Evaluation of a botulinum fragment C-based ELISA for measuring the humoral immune response in primates.

An enzyme-linked immunosorbent assay (ELISA) using botulinum neurotoxin serotype B recombinant fragment C (rBoNTB(HC)) was developed to measure specific humoral immune responses of monkeys vaccinated with a vaccine consisting of rBoNTB(HC). Several fundamental parameters for a bioassay were evaluated. The evaluation results demonstrated that using BoNTB(HC) as the capture antigen led to a specific and sensitive ELISA for botulinum type B antibody with excellent precision, accuracy, and linearity. There was a good correlation (r=0.91) between ELISA titers and neutralization bioassay titers. Experimental results suggested that the ELISA could be useful for detecting botulinum type B antibody levels and may supplement mouse neutralization bioassays during planned clinical manufacturing and clinical trials of rBoNTB(HC) vaccine.     

TNP-LPS induces an IgG anti-TNP immune response in mice.

The trinitrophenylated derivatives of lipopolysaccharide (TNP-LPS) elicit a specific anti-TNP, thymus-independent immune response in mice. After a single injection of antigen, anti-TNP antibodies of IgM and IgG isotypes are detected at the cellular and at the humoral levels, in athymic nude mice as well as in conventional (C57B1/6 × DBA/2)F₁ mice. The immune sera were resolved into IgM and IgG molecules by gel filtration; both fractions showed an anti-TNP activity, thus confirming the data obtained by the cellular analysis.     

NK cell-deficient mice develop a Th1-like response but fail to mount an efficient antigen-specific IgG2a antibody response.

NK cells have been shown to play a role in the modulation of B cell differentiation and Ab production. Using a novel murine model of NK cell deficiency, we analyzed the in vivo role of NK cells in the regulation of Ag-specific Ab production. After immunization with OVA or keyhole limpet hemocyanin in CFA, NK cell-deficient (NK-T+) mice developed an efficient Th1 response and produced significant levels of IFN-gamma but displayed markedly reduced or absent Ag-specific IgG2a production. There were no differences in the levels of Ag-specific IgG, IgG1, and IgG2b between NK-T+ and NK+T+ mice. Furthermore, NK cell-reconstituted, NK+T+ (tgepsilon26Y) mice produced significant amounts of Ag-specific IgG2a after immunization with OVA. These results indicate that NK cells are involved in the induction of Ag-specific IgG2a production in vivo. Moreover, they also demonstrate that the lack of Ag-specific IgG2a Ab production in NK-T+ mice is not associated with the impaired Th1 response and IFN-gamma production.     

Control of IgE and IgG1 antibody production in mice.

The production of IgE and IgG1 was studied in untreated, thymectomized. splenectomized, anti-thymocyte serum-treated, or sublethally X-irradiated mice. Dinitrophenyl Ascaris and ovalbumin were used as antigens, and aluminum hydroxide was used as adjuvant. A suppression of IgE production was observed in adult thymectomized mice, although the kinetic pattern of the antibody response was the same as in control animals. IgG1 antibody production was not affected by thymectomy. Splenectomy did not change either IgE or IgG1 production. A single dose of rabbit anti-thymocyte serum (ATS) given 8 days after immunization inhibited IgE antibody production. The effect of ATS was dose dependent and also varied with the amount of antigen used, the immune response to high doses being more susceptible to the effect of ATS. No alteration in IgG1 production was caused by ATS even when IgE antibody formation was completely inhibited. When preceding immunization, sublethal irradiation enhanced IgE antibody formation and partially suppressed IgG1 production; applied after immunization, irradiation caused an enhancement of IgE production which was inversely proportional to the interval elapsed between the two procedures. On the other hand, the IgG1 antibody production was fairly resistant to the same treatment. The results suggest a clearcut separation between the mechanisms regulating IgE and IgG1 production in mice.     

