The separation of immunoglobulin M from human serum by fast protein liquid chromatography

A fast protein liquid chromatography (FPLC) system was evaluated as a method for rapid separation of serum immunoglobulin M (IgM) from immunoglobulin G (IgG) and immunoglobulin A (IgA). The system incorporates the use of a strong anion exchanger. Evaluation was carried out in 3 ways. The effect of increasing the serum percentage in the 500 microliters volumes loaded on to the column was tested. Samples containing up to 60% serum resulted in only small concentrations of contaminating IgG and IgA in the IgM fraction. Reproducibility was tested by fractionating the same serum sample several times; the coefficient of variation (CV) of the IgM concentration in the IgM fraction was 6%. A number of sera which varied considerably in immunoglobulin concentration were fractionated without any significant adverse effects on the immunoglobulin ratios in the IgM fraction. One serum sample containing a high concentration of IgG and IgA was included. In contrast to gel filtration chromatography, FPLC can separate IgM from IgG and IgA within 6 min. On loading 500 microliters samples containing from 20 to 60% serum, less than 0.01 g/l IgG was detected in the IgM fractions when tested by the radial immunodiffusion method.     

血清唾液酸测定在肺癌、乳腺癌和结肠癌诊断中的价值

目的：探讨血清唾液酸（ＳＡ）水平与肺癌,乳腺癌和大肠癌诊断之间的关系。方法：采用改良间苯二酚快速比色测定法,检测４１名健康人与３１例肺癌、２９例乳腺癌和１６例大肠癌２血清ＳＡ水平,采用方差分析和ｑ检验。结果：肺癌组和大肠癌组血清ＳＡ水平均显著高于乳腺癌组和对照组（Ｐ〈０．０１）,但乳腺癌组与对照组血甭ＳＡ水平差异无显著性（Ｐ〉０．０５）。结论：血清ＳＡ水平检测对于肺癌和大肠癌有一定诊断价值,但对乳     

Serodiagnosis of aspergillosis in falcons (Falco spp.) by an Afmp1p‐based enzyme‐linked immunosorbent assay

Aspergillosis in falcons may be associated with high mortality and difficulties in clinical and laboratory diagnosis. We previously cloned an immunogenic protein, Afmp1p, in Aspergillus fumigatus and showed that anti‐Afmp1p antibodies were present in human patients with A. fumigatus infections. In this study, we hypothesise that a similar Afmp1p‐based enzyme‐linked immunosorbent assay (ELISA) could be applied to serodiagnose falcon aspergillosis. A specific polyclonal antibody was first generated to detect falcon serum IgY. Horseradish peroxidase‐conjugate of this antibody was then used to measure anti‐Afmp1p antibodies in sera collected from falcons experimentally infected with A. fumigatus, and the performance of the Afmp1p‐based ELISA was evaluated using sera from healthy falcons and falcons with documented A. fumigatus infections. All four experimentally infected falcons developed culture‐ and histology‐proven invasive aspergillosis. Anti‐Afmp1p antibodies were detected in their sera. For the Afmp1p‐based ELISA, the mean ± SD OD_450 nm using sera from 129 healthy falcons was 0.186 ± 0.073. Receiver operating characteristics curve analysis showed an absorbance cut‐off value of 0.407. One negative serum gave an absorbance outside the normal range, giving a specificity of 99.2%. For the 12 sera from falcons with confirmed aspergillosis, nine gave absorbance values ≥ cut‐off, giving a sensitivity of 75%. The Afmp1p‐based ELISA is useful for serodiagnosis of falcons with aspergillosis.     

