定量酶联免疫吸附试验用于测定蜜蜂毒液—特异免疫球蛋白G

本文报道了一种定量酶联免疫吸附试验(ELISA)用于测定蜜蜂毒液—特异IgG的方法。此法克服了以往报告的ELISA因稀释曲线不平行而产生的重复性差的缺点。该方法使用蜜蜂毒液(HBV)作抗原,未经透析,用前以PBS稀释。过氧化物酶标记的羊抗人IgG(Per-a-hu-IgG)和经HBV免疫治疗的患者血清(第一抗体)均用10%灭活的正常羊血清(NGS)PBS溶液稀释。测定是用聚乙烯“U”型微量滴板进行。首先加HBV,置37℃1小时冲洗后加人血清白蛋白,37℃1小时,冲洗后再加适当稀释的人血清(第一抗体)室温作用16—24小时后冲洗,最后和Per a hu IgG(第二抗体)     

~3H-TdR掺入法检测SAC诱导全血B细胞转化的实验条件分析

<正> B淋巴细胞转化试验可反映B细胞识别抗原并转化为母细胞的能力。本文报道用~3H-TdR掺入法对含A蛋白的葡萄球菌菌体(SAC)诱导人全血B细胞转化试验的最适实验条件及有关影响因素进行分析。 材料和方法 取肝素抗凝静脉血0.1ml加含SAC(卫生部上海生物制品研究所产品)1mg/ml的1640培养液3ml,作为实验管。另取0.1ml全血加入3ml不含SAC的培养液中,作为对照管,每组二份。置37℃培养24小时。收     

用酶联免疫吸附试验诊断脑囊虫病

本文报道用酶联免疫吸附试验(ELISA)和补体结合试验(CF)对比检测患者血清中抗猪囊虫抗体。 一、试验方法 (一)酶联免疫吸附试验:从猪肉螩虫囊幼中取出囊虫液经处理后为抗原,将1:250稀释的抗原,包被聚苯乙烯塑料板,置37℃3小时,继放4℃过夜。洗涤后,加入待检稀释倍数的不同血清,同时作阴性、阳性及空白对照。加辣根过氧化物酶标记羊抗人IgG结合物,置37℃1小时。洗涤后,加底物溶液(联大茴香胺溶液),置37℃15分钟后,继     

淋巴因子诱导的细胞毒细胞活性检测的研究

本文通过正交设计试验,建立了一种改良的诱生及检测LICC细胞毒活性的培养体系。先将脾脏淋巴细胞(5×10~6/ml)在含有IL-2(0.5U/ml)、CCDF(50％V/V)RPMI1640培养液中预培养72小时,然后加入肿瘤细胞(5×10~4/ml),用~3H-TdR后标法检测LICC细胞毒活性,杀伤时间为72小时。     

全身化脓性感染时T、B淋巴细胞系统的活性

本文研究了全身化脓性感染时,血液中T、B淋巴细胞群的数量和功能活性。检查对象为37名脓毒症患者,并以60名健康人的血液做对照。化脓性感染的诊断经细菌学检查所证实。用Boym方法分离淋巴细胞群。用Mancini单相放射免疫扩散法检查病人和健康人血清中三种主要免疫球蛋白(A、M、G)的数量,用连续稀释法测定抗大肠杆菌和抗葡萄球菌的正常抗体滴度,以便评价B淋巴细胞的功能活性。检查结果证实,全身性化脓性感染时末     

人扁桃腺和外周血IL—2的产生及消炎痛、PGE_2对IL—2产生的影响

本文在建立检测 IL—2活性方法的基础上,对人扁桃腺和外周血 IL—2的产生,及消炎痛和前列腺素 E_2(PGE_2)对 IL—2产生的影响作了初步研究,现将结果报导如下。取手术切除的人扁桃腺淋巴细胞(HTLC)及外周血单个核细胞(PBMC),用含5～10%小牛血清或5%自身血浆的1640液配成2×10~6/ml 细胞悬液,同时加 PHA200μg/ml,37℃5%CO_2培养48小时,     

