Development of a microassay technique for neutrophil adherence

The development of a microassay for neutrophil adherence is described. The nylon fibre column consisted of 10 mg of fibre inserted into a micropipette tip. A vacuum harvester was constructed to aspirate the neutrophil-containing fluid in the columns into disposable test-tubes for quantitation of neutrophil concentrations. In this way, the neutrophil requirement was reduced from 10(7) cells to 10(5) cells. A complete assay can be performed efficiently and rapidly using less than 1 ml of blood.     

Use of monoclonal anti-rat IgE in a radioimmunoassay for antigen specific rat IgE

We describe a new method of preparing C3-coated erythrocytes by coupling C3 to thiol-activated erythrocytes. The procedure involves three steps. Firstly, sheep erythrocytes were treated with N-succinimidyl 3-(2-pyridyldithio) propionate (SPDP) to introduce 3-(2-pyridyldithio) propionyl residues into membrane proteins. Secondly, C3 was cleaved with trypsin or CoVF, Bb enzyme to obtain C3b exposing the SH group (C3b-SH). Finally, the C3b-SH was coupled to the thiol-activated erythrocytes (TA-E) through thiol/disulfide exchange to form the TA-EC3b conjugate. E coated with C3d was prepared by treating TA-EC3b with KSCN inactivated serum and plasmin. Studying the rosette formation between TA-EC3b or TA-EC3d and cells expressing C3b (CR1) and C3d (CR2) receptors and the inhibition thereof with anti-CR1 and anti-CR2 antibodies as well as with C3-sheep E membrane protein complexes, we found that TA-EC3b and TA-EC3d bound exclusively to CR1 and CR2, respectively. In addition, TA-EC3b like EAC1423b bound factors B and H as tested by hemolytic and direct binding assays. The advantage of TA-EC3 for complement receptor and hemolytic assays are the simplicity of the preparation method and the general applicability of the TA-EC3.     

血清学诊断肝纤维化新指标--金属蛋白酶组织抑制因子检测方法学建立及应用评价

目的：自行建立一种改良固相致敏红细胞吸附技术（SPASE）用于快速检测肝硬化病人血清中金属蛋白酶组织抑制因子－1，2（TIMP－1和TIMP－2），同时研究TIMP－1和TIMP－2在肝化病人肝组织中的定位及表达状态，以及肝组织TIMPs与血清TIMPs的相关性。探讨血清中TIMP－1和TIMP－2可否作为肝纤维化的血清学诊断新指标。方法：应用自行建立的SPASE检测1082例肝病病人（其中肝硬化310例）血清标本中的TIMP－1和TIMP－2。用原位杂交及免疫组织化学技术分别检测40例肝硬化病人活检肝组织中TIMP－1和TIMP－2 mRNA及相关抗原的表达，并与病人自身血清检测结果对比。另检测20例正常肝组织作为对照。结果：SPASE法特异性中和抑制试验示中和抑制率均在70％以上，检测310例肝硬化病人血清中TIMP－1和TIMP－2的阳性率分别为79．68％和65．16％。40例肝硬化病人肝组织中TIMP－1和TIMP－2 mRNA及相关抗原表达阳性率为100％，TIMP－1表达强度高于TIMP－2（P＜0．01）；阳性信号主要位于肝细胞胞质内，未见细胞核表达。40例肝硬化肝活检病人血清检测结果，TIMP－1阳性率为100％，TIMP－1阳性率为77．5％（31／40）。正常肝组织无一例有TIMPs相关抗原表达。结论：TIMPs定位于肝硬化病人的肝细胞胞质内，在肝硬化组织内呈程度不等的阳性表达；血清中TIMPs与肝组织中TIMPs表达有明显相关性，因而血清中TIMP－1和TIMP－2可作为肝纤维化较为有用的血清学诊断新指标，尤其是TIMP－1的诊断意义更大。SPASE法检测血清中TIMPs具有较好的特异性。