人巨细胞病毒IgG抗体(ELISA)检测试剂盒稳定性观察

目的观察人巨细胞病毒(human cytomegalovirus,HCMV)IgG抗体(ELISA)检测试剂盒的稳定性。方法将HCMV IgG抗体(ELISA)检测试剂盒分别以不同时间放置于2~8℃和37℃,检测试剂盒的外观、阳性参考品符合率、阴性参考品符合率、重复性和检测限,并对其进行稳定性(实时稳定性、加速破坏稳定性、开瓶稳定性、运输稳定性)的观察及初步应用。结果 3批实验用试剂盒实时稳定性和3批试生产试剂盒加速破坏稳定性、开瓶稳定性、运输稳定性试验中,外观均合格,阴性参考品符合率及阳性参考品符合率均为100%,最低检出限参考品均能检出。3批实验用试剂盒实时稳定性检测重复性良好,CV值均≤15.00%。3批试生产试剂盒在37℃条件下放置6 d后,CV值分别为7.94%、7.16%、4.66%;开瓶后分别置于2~8℃冰箱0w、1w、2w、3w、4w,CV值均≤1 5.00%;运输至上海,CV值分别为2.59%、3.12%、2.37%;运输至海南,CV值分别为4.89%、2.65%、2.33%。样品反复冻融5次后,试剂盒检测稳定性良好。结论 HCMV IgG抗体(ELISA)检测试剂盒具有良好的稳定性,在2~8℃条件下至少可保存15个月。     

一种辅助SNT测定人特异性免疫球蛋白抗体效价的ELISA方法的建立

利用已建立的抗破伤风类毒素和抗狂犬病病毒的抗体半定量检测ELISA试剂,建立检测人特异性免疫球蛋白半成品和成品效价的可靠方法,依靠统计分析技术,推算出抗体ELISA效价,发现其与小鼠血清中和试验(SNT)检测的抗体效价的相关性和一致性极好,可用于ELISA对SNT的稀释范围的确定,提高其准确性,还可部分替代SNT对生产过程进行监控,缩短生产时间。     

Evaluation of quantitative anti-F1 IgG and anti-V IgG ELISAs for use as an in vitro-based potency assay of plague vaccine in mice.

Quantitative anti-F1 and anti-V IgG enzyme-linked immunosorbent assays (ELISAs) were developed to measure the serological response of female Swiss Webster mice after vaccination with the recombinant fusion protein, rF1-V, which is being developed as a plague vaccine. Several fundamental parameters of the ELISA were evaluated: specificity, precision, accuracy, and stability. Experimental results suggested that a potency assay based upon the serological response of female Swiss Webster mice, as measured by quantitative anti-F1 IgG and anti-V IgG ELISAs, might be used to evaluate the rF1-V fusion protein vaccine.     

Morphology of the antibody response to pneumococcal polysaccharide in mice.

In the course of an antibody immune response to pneumococcal polysaccharide - type III (S III) in mice a slight increase was observed in the proportion of plasma cells among the antibody-producing cells, reaching its peak at the time of decline of this reaction. On the basis of ultrastructural resemblance of these plasma cells to primitive reticular cells and in view of other specificities of the immunological response to S III antigen, the author presumes direct reticular origin of anti-S III antibody-producing plasma cells.     

Development and Application of an ELISA for the Detection of Porcine Deltacoronavirus IgG Antibodies

Porcine deltacoronavirus (PDCoV), also known as porcine coronavirus HKU15, was first detected in North America in early 2014 and associated with enteric disease in pigs, resulting in an urgent need to further investigate the ecology of this virus. While assays detecting nucleic acids were implemented quickly, assays to detect anti-PDCoV antibodies have not been available. In this study, an indirect anti-PDCoV IgG enzyme-linked immunosorbent assay (ELISA) based on the putative S1 portion of the spike protein was developed and utilized to determine the prevalence of anti-PDCoV IgG in U.S. pigs. The diagnostic sensitivity of the PDCoV ELISA was 91% with a diagnostic specificity of 95%. A total of 968 serum samples were tested including samples with confirmed infection with PDCoV, porcine epidemic diarrhea virus (PEDV), transmissible gastroenteritis virus or porcine respiratory coronavirus. There was no cross-reactivity with any of the other coronaviruses. Among 355 arbitrarily selected serum samples collected in 2014 and originating from 51 farms across 18 U.S. states, anti-PDCoV IgG antibodies were detected in 8.7% of the samples and in 25.5% of the farms whereas anti-PEDV IgG was detected in 22.8% of the samples and in 54.9% of the farms. In addition, anti-PDCoV IgG antibodies were detected in archived samples collected in 2010, perhaps indicating an earlier undetected introduction into the U.S. pig population. Overall, the obtained data suggest that PDCoV seroprevalence in U.S. pigs is lower compared to PEDV and PDCoV may have been introduced to the U.S. prior to PEDV.     

The vibrational normal modes of beta-barrels in an IgG antibody molecule.