用刀豆素A短时间作用人外周血淋巴细胞形成的稳定E花环

人体外周血T淋巴细胞和羊红细胞形成的E玫瑰花环,在37℃长时间孵育下即容易分离,但由“活化的”T细胞所形成的“稳定”E花环却能抵抗37℃长时间的孵育而不致解离。作者最近的研究证实了稳定E花环不但能由外周血淋巴细胞(PBL)长时期接触刀豆素A(ConA)而形成,并且也能由仅仅1小时的短时接触ConA而形成。不过这一现象需要有量的依赖,用最大浓度的     

环胞素A对IL-4等合成的影响

<正>本文观察环胞素A(C_3A)对哮喘患者PBMC合成IL-4、IL-5、IgE的抑制作用.1 材料和方法1.1 对象 临床确诊为哮喘的病人20例,均为缓解期,1周内均未使用糖皮质激素.β-受体兴奋剂和茶碱类药物.另以10例健康人为对照组.1.2 实验方法1.2.1 单个核细胞(PBMC)的分离和培养:按常规用淋巴细胞分离液分离单个核细胞,并调整细胞浓度为2×10~6/ml,将健康对照和哮喘患者的PBMC加在含有100mg/L PHA的RPMI-1640培养剂中.此外,在PHA(100mg/L)刺激的同时,将哮喘病人的PBMC分别加在含有1×10~(-6)mol/L、2×10~(-6)mol/L、3×10~(-6)mol/L C_sA的RPMI-1640培养剂中.37℃、5%CO_2温育48h,离心,收集上清,-80℃     

干扰素和人淋巴毒素于体外的协同作用:增强淋巴毒素诱导靶细胞的损伤

淋巴毒素(LT)在体外能溶解各种靶细胞,可作为细胞介导免疫介质参与组织破坏反应。干扰素(IFN)有调节淋巴细胞在体外破坏靶细胞的作用。IFN不仅能提高NK细胞的溶解率,而且也能增加NK细胞的循环。最近有人报告,IFN是一种合成蛋白质,它能保护靶细胞。由于IFN对LT诱导靶细胞的损伤有重要的调变作用,所以探讨IFN增强LT诱导靶细胞损伤的作用是有意义的。本文试验用的靶细胞为Hela细胞和WI-38细胞,IFN为人的淋巴细胞所产生的IFN-α及用DNA重组技术由大肠杆菌产生的人IFN-α、LT用从人扁桃体分离的淋巴细胞加PHA-P培养的上清液。LT试验是用形态学和~3H胸腺嘧啶测定细胞活力的方法。作者获得的实验结果如下: (1)Hela细胞加各种剂量的IFN-α预先温育24小时后洗涤,再加入不同稀释度     

分离玫瑰花结与非玫瑰花结淋巴细胞的改良技术

本文介绍了分离玫瑰花结与非玫瑰花结淋巴细胞的改良技术。普遍认为,因人T淋巴细胞有羊红细胞受体,所以能与新鲜的SRBC形成E花结。形成花结的基本过程包括用聚蔗糖—泛影葡胺混合液分离全血得到单个核细胞;使之与神经氨酸酶处理过的SRBC在37℃一同孵育5分钟。随后离心,再于4℃孵育1小时。为了分离E花结与非E花结细胞,将细胞小团重混悬。重混悬过     

Monoclonal antibody testing of lymphocytes after overnight storage

Often monoclonal antibody testing of lymphocytes is not performed until the day after blood collection for reasons of convenience or due to the need to transport the blood to other facilities. In order to determine whether accurate results can be obtained on the day after collection, we compared results obtained after storage overnight at 4° C or 22°C with results obtained with fresh lymphocytes. Lymphocytes from 24 normal individuals were evaluated with 10 monoclonal antibodies using an immunofluorescence technique with analysis by flow cytofluorometry. There were markedly altered results obtained with lymphocytes separated on the day after collection from the whole blood stored at 4° C. Lymphocytes separated from whole blood stored at 22° C showed moderate changes in reactivity with some monoclonal antibodies. Lymphocytes that were separated from flesh blood and then stored at 4°C or 22°C showed results similar to fresh lymphocytes. These results underscore importance of proper processing of blood samples to avoid misinterpretation of results.     

