Antisperm Antibody Detection: 2. Clinical,Biological, and Statistical Correlation Between Methods

PROBLEM: In clinical andrology, the detection of antisperm antibodies (ASA) is regarded as one of the most important steps in the study of male infertility. This practice is generally accepted even though there is still some disagreement about the true meaning of antisperm immunity, and there remains a good deal of controversy about the test regarded as the most suitable for the detection of antibodies directed against sperm antigens. International Workshops have tried to standardize universally accepted protocols. A panel of three or four methods is generally advised to provide a correct and complete screening of patients with antisperm immunity. The aim of the present paper is to report our studies on the correlation between direct methods (IBT, MAR test) and indirect methods (gelatin agglutination test [GAT], and tray agglutination test [TAT]) and to establish whether biological models can explain the antibody tests results. An attempt was also made to establish a “predictive threshold” to explain even apparently discordant direct and indirect results.     

Study of Antisperm Antibodies Bound to the Sperm Cell Surface and Their Relationship to Circulating ASA

PROBLEM : Direct and indirect tests for antisperm antibody (ASA) detection are commonly used in laboratories for the diagnosis of male immunological infertility. Even though the two kinds of tests study the same immunological phenomena, frequently no perfect correlation is found even on large series of patients and with precise statistical models. The aim of the present investigation was to try to establish whether biological models can explain the antibody test results and if a predictive threshold can be established for expected positivity/negativity. METHOD : The data relate to 667 patients, who were screened using the Direct Immunobead test (d-IBT) for ASA bound to the sperm surface and with the gelatin and tray agglutination test (GAT and TAT) in sera for circulating ASA. Correlations were studied and cases of no correlation were further analyzed. RESULTS : The number of patients with a clinically significant positivity to d-IBT (binding percentage ≥ 20%) was 134 (20.1 % of total patients). The analysis of the results of direct and indirect tests shows that the discordances are due to differences in the Ig class of immunization or to the site of epitopes involved. In fact, as far the Ig class is concerned, if d-IBT shows a prevalent or exclusive IgG or IgA positivity, this results in a poor correlation between binding percentage to d-IBT and GAT-TAT titers. If both the Ig classes are involved, the level of positivity of the two kinds of test are strictly related. As far as the site of Ag-Ab reaction on the sperm surface is concerned, the wide immunization involving all the surface sperm antigens (mixed), shown by d-IBT, is related to higher indirect test titers. CONCLUSION : Local and systemic antisperm immunizations are strictly related and a predictive threshold of expectation can be established to explain even apparently discordant direct and indirect results.     

Multivariant Analysis of Men from Infertile Couples With and Without Antisperm Antibodies

PROBLEM: Research studies in animal and human systems have demonstrated conclusively that antisperm antibodies can interfere with fertilization. In the male, autoantibodies to sperm can be detected both in the sera and seminal plasma. METHOD OF STUDY: Ninety-seven men who were tested for antisperm antibodies as a part of an infertility evaluation were identified. Complete medical history was obtained, including information related to events suspected of being associated with antisperm antibodies. History of surgery (varicocele repair, hernia repair, and vas reversal) and infection (epididymitis, sexually transmitted disease, and orchitis) were compared with semen parameters (motility less than 60%, concentration less than 20 times 10⁶, and volume less than 2 cc). These were compared to antisperm antibody results of mixed agglutination reaction (MAR) and direct immunobead binding test (IBT) for immunoglobulin G (IgG). Statistical analysis was performed using Fishers exact two-tailed test. RESULTS: As expected, prior vas reversal was significantly associated with the presence of antisperm antibodies (P = 0.0002) by MAR or IBT with a fivefold increased relative risk (95% confidence interval, 1.97-12.38). Other surgeries manipulating the cord structures independent of vas reversal were not associated with antisperm antibodies (P = 0.09). Prior infections, independent of vas reversal, were significantly associated with antisperm antibodies by MAR (P = 0.04) with a 3.8-fold increased relative risk (95% confidence interval, 1.06–13.87) but not by IBT. Sperm concentration less than 20 times 10⁶, motility less than 60%, and a volume less than 2 cc were not associated with antisperm antibodies by MAR or IBT. CONCLUSION: These findings suggest that manipulation of the cord structures excluding the vas were not associated with antisperm antibodies; however, vas reversal and prior infection are significant risk factors for the development of antisperm antibodies.     

