血脂调节剂的研究——Ⅰ.HMG-CoA还原酶抑制剂M-8614A及MH-2688B产生菌的筛选

利用酶联免疫吸附测定法,酶标β-羟基β-甲基-戊二酸单酰铺酶A(HMG-CoA)还原酶抑制剂Compactin抗体,定向筛选血脂调节剂。从青霉M-8614菌株发酵液中分离到M-8614A。该物质理化性质及波谱解释表明与Mevastatin为同一物质。 M-8614菌株用亚硝酸盐等诱变剂处理,从诱变株MH-2688发酵液中分离到MH-2688B。该物质理化性质及波谱解释表明与Lovastatin为同一物质。     

ELISA检测襄虫病人血清循环抗原及其在疗效考核方面的应用

用快速双抗体夹心ＥＬＩＳＡ检测囊虫病人血清中的循环抗原，其阳性率为６７．５％；正常人血有的假阳性率为５．３３％；包虫、肝吸虫和弓形虫病人血清的交叉反应率分别为６．９％、７．５％和１２．５％。结果表明．该法与常规双抗体夹心ＥＬＩＳＡ相比具有较为敏感、快速、稳定和节省血清、试剂用量的优点，但其特异性有待进一步提高。用快速双抗体夹心ＥＬＩＳＡ和间接型ＥＬＩＳＡ检测第１至１０疗程囊虫病人血清中的循环抗原和抗体，表明检测循环抗原考核囊虫病人的治疗效果优于检测抗体     

斑点—酶联免疫吸附试验检测卡氏肺孢子虫

应用完整的卡氏肺孢子虫包囊作抗原，以４×１０＾７／ｍｌ包裹点样，作斑点－酶联免疫吸附试验检测肺印片卡氏肺孢子虫包囊阳性的ＢＡＬＢ／ｃ小鼠血清２０份，同时检测包囊阴性的小鼠血清２０份，结果除一份小鼠血清ＩｇＧ阴性反应外，均为阳性反应，阳性率为９５％；而正常对照血清全为阴性反应。另外，检测了３份纤支镜刷检物中包囊阳性的病人血清，均为阳性反应；５０例健康孕妇血清的检查，抗体阳性率为１２％。     

Elevation of plasma thioredoxin levels in HIV-infected individuals

Thioredoxin (Trx), a ubiquitous protein intimately involvedin redox and protein disulfide reductions, has been shown tobe released from cells and to have cytokine-like activities.In addition, Trx has been implicated in the redox regulationof immunological responses and shown to be deficient in tissuesfrom AIDS patients. In studies presented here, plasma Trx levelswere measured by ELISA in plasma samples from HIV-infected individuals(n = 136) and HIV-negative controls (n = 47). To account forthe release of Trx into plasma due to hemolysis, the Trx measurementswere corrected according to the level of hemoglobin in the plasmasample. Data presented show that, in contrast to tissue Trxlevels, corrected plasma Trx levels are significantly higherin HIV-infected individuals than in controls (P < 0.0001).Furthermore, {small tilde}25% of the HIV-infected individualsstudied have plasma Trx levels greater than the highest levelfound in controls (37 ng/ml). Detailed multiparameter FACS analysisof peripheral blood mononuclear cells (PBMC) from the infectedindividuals demonstrates that those with higher plasma Trx levels(37 ng/ml or greater) tend to have lower overall CD4 counts.In addition, increases in plasma Trx levels correlate with decreasesin monochlorobimane staining (indicative of lower intracellularglutathione levels in PBMC) and with changes in surface antigenexpression (CD62L, CD38 and CD20) that occur in the later stagesof HIV infection. These correlations suggest that elevationof plasma Trx levels may be an important component of advancedHIV disease, perhaps related to the oxidative stress that oftenoccurs at this stage.     

Flow cytometry analysis of platelet antibodies

We have devised assays to detect both circulating alloantibodies to platelets (indirect assay) and platelet-association IgG and IgM (direct assay) using a flow cytometric technique. A variety of patients with immune thrombocytopenia were studied. Employment of a confocal lens in the flow cytometer increased the discrimination power of the instrument. Patients with autoimmune thrombocytopenia (idiopathic thrombocytic purpura [ITP], systemic lupus erythematosus (SLE), lymphoma, leukemia, and drug-induced thrombocytopenia showed a significant increase in platelet-associated antibody. Circulating antibodies to platelets (alloantibodies) were demonstrated in cases of platelet refractoriness and neonatal isoimmune purpura. Day-today precision of the assays ranged from 3% to 6% (coefficient of variation). No interference was shown in the presence of hemoglobin (5 g/L), triglycerides (10 g/L), or polyclonal and monoclonal immunoglobulinemia (50 g/L: IgG, IgA, IgM). The sensitivity of the direct assay was 500 attograms of IgG or IgM platelet.     

巨细胞病毒感染捕获ELISA分析方法的建立和应用

目的：建立CMVIgM抗体捕获ELISA法并用聚合酶链反应（PCR）技术对此方法进行了评价。评价：采用巨细胞病毒（CMV）AD－169株感染人胚肺细胞的方法制备CMV抗原，并采用冻化抗制备法及甘愿酸法制备抗原，羊抗人IgM（μ链）McAb包被酶标板，建立CMVIgM抗体捕获ELISSA法。结果：不同浓度抗μ链McAb包被，包被时间、包被液、封闭液等不同条件，以有抗原浓度、抗CMV抗体酶结合物浓度对此法的建立有不同的影响。PCR法与用最佳条件配合后的本方法CMVIgM抗体检出率无显著差异（P＞0．05）。结论：本法测定CMV－IgM抗体的敏感性和特异性较高，简便、适用，易于推广，可广泛应用于临床。     