Stability of cryopreserved and stored peripheral blood lymphocytes

The determination of the T and B subpopulation percentages and lymphocyte blastogenic response functions are useful tools to evaluate and monitor disorders associated with immunodeficiencies, lymphoproliferations, malignancies, infections, inflammations, and autoimmune diseases. Comparative studies of cryopreserved lymphocytes frozen at - 190 degrees C over a period of one year obtained by lymphocytapheresis of normal donors and fresh lymphocytes found no significant changes of lymphocyte subpopulations or blastogenic response functions. These cryopreserved lymphocytes can therefore be used as a reproducible quality control reagent. Comparisons of the stability of T and B populations of fresh, one, two, three, four, and five day old culture medium (RPMI 1640) stored separated peripheral blood lymphocytes and culture medium (RPMI 1640) stored one, two, three, four, and five day old whole peripheral blood revealed that culture medium prolonged the viability to approximately three days. It seems feasible for smaller clinics and hospitals that lack the facilities to run tests on lymphocytes to mix their heparinized blood with culture medium and then transport it to laboratories where the tests can be performed.     

Immunological responsiveness of frozen-thawed human lymphocytes.

Mononuclear cells (10--20 X 10(6)) obtained from human peripheral blood by a standard Ficoll-Hypaque technique were suspended in RPMI 1640 media at 4 degrees C containing 10% foetal calf serum and 7-5% dimethyl sulphoxide (DMSO). Two-millilitre aliquots were cooled at -1 degree C/min in a Cryoson BV-4 programmed freezing system to -30 degrees C, then -5 degrees C/min to -80 degrees C and stored in liquid nitrogen vapor. On the day of testing, cell suspensions were thawed rapidly in a 37 degree C water bath. DMSO was diluted slowly out of the sample and cells resuspended in fresh RPMI 1640. It was found that frozen stored human lymphocytes (FSHL) demonstrated all the characteristics of fresh unfrozen cells. These included their ability to form spontaneous rosettes with sheep erythrocytes ('E' rosettes) and sheep erythrocyte--antibody--complement rosettes ('EAC' rosettes). The presence of surface immunoglobulins and Fc receptors were shown by membrane immunofluorescence to be comparable. In addition, the results show that FSHL respond to mitogens, specific antigens; act as both stimulators and responders in the mixed lymphocyte culture reaction; and exhibit cell-mediated lymphocytotoxicity following in vitro sensitization, or against antibody-coated target cells.     

Effects of anticoagulant, serum, and temperature on the natural killer activity of human peripheral blood mononuclear cells stored overnight.

The effects of immediate versus delayed cell separation, storage temperature, presence of serum, and type of anticoagulation on the natural killer (NK) cytotoxicity of human mononuclear cells were assessed. The NK cytotoxicity of Ficoll-Hypaque-separated peripheral blood mononuclear cells (PBMC) was tested in a 3-h chromium-51 release assay with K562 cells at various effector/target cell ratios. The NK activities of PBMC from blood anticoagulated with either heparin or EDTA and then immediately separated and assayed were not different (42.9 +/- 2.5% for heparin and 40.3 +/- 4.6% for EDTA). When these separated cells were cultured in medium with 10% fetal calf serum and stored at 4,25, or 37 degrees C for 18 h before the assay, there was a significant increase in cytotoxicity. PBMC from blood stored in heparin or EDTA for 18 h before separation had reduced NK cytotoxicity, particularly if they were kept at 37 degrees C. When separated PBMC were cultured in medium with 10% human AB serum, however, samples held at 25 and 37 degrees C decreased in cytotoxicity but samples held at 4 degrees C maintained the cytotoxicity demonstrated at the baseline level with fresh cells. We recommend that heparinized blood be used for NK assays and that the PBMC be isolated immediately and held overnight at 4 degrees C in medium with 10% AB serum if the assay must be delayed. The NK cytotoxicity under these storage conditions most closely matches the results obtained when the PBMC are isolated and tested on the same day. IF PBMC isolation must also be postponed, it is best to store the blood in heparinized tubes at 25 degrees C to prevent loss of cytotoxic function.     