Detection of Antisperm Antibodies on the Surface of Motile Spermatozoa. Comparison of the Immunobead Binding Technique (IBT) and the Mixed Antiglobulin Reaction (MAR)

ABSTRACT: Investigators testing for antisperm antibodies have recently focused on tests that detect the Ig classes of the sperm-bound antibodies. The aim of this study was to compare the sensitivity of two of these tests, viz. the immunobead binding technique (IBT) and the mixed antiglobulin reaction (MAR). Twenty-one male or female sera were tested for IgG and IgA antisperm antibodies with the IBT and the MAR. The sera were selected on the basis of the IBT results, and the MAR was carried out without knowledge of these results. For IgG antisperm antibodies, there was a highly significant correlation between the two tests (P = 0.0043), whereas, for IgA antisperm antibodies, the correlation was poor (P = 0.2951), because the IBT revealed a positive reaction for IgA in sera in which no such antibodies could be detected by the MAR.     

The Effect of Sera Antisperm Antibodies in the Female Partner on In Vivo and In Vitro Pregnancy and Spontaneous Abortion Rates

PROBLEM: To determine the incidence of antisperm antibodies (ASA) in female sera from infertile couples or those suffering from recurrent abortions. Also to determine if the pregnancy and/or abortion rates are any higher in those positive versus those negative for ASA. METHOD: All registered patients had sera drawn and ASA measured by indirect immunobead test on initial study. Pregnancy and abortion rates were determined for patients undergoing in vivo or in vitro therapy. RESULTS: There was a low incidence of ASA in patients having in vivo or in vitro treatment. There was no decrease in pregnancy rates (PRs) or increase in spontaneous abortions (SAB) in those positive for ASA. CONCLUSION: Antisperm antibodies in female sera do not seem to be etiologic in causing infertility or SAB. Future studies might consider changing the antigen source from donor sperm to husband's sperm.     

人血清布鲁氏菌病两种检测方法比较

目的 对比分析虎红平板凝集实验（RBPT）和试管凝集实验（SAT）两种检测方法的实验结果,为提高布鲁菌病实验室检测水平提供依据。方法 同时采用RBPT和SAT两种方法对2016年1月~2017年12月到锦州市疾控中心布病免疫门诊就诊的高危人群2873份人血清进行布鲁菌病实验室检测,对检测结果进行对比分析。结果 RBPT的阳性率为53.11%（1526/2873）,SAT的阳性率为52.41%（1506/2873）。RBPT与SAT总体阳性进行比较:敏感度为98.47%（1483/1506）,特异性为96.85%（1324/1367）,符合率为97.70%（2807/2873）,两种检测方法符合率很高;RBPT与SAT 1:100++及以上阳性者比较,符合率为72.96%（2096/2873）,两种检测方法符合率不高。结论 RBPT不能代替SAT用于布鲁菌病的临床诊断,对SAT凝集低滴度者应跟踪调查。     

Detection of sperm antibodies on unwashed spermatozoa with the immunobead test: a comparison of results with the routine method and seminal plasma TAT titers and SCMC test