The RD1-encoded antigen Rv3872 of Mycobacterium tuberculosis as a potential candidate for serodiagnosis of tuberculosis

Tuberculosis (TB) infections in India account for one-third of the global burden, making it important to develop speedy, cost-effective diagnostic tools. This study evaluated recombinant RD1-encoded antigens of Mycobacterium tuberculosis as tools for serodiagnosis by determining the immunological reactivity of these proteins against sera from healthy, bacille Calmette-Guérin (BCG)-vaccinated and TB-infected individuals from Kolkata. Rv3872, Rv3875 (ESAT-6) and Rv3878 were able to discriminate healthy BCG-vaccinated controls from TB patients. Rv3872 showed the highest level of antibody response in comparison with other antigens, and also showed statistically significant differences between pulmonary (p <0.0001) or extra-pulmonary (p <0.001) TB patients and healthy BCG-vaccinated individuals. The levels of antibody were measured using 20-mer overlapping peptides spanning the entire Rv3872 sequence. The immunological reactivity against a mixture of two peptides (P8 and P9) encompassing amino-acids 57-84 correlated well with that obtained using full-length Rv3872. This result was explained by the fact that two of the predicted regions of high antigenicity lie within amino-acid residues 57-85 of Rv3872. The high sensitivity and specificity of Rv3872, as well as the mixture of two synthetic overlapping peptides derived from Rv3872, highlight their potential and argue in favour of their use in serodiagnosis of both pulmonary and extra-pulmonary TB.     

Serodiagnosis of Chlamydia pneumoniae infection using three inclusion membrane proteins

The Chlamydia pneumoniae genome‐encoded open reading frames Cpn0146, Cpn0147, and Cpn0308 were expressed as recombinant proteins for detecting C. pneumoniae‐specific antibodies in samples from three groups of individuals including 183 with C. pneumoniae‐associated respiratory infection (group I), 60 healthy blood donors (group II), and 32 with no known respiratory infection (group III). The recombinant Cpn0146 was recognized by 71 (38.8% positive recognition rate), 15 (25%) and 1 (3.1%), Cpn0147 by 75 (40.9%), 14 (23.3%), and 2 (6.3%), and Cpn0308 by 82 (44.8%), 16 (26.7%), and 0 (0%) samples from groups I, II, and III, respectively. The positive recognition rates with any of the three antigens were significantly higher in group I than those in groups II and III, suggesting that more individuals from group I were likely infected with C. pneumoniae. This conclusion was confirmed with a commercially available whole organism‐based ELISA kit (Savyon Diagnostics Ltd., Ashdod, Israel), which detected C. pneumoniae antibodies in 98 (64.1%), 26 (43.3%), and 4 (12.5%) samples from group I, II, and III, respectively. Comparing to the commercial kit, the recombinant antigen‐based detection assays displayed >97% of detection specificity and >87% of sensitivity, suggesting that these recombinant antigens can be considered alternative tools for aiding in serodiagnosis of C. pneumoniae infection. J. Clin. Lab. Anal. 24:55–61, 2010. © 2010 Wiley‐Liss, Inc.     

神经梅毒11例临床特征分析

目的总结神经梅毒的临床特征及早期诊断依据。方法回顾性分析11例神经梅毒患者的临床分型、临床表现、脑脊液检查及影像学表现。结果所有患者血清快速血浆反应素实验（RPR）和梅素螺旋体明胶凝集实验（TPPA）阳性,其中9例脑脊液检查RPR和TPPA均阳性;2例RPR阴性,TPPA阳性。影像学表现多样化,缺乏特异性。结论神经梅毒的诊断目前无统一的金标准,应结合临床表现、脑脊液检查及影像学检查综合分析,特别是脑脊液的检查尤为重要。     

合并丙型肝炎病毒感染的系统性红斑狼疮患者临床与免疫学特征

目的探讨系统性红斑狼疮(SLE)患者丙型肝炎病毒(HCV)感染的发生率及其临床意义。方法用ELISA-3、RIBA-3及PCR检测134例SLE患者和200名正常献血者的HCV感染情况,并分析临床与免疫学其特征。结果15名SLE患者(13 %)和2名正常对照(1%)HCV抗体阳性(P<0.001)。与未合并HCV感染的SLE患者相比,合并HCV感染的SLE患者皮肤表现、dsDNA抗体阳性发生率低(P=0.01,P<0.001),肝脏损害、低补体血症、冷球蛋白血症发生率高(P<0.001,P<0.05,P=0.03)。结论SLE患者合并HCV感染的发生率显著高于正常人群,HCV抗体阳性SLE患者显示出特异的临床和免疫学表现。     