血小板聚集实验诱导剂储存条件及配制

目的 探讨血小板聚集诱导剂花生四烯酸(从)和二磷酸腺苷(ADP)的储存条件.方法 随机收集101例门诊体检健康志愿者静脉血标本,枸橼酸钠抗凝,离心分离富含血小板血浆(PRP)和贫血小板血浆(PPP),采用血浆比浊法,分别用不同储存条件从和ADP作为诱导剂,上机测血小板聚集率.结果 AA-50℃储存0月和7月诱导血小板聚...     

Effects of storage and radiation on human neutrophil function in vitro

To better understand the process of time-related functional deterioration which occurs in human polymorphonuclear leukocytes (PMNs), we examined the effects of in vitro storage on multiple functional parameters of human PMNs. Single-donor, phlebotomy-collected PMNs were stored at both room temperature and 37°C for 24 and 48 h, then compared to fresh cells from the same donor. Similar numbers of cells were recovered from each storage condition. Cell viability decreased after 37°C storage for 48 h. Cells stored at room temperature for 24 h showed significant depression of multiple functions (bactericidal activity, chemotaxis, aggregation, superoxide production, and oxygen consumption) compared to fresh cells. They contained less vitamin B₁₂ binding protein activity than fresh cells, and by fluorescenceactivated cell-sorter analysis, their forward light scatter and membrane depolarization responses were abnormal. For all parameters examined, cells stored at 37°C were more abnormal than cells stored at room temperature. Stored cells from a patient with myeloperoxidase deficiency lost bactericidal and chemotactic activity after storage at 37°C for 24 h, but cells from a patient with chronic granulomatous disease retained their original bactericidal and chemotactic activity after 37°C storage for 24 h. Radiation, in doses used to prevent graft vs. host disease in leukocyte-transfusion recipients (2500–5000 rads) caused a significant decrease in the mean percentage of continuous flow centrifugation leukapheresis (CFCL) collected PMNs capable of reducing nitroblue tetrazolium. Human PMNs show deterioration of multiple in vitro functions when they are stored and are susceptible to damage by radiation when they are collected by CFCL.     

Artifactual changes in the haematocrit buffy coat of stored anti-coagulated blood samples of farm animals

The buffy coat percentage (BCP) of centrifuged anti-coagulated blood is routinely clinically used to estimate the total leukocyte count (TLC). There had been observations in our clinical laboratory of BCPs that do not correlate with TLC, especially in blood samples in which centrifugation were delayed. This study therefore investigated the artifactual changes that occur in the BCP of stored anti-coagulated blood samples of farm animals. The BCPs of blood samples from a total of 16 cattle, 18 goats, 15 pigs, and 16 chickens were determined immediately upon blood collection to obtain the baseline value (BV). The blood samples were then divided into three parts and stored at 5°C (3–7°C), 30°C (27.5–32.5°C), and 37°C (35.5–38.5°C). Further BCP determinations on the samples were carried out at 24-h intervals for 72 h (3 days). Results showed that there were statistically significant increases (p < 0.05) in BCP of cattle blood samples at all temperatures of storage as from the 48th hour of storage onwards, in goat blood samples stored at 5°C and 37°C at the 72nd hour of storage and as from the 48th hour of storage in the goat blood samples stored at 30°C, in pig blood samples as from the 24th hour of storage onwards for all the storage temperatures, and in chicken blood samples stored at 30°C and 37°C as from the 48th hour of storage onwards. However, clinically significant increases (BCP > 1.5%) occurred only in cattle and chicken blood samples stored at 30°C and 37°C at hour 72 of storage and in pig blood samples stored at 30°C and 37°C as from the 48th hour of storage onwards. It was concluded that statistically significant increases in BCP occur in the blood of cattle, goats, pigs, and chicken during storage, but clinically significant increases that could lead to a false notion of leukocytosis do occur in the BCP of stored anti-coagulated blood of cattle, pigs, and chicken when the blood is stored/kept at 30°C and 37°C.     