This study investigated whether the immunobead test (IBT) could, for the purposes of simplicity and saving time, be applied directly on an unwashed semen sample instead of washed spermatozoa. These two methods were performed simultaneously on the semen samples of 15 men with a positive MAR test and 10 men with a negative MAR test. A possible association was found between the unwashed samples, showing positive IB binding (greater than 20%) on the tail and/or head and the seminal plasma TAT titers (P less than .00001, Fisher's exact test). In all cases with IB binding of greater than or equal to 20% on unwashed spermatozoa, positive seminal plasma TAT titers (greater than or equal to 32) and SCMC tests (greater than or equal to 50%) were found. In all cases where the binding of the beads was mainly located on the tailtips of the washed or unwashed spermatozoa, negative seminal plasma TAT titers and SCMC tests were found. Coating of the head and/or upper tail regions in both methods was always associated with high TAT titers and a strong-positive SCMC test. It is concluded that the IBT for IgA, but not for IgG, can be performed directly on unwashed semen and that the position of IB binding on the spermatozoa is of prognostic importance with regard to the expected outcome of the SCMC test and seminal plasma TAT titers.     

酶联免疫吸附试验检测梅毒螺旋体抗体的分析性能评价

目的评价ELISA检测梅毒螺旋体（TP）抗体的分析性能,探讨未知诊断的定性实验的性能评价方法。方法参照临床实验室标准化协会（CLSI）发布的EP12-A2文件对ELISA法测定TP的临界值±20%范围的重复性进行分析,并与TP明胶凝集试验（TPPA）结果进行一致性比较。结果 ＋20%浓度临界值检测阳性率为≥95%,CV为14.8%;-20%浓度临界值检测阴性率≥95%,CV为19.6%;两种方法的一致程度百分比为99.6%,一致程度95%可信区间为98.8%~99.6%。结论 TP-ELISA法检测TP抗体的临界值±20%浓度范围之外标本,可得到可靠的检测结果。TP-ELISA法与TPPA法结果一致性好,可代替其作为TP诊断试验。     

Detection of antibodies to opioid and glutamate receptors by latex agglutination and enzyme immunoassay

We studied adsorption capacity of 5 latexes to synthetic peptide fragments of μ- and δ-opioid receptors and to GluR1 and NR2A subunits of glutamate receptor. Levels of autoantibodies to opioid receptors in the latex agglutination test and enzyme immunoassay were in good correlation. The level of autoantibodies to opioid receptors measured by these methods was increased in patients with opium narcomania, while the content of autoantibodies to the glutamate receptor subunits was increased in epileptics.__________This revised version was published online in July 2005 with the addition of the issue titleTranslated from Byulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 139, No. 1, pp. 94–97, January, 2005     

ELISA方法检测MP-IgM与冷凝集试验对小儿肺炎支原体感染诊断价值的比较

目的 通过ELISA法与冷凝集试验两种方法检测小儿肺炎支原体肺炎(MPP)患者的血清,分析两种检验方法在小儿肺炎支原体感染的诊断的价值.方法 选择本院儿科收治确诊的157例肺炎支原体感染患儿,分别采用血清ELISA和冷凝集两种检测方法,分析探讨两组阳性率.结果 ELISA法检测MP-IgM阳性有142例,阳性检出率为90.4％;冷凝集试验方法检测阳性为126例,阳性检出率为80.3％,各年龄段阳性检出率差异具有统计学意义,两种检测方法学差异具有统计学意义(P＜0.05).结论 ELISA法与冷凝集试验相比,敏感性更高,具有良好的实验室诊断价值.     

Assessment of Circulating Agglutinating Anti-Sperm Antibodies in Buffalo Cows With Unexplained Infertility and an Attempt to Identify Buffalo Sperm Isoantigens

The presence of circulating agglutinating anti-sperm antibodies as cause for unexplained infertility of artificially inseminated buffalo cows was assessed. An attempt to identify buffalo sperm isoantigens was also made. The following methods were applied for that purpose: the tray agglutination test (TAT), SDS-polyacrylamide gel electrophoresis, and immunoblotting. The results obtained showed that three of 90 sera from buffalo cows with unexplained infertility were positive in TAT (3.3%) and their titers were low. A total of 27 spermatozoal polypeptides reacted positively with the IgG-isoantibodies of one of the sperm agglutinating sera in immunoblotting. Of the control sera 12 also revealed individual variations in the number of positive fractions on the blots. On the basis of comparing the blot with the positive sperm agglutinating serum to the blots of the positive controls, two buffalo sperm isoantigens were identified, with the respective molecular weights of 40 kDa and 120kDa. In conclusion, circulating agglutinating anti-sperm antibodies are very rarely detected in buffalo cows with unexplained infertility after several artificial inseminations.     