Antibody reactivity to a synthetic peptide (P62) of the Epstein-Barr nuclear antigen in sera of patients with X-linked lymphoproliferative syndrome

An enzyme-linked immunosorbent assay (ELISA) was used to measured IgG antiboody titers againt a synthetic peptide whose sequence was derived from the glycine-alanine repeating region of Epstein-Barr virus nuclear associated antigen 1 (EBNA-1). Antibody titers were determined in sera from 15 normal subjects, sera from 21 normal male siblings of X-linked lymphoproliferative syndrome (XLP) patients, from 20 XLP patients comprising a total of 42 samples, and ten samples before and ten samples after gamma-globulin therapy in ten patients with XLP. Data analysis demonstrated that while there are differences between the ELISA and ACIF, they appear to measure a similar response as demonstrated by their correlation coefficient (0.77) and the GMT to EBNA observed by both methods. No cross-reactivity of cytomegalovirus antibodies to the EBNA-1 peptide was observed by immunobv using adsorption against AD-169 infected MRC-5 cells.. However, non-specific binding was observed if samples were not pre-incubated in a 10% goat serum PBS-Tween 20 solution. This pre-treatment removed the non-specific binding that falsely elevated GMT in approximately 15% of both normal and XLP samples in ELISA. The ELISA system appears to be a sensitive, reproducible and objective test that may be useful for assessing the antibody responses of patients to the EBNA-1 protein.     

A cyclic peptide–polymer probe for the detection of Clostridium botulinum neurotoxin serotype A

A Botulinum neurotoxin serotype A (BoNT/A) ELISA detection system was developed based upon an 11-mer cyclic peptide, termed C11-019, that was identified through peptide phage display technology. The assay employs a sandwich format using the C11-019 cyclic peptide attached to a PEMA (poly(ethylene maleic anhydride)) matrix as the capture phase and anti-BoNT/A polyclonal antibodies as the detection phase. Results reported demonstrate that the C11-019 peptide–polymer can specifically bind to BoNT/A with no cross-reactivity to other serotypes examined in assay buffers and a variety of body fluids and foodstuffs. When a highly sensitive chemiluminescent substrate was engaged, the detection of 1 pg/mL could be readily achieved within 3 h with a linear range of 0.1–1 ng/mL. These results demonstrate that an inexpensive peptide–polymer-based capture ELISA system can be used for rapid, sensitive and highly specific BoNT detection.     

Hepatic UDP-glucose 13C isotopomers from [U-13C]glucose: a simple analysis by 13C NMR of urinary menthol glucuronide.

Menthol glucuronide was isolated from the urine of a healthy 70-kg female subject following ingestion of 400 mg of peppermint oil and 6 g of 99% [U-(13)C]glucose. Glucuronide (13)C-excess enrichment levels were 4-6% and thus provided high signal-to-noise ratios (SNRs) for confident assignment of (13)C-(13)C spin-coupled multiplet components within each (13)C resonance by (13)C NMR. The [U-(13)C]glucuronide isotopomer derived via direct pathway conversion of [U-(13)C]glucose to [U-(13)C]UDP-glucose was resolved from [1,2,3-(13)C(3)]- and [1,2-(13)C(2)]glucuronide isotopomers derived via Cori cycle or indirect pathway metabolism of [U-(13)C]glucose. In a second study, a group of four overnight-fasted patients (63 +/- 10 kg) with severe heart failure were given peppermint oil and infused with [U-(13)C]glucose for 4 hr (14 mg/kg prime, 0.12 mg/kg/min constant infusion) resulting in a steady-state plasma [U-(13)C]glucose enrichment of 4.6% +/- 0.6%. Menthol glucuronide was harvested and glucuronide (13)C-isotopomers were analyzed by (13)C NMR. [U-(13)C]glucuronide enrichment was 0.6% +/- 0.1%, and the sum of [1,2,3-(13)C(3)] and [1,2-(13)C(2)]glucuronide enrichments was 0.9% +/- 0.2%. From these data, flux of plasma glucose to hepatic UDPG was estimated to be 15% +/- 4% that of endogenous glucose production (EGP), and the Cori cycle accounted for at least 32% +/- 10% of GP.     

Fumonisins as a possible contributory risk factor for primary liver cancer: A 3-year study of corn harvested in Haimen, China, by HPLC and ELISA

Employing HPLC fluorometry, gas-liquid chromatography (GLC) and a novel enzymelinked immunosorbent assay (ELISA) based on a monoclonal antibody, 40 corn samples, each collected in 1993 from agricultural stocks for human consumption in Haimen (Jiangsu County) and Penlai (Shandong Province), high- and low-risk areas for primary liver cancer (PLC) in China, respectively, were analysed for fumonisins (FBs), aflatoxins (AFs) and trichothecenes. Levels and positive rates of FBs and deoxynivalenol (DON) were significantly higher in Haimen than in Penlai. ELISA of the 40 corn samples harvested in the two areas in 1994 revealed that FB contamination levels and rates in these areas were comparable to those observed in 1993 in Haimen. ELISA analysis of 1993 and 1994 products revealed a wide occurrence of AFB₁ but the positive rates as well as levels were not significantly different between these areas. ELISA of the same sample number of corn harvested in 1995 revealed that FB contamination in Haimen was significantly higher than in Penlai. These 3-yearly surveys of corn samples (240 in total) demonstrated that corn harvested in Haimen was highly contaminated with FBs and that the contamination level, as well as positive rate in 1993 and 1995, were 10–50-fold higher than those in Penlai, suggesting FBs as a risk factor for promotion of PLC in endemic areas, along with the trichothecene DON. Co-contamination with AFs, potent hepatocarcinogens, was assumed to play an important role in the initiation of hepatocarcinogenesis.