Based on the quasi-continuity model, and using the method of group theory, we studied the normal vibrations of the VL- and the CHL-beta-barrels in an IgG molecule. We put emphasis on the Raman- and the infrared-active normal modes. The Raman modes we obtained include both the breathing motion mode (or the dominant low-frequency mode) which corresponds to the maximum peak in the Raman spectrum, and the normal modes that correspond to the lower peaks. Our calculated vibration frequencies are found to be in good agreement with the experimental results observed by Painter et al. (Biopolymers 20 (1981) 243). The method and work presented in this paper may improve Chou's quasi-continuity theory in calculating the vibrational modes of a beta-barrel protein.     

Late acting T cell depression of high avidity IgG antibody secretion in the secondary response.

Activated T lymphocytes (ATC) suppressed the expression of high avidity plaque-forming cells (PFC) in the secondary immune response when given in vivo 1 day before assay or when mixed in vitro with hyperimmune cells for 90 min before assay. This late acting suppression is a cell dose-dependent, carrier-spedific, T cell-mediated phenomenon. The target of this activity if probably the antibody-secreting cell.     

检测人血清中肺炎球菌10A型荚膜多糖IgG抗体的包被抗原适用性研究

目的 确定用于23价肺炎多糖疫苗免疫后临床血清样本检测的包被用10A型肺炎球菌荚膜多糖(Pn10A)。方法 根据WHO推荐的检测人血清中肺炎球菌荚膜多糖IgG抗体含量的ELISA(PnPSELISA),包被不同来源(ATCC、A公司、B公司、5个公司混合)的10A多糖[Pn10A(ATCC)、Pn10A(A)、Pn10A(B)、Pn10A(mix)],检测38份血清中Pn10AIgG抗体的几何平均浓度(GMC)和相同样本免疫前、后的阳转率(免疫后/免疫前≥2为阳转),确定用于临床血清检测的包被Pn10A。结果 用Pn10A(ATCC)、Pn10A(A)、Pn10A(B)包被检测38份相同样本免疫前、后血清中Pn10AIgG抗体的GMC和阳转率,Pn10A(A)与Pn10A(ATCC)包被的检测结果差异无统计学意义(P>0.05),数据一致性好(r>0.9);Pn10A(B)与Pn10A(ATCC)包被的检测结果差异有统计学意义(P<0.05),数据一致性差(r<0.8);再以Pn10A(A)、Pn10A(ATCC)、Pn10A(mix)包被检测另46份相同样本免疫前、后血清中Pn10AIgG抗体的GMC和阳转率,Pn10A(A)、Pn10A(mix)与Pn10A(ATCC)包被的检测结果差异无统计学意义(P>0.05),数据一致性好(r>0.9),免疫前、后GMC值相近,阳转率相近,差异无统计学意义(P>0.05)。结论 Pn10A(A)、Pn10A(mix)与Pn10A(ATCC)均可以作为包被多糖用于检测人血清中肺炎球菌Pn10AIgG抗体;但从长久使用相同抗原检测大批量临床样本的需求考虑,Pn10A(mix)更具有足量、经济的优势。     

人巨细胞病毒IgG酶联免疫检测试剂盒的研制

为研制人巨细胞病毒 (HCMV)IgG酶联检测试剂盒 ,将HCMV接种人二倍体细胞 ,收获的病毒经纯化后用作包被抗原 ,辣根过氧化物酶 (HRP)标记羊抗人IgG为检测抗体 ,采用间接ELISA制备酶联免疫检测试剂盒并进行检定。该试剂盒操作简便、特异性强 ,稳定性、线性及精密性符合体外诊断试剂的要求。     

The IgM and IgG antibody responses in iron-deficient and iron-loaded mice

The humoral immune response was evaluated in male CD-1 mice fed the iron deficient (7 ppm Fe), iron sufficient (120 ppm Fe), and high-iron diets (3000 or 5000 ppm Fe) for 54 d. The IgM and IgG antibody responses against sheep erythrocytes (SRBC) determined by hemolytic plaque assay were suppressed by 65.4 and 51.2%, respectively, in the iron deficient mice. Subclinical iron deficiency was manifested by a marked reduction in hepatic iron concentration without any changes in hematocrit or body weight gain. In contrast, consumption of high-iron diets caused a marked accumulation of iron in the liver and a twofold reduction in the IgM antibody response without alteration in the IgG response. The suppression of the IgG antibody response in the iron deficient mice, however, did not result in a compensatory increase in delayed type hypersensitivity response.