抗HBsAg和抗红细胞双特异Diabody的构建及表达

目的构建及表达抗乙肝病毒表面抗原（ＨＢｓＡｇ）和抗红细胞（ＲＢＣ）双特异Ｄｉａｂｏｄｙ，用于血清中ＨＢｓＡｇ的快速检测。方法将抗ＨＢｓＡｇ的人抗体轻重链可变区基因与抗红细胞的鼠抗体重轻链可变区基因交叉配对构建成两个杂合Ｄｉａｂｏｄｙ基因，并将两个配对基因组建在一个表达载体中，成为双特异Ｄｉａｂｏｄｙ表达载体，并在大肠杆菌中分泌表达。结果经ＥＬＩＳＡ检测和红细胞凝集实验测定，证明所表达的双特异性Ｄｉａｂｏｄｙ具有抗ＨＢｓＡｇ和抗ＲＢＣ双重功能，并可使含有ＨＢｓＡｇ的ＲＢＣ悬液产生血球凝集。结论所表达的双特异性Ｄｉａｂｏｄｙ具有双重抗体活性，可用于血清中ＨＢｓＡｇ的快速检测     

Antibodies to Spermatozoa: XI. The Use of Immunobeads for the Detection of Sperm Antibodies in Serum

ABSTRACT: Two procedures were developed and evaluated that used either larger or smaller volumes for the detection of sperm antibodies in serum by means of an indirect immunobead test (IBT). The immunobeads, coated with rabbit antibody to each of the major human immunoglobulins (IgG, IgA, or IgM), were mixed with preparations of donor sperm, onto which antibody had been coated by passive transfer from various serum samples. The results of the IBT could be evaluated in various ways: (1) positive or negative; (2) if positive, whether binding is to the tail, the head, or the head and tail of the sperm cells; (3) if positive, whether binding is by IgG, IgA, or IgM. The diverse IBT results were obtained from a group of 50 serum samples; these sera were also tested by two sperm agglutination methods; the gelatin agglutination test (GAT) and the tube-slide agglutination test (TSAT). There was an excellent agreement between the IBT and the GAT; it was not as good between the IBT and the TSAT. However, considering both agglutination methods together, 90% of the IBT-positive sera were agglutination-positive. In terms of morphological sites, tail binding occurred in 27 of 31 sera, head binding in 12 of 31 sera, and head-tail binding in 15 of 31 sera. The number with tail binding was very close to the number that were GAT-positive (26). As for the immunoglobulins, the most frequent class was IgG. IgA was 83% as frequent and IgM was only 25% as frequent as IgG. In a larger group with only IgG and IgA, of 31 IBT-positive sera, 26 showed IgG and 23 showed IgA; 18 showed both. Hence, only eight showed IgG exclusively, and only five showed IgA exclusively. One final point is that several sera with GAT titers of only 4 were IBT-positive, adding strength to the concept that such a low GAT titer does have antibody significance.     