Artifactual changes in hematological variables in equine blood samples stored at different temperatures and various anticoagulants

The objective of this study was to determine the effect of storage time, temperature, and anticoagulant on hematologic parameters in equine blood samples. Blood samples were obtained from 50 clinically healthy warm-blooded horses at two major equestrian complexes in Tehran, Iran. The samples were collected in three different tubes containing EDTA, sodium citrate, or heparin and were analyzed within 4?h of collection. Blood samples collected into EDTA-containing tubes were stored at 4°C or 24°C. Each sample was analyzed again at 24, 48, and 72?h after collection. The statistically significant (P?<?0.05) alterations included decreased RBC count and increased hemoglobin concentration [Hgb] in blood samples stored at 24°C after 48 and 72?h; increased hematocrit in blood samples stored at 24°C after 24?h; decreased hematocrit in blood samples stored at 4°C after 72?h; decreased MCHC in blood samples stored at 4°C after 72?h; and decreased total WBC count in blood samples stored at 24°C after 48?h. Although there was no significant difference in hematologic analytes between heparinized and EDTA-anticoagulated blood samples, most of the hematologic analytes were decreased significantly (P?<?0.05) in sodium citrate-containing blood samples, compared with blood samples stored in EDTA. The results of this study suggest that within certain limitations for some hematologic analytes, equine blood samples stored in EDTA at 4°C for up to 72?h may be suitable for hematologic testing.     

Comparison of T and B cell analyses on fresh and aged blood

We have compared T and B cell analyses using whole blood, separated lymphocytes, and separated, frozen, then thawed lymphocytes to see how long blood can be kept before separation and analysis. We also examined the effect of various anticoagulants and the effect of diluting blood in culture media on T and B cell analysis over time. We found that the whole blood method is a very reliable method for T and B cell analysis, even 4 days after the blood is drawn, provided that heparin or ACD is the anticoagulant used. Separated lymphocytes and cryopreserved lymphocytes from blood that was separated within 24 h of collection was satisfactory; however, results were less consistent if separation was delayed more than 24 h. For lymphocyte separation, blood collected in heparin or ACD held up better over time than did blood collected in EDTA, and dilution with either RPMI 1640 or McCoy's medium gave better lymphocyte separation in older blood.     

Effects of Duration of Storage and Storage Temperature on Cell Counts of Bovine Blood Samples as Determined by an Automated Haematology Analyser

Analysis of blood cells is an important part of many scientific investigations in the field of cattle herd health. Over the last 30 years, automated blood analysis has all but replaced manual counting of blood cells using counting chambers. The present study investigated the effects of prolonged storage and storage temperatures on cell counts as determined by a haematology analyser. Blood samples from 20 clinically healthy cows were repeatedly analysed with a Cell-Dyn 3500 (Abbott Diagnostika, Delkenheim), within 24 hours after collection and after storage at either 4° C or 20° C. The counts of most blood cells were more stable in samples stored at 20° C than those stored at 4° C. For at least 8 h, the counts of all analysed cell types, with the exception of lymphocytes, remained within ±3 standard deviations that were calculated for fresh samples, provided that the blood was stored at 20° C. Correspondence and offprint requests to: Dr Ulrich Bleul, Klinik für Fortpflanzungskunde, Universit?t Zürich, Winterthurerstrasse 260, 8057 Zurich, Switzerland.