Comparison of the neutralizing and ELISA antibody titres to measles virus in human sera and in gamma globulin preparations

Measles virus infection as well as measles vaccination induces a long-lasting immune protection. Specific antibodies have been proven to be associated with this immune protection, since measles immunity can be transferred by immune globulin application (passive immunisation). The neutralisation test (NT) is regarded as the gold standard method for measles immunity because it measures functional neutralising antibody, while with the ELISA, which is often based on cell culture grown native virus antigens, predominantly antibodies to the nucleoprotein antigen were detected. To compare the results of NT and ELISA 199 individual sera and 364 gamma globulin samples, which were made from plasma pools, were tested. Qualitative results showed that the sensitivity of the ELISA was 141/144 (97.9%) and specificity was 48/55 (87.3%) when compared to the NT and focused to the patient samples. For the gamma globulin samples the sensitivity and specificity was 100%. As expected no measles NT negative plasma pool samples were found. The present study showed that with increasing NT-titre, the ELISA-values also rise. False negative ELISA results were obtained in 1.5% of patient sera, mainly containing low levels of neutralising antibody. In both antibody tests seropositive specimens revealed a quite good to moderate correlation. Taken together, the measles IgG ELISA is adequately for immunity testing and identifying of seronegative individuals for vaccination.     

Detection of transmissible gastroenteritis virus neutralising antibody in cats

Summary High titres of neutralising activity to transmissible gastroenteritis virus (TGEV), a porcine coronavirus, were found in sera and peritoneal fluids from cats infected with feline infectious peritonitis (FIP). A small proportion of cats, from a hospital population unaffected by FIP, also had neutralising activity. Procedures to remove non-specific viral inhibitors, including treatment by heat inactivation, trypsin, sulphydryl reagent and kaolin absorption were unsuccessful. The active component was unable to neutralise another porcine coronavirus, haemagglutinating encephalomyelitis virus or the porcine enterovirus, Talfan. Gel filtration of feline sera and peritoneal fluid demonstrated high levels of the neutralising activity in the area corresponding to 7S IgG, which could be removed by absorption with specific anti-IgG serum and these properties are suggested to be consistent with those of antibody. These findings imply that there is a coronavirus in cats which is antigenically related to TGEV and its possible nature is discussed.With 4 Figures     

Development and characterisation of neutralising monoclonal antibody to the SARS-coronavirus

There is a global need to elucidate protective antigens expressed by the SARS-coronavirus (SARS-CoV). Monoclonal antibody reagents that recognise specific antigens on SARS-CoV are needed urgently. In this report, the development and immunochemical characterisation of a panel of murine monoclonal antibodies (mAbs) against the SARS-CoV is presented, based upon their specificity, binding requirements, and biological activity. Initial screening by ELISA, using highly purified virus as the coating antigen, resulted in the selection of 103 mAbs to the SARS virus. Subsequent screening steps reduced this panel to seventeen IgG mAbs. A single mAb, F26G15, is specific for the nucleoprotein as seen in Western immunoblot while five other mAbs react with the Spike protein. Two of these Spike-specific mAbs demonstrate the ability to neutralise SARS-CoV in vitro while another four Western immunoblot-negative mAbs also neutralise the virus. The utility of these mAbs for diagnostic development is demonstrated. Antibody from convalescent SARS patients, but not normal human serum, is also shown to specifically compete off binding of mAbs to whole SARS-CoV. These studies highlight the importance of using standardised assays and reagents. These mAbs will be useful for the development of diagnostic tests, studies of SARS-CoV pathogenesis and vaccine development.     

Complement-independent neutralising monoclonal antibody with differential reactivity for strains of human cytomegalovirus

A mouse monoclonal antibody with complement-independent neutralising activity against cytomegalovirus (CMV) and reactive with the 86 kilodalton (kDa) viral glycoprotein H is described. Neutralisation tests against a range of different strains of CMV showed significant crossreactivity, but clear differences were evident between the two prototype viruses AD169 and Davis, and particularly between AD169 and several low-passage recent clinical isolates; CMV present in urine was neutralised weakly if at all.     

