freeze–thaw cycling, and substitution of plasma in place of serum) were simulated in a laboratory environment and then run concurrently against a gold standard sample (storage at −80 °C).The mishandling treatment groups that simulated high levels of haemolysis had significantly lower optical density (OD) readings when compared to the gold standard. However, the magnitude of the effects was relatively small and only samples with OD values close to the cut-off changed state from positive to negative. Heat treatment had a minor, but non-significant, effect on OD values. Findings from this study suggested that immunoglobulin G antibody was stable in the face of most common sample mishandling events.