Comparison of ELISA and immunoblotting techniques for the detection of cherry mottle leaf virus

Formaldehyde treated cherry mottle leaf virus (ChMLV) and the isolated coat protein were used successfully for the production of polyclonal and monoclonal antibodies. The monoclonal antibodies had a titre of 1:51 200 and consisted of IgG1 and IgG2. The antibodies reacted with all 11 isolates of ChMLV, from five locations in Canada and the USA, included in this study. Several serological procedures were assessed to compare their sensitivity for detecting ChMLV. Plate-trapped antigen ELISA (PTA-ELISA) and dot-blot immunobinding assay (DBIA), using virus specific MAbs, were the most sensitive tests in this study. Triple antibody sandwich ELISA (TAS-ELISA) and Western blot were found to be less sensitive. Dilution of the samples appeared to increase the sensitivity of both PTA-ELISA and Western blot detection. Young leaves and flowers of Prunus avium were the best tissue for detecting the virus which could also be detected in the fruit and leaves of P. tomentosa. April and May were optimal for detection of the virus in the field, whereas both April to May and August to September were optimal for screenhouse-grown plants.     

Electro-immunoblotting characterization ofPseudo-nitzschia multiseries andP. pungens antigens recognized by antibodies directed against whole cells

Separate polyclonal antibodies have previously been developed against the domoic-acid-producingPseudonitzschia multiseries (=Pseudo-nitzschia pungens f.multiseries) and the non-toxicP. pungens (=P. pungens f.pungens). These antibodies bind to the surface of the diatoms as shown by immunofluorescence studies. Here we examine the molecular nature of the antigens by Western blotting (electro-immunoblotting) analysis. The major antigens for both polyclonal antibodies migrated as high molecular-weight diffuse bands, mostly remaining in the stacking gel, using an SDS-PAGE system. The antibodies prepared againstP. multiseries strongly labelled the high molecular-weight antigens of allP. multiseries strains tested and showed little reactivity towardsP. pungens extracts on Western blots.P. pungens antibodies strongly labelled high molecular-weightP. pungens antigens and faintly labelled a fewP. multiseries antigens. The selectivity of the antibodies for their respective species correlates with the results of the immunofluorescence experiments, suggesting that the antigens examined in this study are responsible for the selective labelling in immunofluorescence studies. The electrophoretic mobility and the antibody labelling of antigens were not altered by proteolytic digestion of cell pellets. However, disruption of carbohydrates in the pellets by treatment with periodic acid resulted in loss of the antigen. These data suggest that the major antigens of toxicP. multiseries and non-toxicP. pungens are high molecular-weight (°>100kDa) polysaccharides located on the surface of these diatoms.Author for correspondence     

用人工合成的丁型肝炎病毒抗原多肽检测丁肝病毒IgM抗体及其初步应用

用人工合成的丁型肝炎病毒抗原（ＨＤＶ－Ａｇ）肽建立了检测抗ＨＤＶ－ＩｇＭ抗体的ＥＬＩＳＡ方法,本法操作简便、快速,重复性好,特异性强,与抗ＨＡＶ－ＩｇＭ、抗Ｈｋ－ＩｇＭ、抗ＨＢｓ－ＩｇＭ、抗ＨＣＶ－ＩｇＩ、抗ＣＭＶ－ＩｇＭ、抗ＲＶ－ＩｇＭ、类风湿因子（ＲＦ）及抗核抗体（ＡＮＡ）阳性血清均不起反应,且可被２－巯基乙醇阻断而不起反应。经初步临床应用,３１例正常人血清抗ＨＤＶ－ＩｇＭ全部阴性,２８例慢活肝患者检出率为３２．１％（９／２８）,１７例慢迁肝患者血清阳性率为１１．８％（２／１７）１８例肝癌和肝硬化病人血清阳性率为２２．２％（４／１８）这三组病人与正常对照者相比较均有显著性差异（Ｐ＜０．００１）。此外,抗ＨＤＶ－ＩｇＭ阳性血清的ＡＬＴ值均明显高于正常参考范围,提示在ＨＤＶ感染过程中,患者肝细胞进一步受损。实验结果证明,抗ＨＤＶ－ＩｇＭ是诊断ＨＤＶ感染的重要指标,对ＨＤＶ感染早期诊断具有重要价值。     