Assay of measles virus hemolysin and its antibody

Summary An assay method of measles virus hemolysin (HL) was developed utilizing spectrophotometric measurement of released hemoglobin. The method was based on the linear relationship, in a certain range of HL dilution, between the percentage of hemolyzed erythrocytes and the HL dilution as expressed in a logarithmic scale, and a HL unit was defined. The hemolysis inhibition (HLI) by measles immune serum was represented by an S-shaped symmetric curve with a constant amount of HL when the per cent HLI was plotted against the serum dilution expressed in a logarithmic scale, and the HLI antibody titer was expressed by the serum dilution showing 50% inhibition. The results of HLI test on children, though preliminary in nature, seemed to show that the test is satisfactory enough for routine use. These assay methods will be obviously very useful in studies of measles, in particular enable us to perform rational studies of HL and its activity, which would in turn contribute to the understanding of the nature of the virus and its infection.     

A hybridoma secreting a neutralising monoclonal antibody to egg drop syndrome 1976 virus

A hybridoma (HPRS/AM/1) secreting neutralising antibody to Egg drop syndrome 1976 virus strain D61 was established. This monoclonal antibody also neutralised two other strains tested thereby showing that all the strains possess at least one common antigenic determinant which is important in infection. However, this epitope was not involved in haemagglutination. The antigen was identified in the nuclei of infected chick kidney cells by an indirect fluorescent antibody test.     

Effects of local and systemic immunization on serum antibody titres in splenectomized chickens

The role of spleen in local (per anum) and systemic (intravenous = i.v.) immunization was studied by splenectomizing chickens and immunizing them twice with sheep red blood cells (SRBC) and Brucella abortus. The number of germinal centers in spleen and the splenic weight were also recorded. Splenectomy had no effect on the serum titres evoked by the per anum immunization but it affected the anti-SRBC and, to a lesser extent, anti-Brucella titres in serum after i.v. immunization. The effect of splenectomy was less obvious both in the secondary response and when the chickens were younger at operation and the time between splenectomy and immunization was increased. The i.v. immunizations induced germinal centre formation in the spleen whereas immunization per anum did not significantly do so. Also the splenic weight in the i.v. immunized chickens was higher than that in the per anum immunized birds. The present study shows that the spleen is the major site of antibody production against i.v. administered antigens in chickens. Antibody production to antigens applied per anum occurs mainly in other lymphatic organs, most probably in the gut-associated lymphoid tissues.     

急性冠脉综合征病人肺炎衣原体抗体滴度的临床价值

目的：探讨肺炎衣原体TWAR IgG,IgM抗体滴度对急性冠脉综合征（ACS）的临床预测价值。方法：应用间接微量免疫荧光法，测定了102例ACS组病人和60例对照组受试者血清TWAR IgG,IgM,抗体滴度，并随访6个月。结果：ACS组TWAR IgG既往感染予性率和平均几何滴度均显著高于对照组，P＜0．01，高抗体水平发生ACS风险相对增高，ACS病人随访中，非感染者心血管事件发生率较感染者明显降低，P＜0．05，结论：肺炎衣原体感染与ACS有关，TWAR IgG抗体滴度对判断ACS病人预后有较好的临床价值。     

A sensitive assay for detection and measurement of neutralising antibody to human immunodeficiency virus.

An assay based on the inhibition of syncytium formation in C8166 cells was developed to measure low levels of neutralising antibody (NT-AB) to human immunodeficiency virus (HIV) and to detect cross-reactivity between virus strains. The relationship between virus challenge and antibody titre was represented by a tripartite curve which was essentially linear over moderate levels of virus input. Based on these findings, antibody titres were standardised against 100 TCID50 of challenge virus. However, lower virus inocula were found to detect minimum levels of antibody. Reproducibility of antibody titres between tests was high, with variation generally lying within one dilution step. The improved sensitivity of the technique allowed detection of NT-ABs in animals immunised with immune-stimulating complexes (ISCOMS) incorporating HIV antigens. Consistent levels of cross-reactivity between HIV strains was demonstrated, indicating the presence of distinct viral groups, from which dominant isolates may be chosen for use in vaccination studies.     

Enzyme-linked immunosorbent assay for detection of measles antibody.