Detection of anti-cord factor antibodies in intestinal tuberculosis for its differential diagnosis from Crohn's disease and ulcerative colitis

We have developed a diagnostic method for pulmonary tuberculosis by detecting antibody to cord factor using enzyme-linked immunosorbent assay (ELISA). This study was to evaluate the usefulness of our method for a diagnosis of intestinal tuberculosis, and especially its ability to differentiate this disease from other inflammatory bowel diseases. Antibodies of the immunoglobulin G class against cord factor (trehalose-6,6'-dimycolate) from 27 patients with intestinal tuberculosis, 16 patients with Crohn's disease (CD), and 27 patients with ulcerative colitis (UC) were tested by ELISA with cord factor purified fromMycobacterium tuberculosis H37Rv as the antigen. Twenty-three of the 27 patients with intestinal tuberculosis (85%) showed elevated values distinct from healthy controls. None of the patients with CD showed an elevation of antibody titers. Of the 27 patients with UC, 26 (96%) did not show any anti-cord factor antibody elevation. We conclude that this method is simple and results are reproducible. The results of our study justify undertaking the detection of anti-cord factor antibodies to diagnose intestinal tuberculosis.     

The production and comparative evaluation of native and recombinant antigens for the fast serodiagnosis of cystic echinococcosis with dot immunogold filtration assay

Clinical diagnosis and post‐surgery assessment of cystic echinococcosis depend on laboratory serodiagnosis and ultrasound examinations. This study aims to produce the recombinant antigen (rAgB) and compare its diagnostic effect with natural antigens (crude fluid antigen, protoscolex antigen). After rAgB, crude fluid antigen, protoscolex antigen were produced, and the diagnostic accuracy was evaluated with dot immunogold filtration assay (DIGFA) by the sera from the following groups: surgically confirmed cystic echinococcosis patients (n = 113), alveolar echinococcosis patients (n = 46), other parasitic diseases (n = 49), nonparasitic hepatic diseases (n = 63) and healthy people (n = 121). In diagnosing cystic echinococcosis, the sensitivity of recombinant AgB was 77·9% and the specificity was 98·3%. The crude fluid antigen B showed a sensitivity of 92·9% and specificity of 81·0%. The protoscolex antigen had sensitivity of 87·6% and specificity of 90·9%. The recombinant AgB indicates the advantage of no cross‐reaction with other parasite diseases or nonparasite hepatic diseases. Recombinant antigen B can improve the specificity but decrease the sensitivity. The combination of native and recombinant antigens will improve the overall performance of serodiagnosis of cystic echinococcosis.     

成虫切片间接免疫酶染法诊断隐性血吸虫病的探讨

用血吸虫成虫冰冻切片间接免疫酶染法(IIP-AWA)检测血清抗血吸虫成虫抗体(AWAb)。5年前已消灭血吸虫病的上海县COPT阳性血清229份和有治疗史COPT阴性血清135份的阳性率各为96.9％和5.2％;122份粪检阳性患者血清均为强阳性;80份健康人血清均阴性。阳性血清滴度≥1:16时,成虫切片多数呈全虫弥漫型显色。149例血清阳性者经吡喹酮治疗后的阴转率,1年内IIP-AWA为80％,明显高于COPT(60.1％);2.5年两者无差别(85.5％与83.6％)。未转阴者滴度明显下降,染色多呈局限型(肠管显色)。表明血清AWAb阳性者,体内可能仍有成虫残存。本法对清查隐性血吸虫病患者(轻度感染,无临床表现者)有实用价值。