鼠肺支原体单克隆抗体的研究

通过杂交瘤技术建立了３株抗鼠肺支原体单克隆抗体细胞株,它们分别是ＢＡ１１,ＢＤ７和Ｂ６１２．试验表明：３株细胞所分泌的抗体均属ＩｇＧ１亚类,都是鼠肺支原体的特异性抗体,与多种其它支原体无交叉反应。     

猴B病毒相关抗体及B病毒抗体检测的比较研究

本文比较了用ＨＳＶ－１（ＩＥＡ）和Ｂ病毒（ＩＦＡ）的方法检查猴Ｂ病毒相关抗体和Ｂ病毒抗体的结果：ＩＥＡ检查出的Ｂ病毒相关抗体阴性猴,经ＩＦＡ检查,全部为Ｂ病毒抗体阴性。用ＩＦＡ检查了Ｂ病毒相关抗体阴性的恒河猴,在单笼隔离饲养六个月后,有９８．３％的动物Ｂ病毒相关抗体仍为阴性,表明ＩＲＡ的阴性结果有较好的一致性。本文讨论了建立无Ｂ病毒感染猴群的可行性。     

不同月龄婴儿接种DTP后血清中百日咳抗体水平的观测

本文对比观察了２１３例２月龄、３月龄婴儿接种ＤＴＰ后百日咳抗体水平的变化,探讨了母传抗体对免后抗体增长的影响。结果表明２月龄、３月龄婴儿ＤＴＰ免疫后１个月和３个月血清中百日咳抗体达保护水平的百分率无显著性差异（ｘ￣２＝０．０３６,ｐ＞０．９;ｘ￣２＝０．３２７,ｐ＞０．５）,免后１个月抗体ＧＭＴ无显著性差异（ｔ＝０．１７,ｐ＞０．５）,免后３个月抗体ＧＭＴ３月龄组高于２月龄组（ｔ＝２．２２,ｐ＜０．０５）。我们还发现免前抗体水平与免后１个月ＧＭＴ虽有负相关（ｒ＝—０,７５４）的倾向,但总体上对抑制免后抗体应答不明显,因而建议将儿童ＤＴＰ基础免疫的起始月龄提前至２月龄进行。     

Intra-acrosomal 155,000 dalton protein increases the antigenicity during mouse sperm maturation in the epididymis: A study using a monoclonal antibody MC101

We found an intra-acrosomal antigen of about 155,000 daltons (155 kDa) in a survey using the monoclonal antibody MC101 raised against mouse cauda epididymal spermatozoa. Morphological studies by means of indirect immunofluorescence and immunogold electron microscopy localized the antigen to the cortex region of the anterior acrosome. Avidin biotin complex immunocytochemistry initially demonstrated a faint signal at the anterior acrosome in the testis spermatozoa that increased in intensity as the sperm moved toward the distal epididymis. This incremental immunoreactivity was also confirmed by immunoblotting following one-dimensional SDS-PAGE. The 155 kDa protein band was immunostained, and it was much more intense in the cauda epididymal than in the caput and corpus epididymal spermatozoa. Only a trace or no immunostain was evident in the caput or testis spermatozoa. The antigen localization did not change during passage through the epididymis, being confined at the cortex region of the anterior acrosome. The epididymal epithelial cells were not immunostained. These findings suggested that the 155 kDa protein is biochemically modified, further implying that the biochemical alteration of intra-acrosomal material is involved in sperm maturation in the epididymis. © 1995 wiley-Liss, Inc.     

The biotechnology and applications of antibody engineering

The exquisite specificity of monoclonal antibodies (MAb) has long provided the potential for creating new reagents for the in vivo delivery of therapeutic drugs or toxins to defined cellular target sites or improved methods of diagnosis. However, many difficulties associated with their production, affinity, specificity, and use in vivo have largely confined their application to research or in vitro diagnostics. This situation is beginning to change with the recent developments in the applied molecular techniques that allow the engineering of the genes that encode antibodies rather than the manipulation of the intact antibodies themselves. Techniques, such as the polymerase chain reaction, have provided essential methods with which to generate and modify the genetic constituents of antibodies, allow their conjugation to toxins or drugs, provide ways of humanizing murine antibodies, and allow discrete modular antigen binding components to be produced. More recent developments of in vitro expression systems and powerful phage surface display technologies will without doubt play a major role in future antibody engineering and in the successful development of new diagnostic and therapeutic antibody-based reagents.