三种初筛方法检测HIV抗体的结果比较及评估

目的比较酶联免疫吸附试验（ELISA）、明胶颗粒凝集试验（PA）、胶体硒免疫层析法（ICA）检测人类免疫缺陷病毒抗体的结果，了解这三种方法的优劣，探讨临床实验室初筛诊断HIV方法的灵敏性和准确性。方法用卫生部HIV质控血清和免疫印迹法（WB）确认的HIV抗体阳性标本和阴性标本。分别用ELISA法、PA法及ICA法检测；用免疫印迹法确认的HIV阳性的标本作倍比稀释后，再分别用PA法、ELISA法及ICA法检测。结果80例经免疫印迹法确认的HIV阳性患者，ELISA法、PA法、ICA法的检出符合率分别为100％、98．8％、97．5％；经稀释法检测。ELISA法较PA法和ICA法敏感。其检测的准确性分别为97．5％、97．5％、92．5％。结论筛查HIV抗体方法最好选用ELISA法或PA法。以防漏检。     

确证TRFIA检测乙肝病毒表面抗体结果的研究

建立简易时间分辨免疫荧光法(TRFIA)检测乙型肝炎病毒表面抗体(抗-HBs)的中和试验方法,验证TRFIA检测结果.收集乙型肝炎病毒表面抗原(HBsAg)异常值血清,确定TRFIA中和抑制试验中和比为0.25-1.26ng/mL∶1mIU/mL,对抗-HBs的定量结果进行确认.结果显示,使用TRFIA定量检测的结果与...     

抗阴道毛滴虫单克隆抗体的制备及其特性

用杂交瘤技术建立了6株能稳定分泌抗阴道毛滴虫单克隆抗体的细胞株,选取其中3株进行了分析。3株单抗的效价分别是:ⅣA12B1和ⅣA22B1为250000ⅡB2A1为1250000。ⅣA12B1和ⅡB2A为IgG3抗体,ⅣA2281为IgGl抗体。位点分析及免疫酶染色发现,ⅣA12B1和ⅣA22B1抗同一抗原决定簇,所针对的抗原主要分布于膜上;ⅡB2A1针对的抗原主要分布于胞浆中。3株单抗针对的抗原均为糖蛋白。特异性试验发现,这3株单抗与人白细胞、阴道上皮细胞、白色念珠菌、大肠杆菌及人毛滴虫均无交叉反应。用制备的单克隆抗体,建立了试断滴虫性阴道炎的胶乳凝集试验(LAT)。研究表明,LAT是一种敏感、特异、简单和快速的方法。     

糖化白蛋白测定及其在临床的应用进展

本文主要介绍了人血清白蛋白糖化位点、糖化速率,及糖化白蛋白的测定方法.2015年中国血糖监测临床应用指南中明确指出,糖化白蛋白能够反映2～3周血糖水平,在糖尿病监测中可以作为糖化血红蛋白的有益补充,现对糖化白蛋白的检测方法及临床应用进行阐述.     

Identification of Raoultella spp.: Comparison of Three Methods

Background: Raoultella is a Gram-negative bacteria, which commonly occur in the natural environment such as water, soil and on plants. In recent years, Raoultella spp. gained more interest. There is also an increasing number of publications describing mainly clinical cases involving these bacteria. Identification of Raoultella spp. is difficult due to a phylogenetic relationship with Klebsiella spp. Purpose: Available biochemical tests do not always allow for their identification to species. Thus, the aim of this study was to evaluate selected methods of identification of Raoultella spp. and their differentiation from genus Klebsiella. Materials and Methods: In this evaluation three methods were used such as manual test ID32E (bioMérieux), automatic test VITEK2 Compact (bioMérieux) and matrix-assisted laser desorption ionisation-time of flight mass spectrometry (MALDI-TOF MS) method (Bruker). Results: Good identification of the species was obtained for 81.4% of the strains in the ID32E test, 93.3% in VITEK2 Compact test, and 97.4% in MALDI-TOF MS method, respectively. Conclusion: It was established that MALDI-TOF MS method is reliable in identifying genus Raoultella.     

Detection of four novel alleles: DRB1*1130, DRB1*13072, DRB1*1315 and DRB1*1331

Four new DR52-associated DRB1 alleles are described. One allele, DRB1*1130, is a hybrid between a DRB3*02 allele and a DRB1*11011 allele. The other alleles, DRB1*13072, DRB1*1315, and DRB1*1331, are simple reshufflings of known polymorphic motifs.