An enzyme-linked immunosorbent assay (ELISA) was developed for the detection of measles immunoglobulin G antibody (MEASELISA). This assay was found to be comparable to the measles hemagglutination inhibition (HAI) test. Approximately 500 sera from three centers were tested by MEASELISA and the HAI test. MEASELISA demonstrated values of greater than 99% for sensitivity, specificity, and accuracy. Values were very precise, with a mean coefficient of variation of 5.4%. MEASELISA values were shown by linear regression analysis to increase as HAI titers increased. A coefficient of determination of 1.00 was obtained from test center three. MEASELISA values were found to be linearly related (r2 greater than 0.97) to MEASELISA titers, thus enabling quantitation of measles antibody from a single value. Also, data are presented that show MEASELISA to be equivalent to complement fixation for evaluating paired sera for the presence of a significant increase in antibody levels to measles virus.     

Induction of humoral neutralising and haemagglutination-inhibiting antibody by the spike protein of avian infectious bronchitis virus

The spike (S), membrane (M) and nucleocapsid (N) proteins of avian infectious bronchitis virus strain M41 (IBV-M41) were separated by sucrose gradient sedimentation after dissociation of the virus by non-ionic detergent. Groups of four chickens were inoculated intramuscularly with 20 microg of S, M or N in Freund's complete adjuvant and at 4 and 7 weeks later with 20 microg, of S, M or N protein in Freund's incomplete adjuvant. Chickens were bled at 4, 7, 10 and 13 weeks after the first vaccination and the sera analysed for serum virus neutralising (SN) and haemagglutination-inhibiting (HAI) antibody. Sera from all four chickens inoculated with S contained SN and HAI antibody. Maximum titres, in the range 5 to 9 log(2), were attained after one, two or three injections in one, two and one chickens respectively. The SN and HAI titres rose in parallel. Neither M nor N induced detectable SN or HAI antibody. None of the chickens resisted challenge to the respiratory tract at 6 weeks after the final vaccination. Sera from chickens which had been infected twice with live IBV-M41 immunoprecipitated more radiolabelled S than N protein and little or no M. Similar results were obtained with sera from guinea pigs which had been inoculated intramuscularly with inactivated virus.     

Antisperm antibody titres, immune complex deposition and immunocompetence in long-term vasectomized mice

Experiments were performed to determine sperm antigen-specific and non-specific immunological changes in long-term vasectomized BDF₁ mice. Circulating antisperm antibodies, detected by immunofluorescence assay, were observed as early as 1 month post-vasectomy; antisperm titres increased with time and were highest in animals that had been vasectomized for over 2 years. Several aged sham-vasectomized mice also had significant antisperm antibody titres, but the development of antisperm antibodies was significantly different from that of the vasectomized group. Tests of general immunocompetence, performed on vasectomized and sham-vasectomized mice at various intervals up to 2·5 years post-surgery, revealed a decrease in mitogenic responsiveness over time in both groups, but no difference between age-matched groups in lymphocyte responses to mitogenic or allogeneic stimulation in vitro, or to in vivo challenge with picryl chloride (delayed hypersensitivity response) or immunization with foreign antigen (humoral response). Most vasectomized mice developed epididymitis and epididymal sperm granulomas by 9 months post-surgery. Patchy regions of hypospermatogenesis were observed in some testes as early as 3 months post-vasectomy, and spermatogenesis was markedly impaired in all long-term vasectomized mice examined. Orchitis lesions characterized by immune complex deposition and lymphocytic infiltration were found in testes from three out of 14 long-term vasectomized mice, as compared to none of the sham-operated group. Studies of vasectomy-associated immune complex deposition in the kidney were inconclusive because aged animals from both vasectomized and sham-vasectomized groups had similar patterns of immunoglobulin and complement deposition in the glomerular basement membranes.     

Inverse relation of serum Helicobacter pylori antibody titres and extent of intestinal metaplasia.

AIMS: To clarify the relation between the serum titre of anti-Helicobacter pylori (H pylori) antibody and the extent of intestinal metaplasia of the gastric mucosa. METHODS: The serum anti-H pylori IgG titres of 95 asymptomatic individuals (mean age 65 years) undergoing an annual health examination were measured and compared with the extent of intestinal metaplasia (absent, moderate, or extensive), determined by examination of multiple endoscopic mucosal biopsy specimens. Serum pepsinogen I (PGI) levels, as a marker for gastric atrophy, were also measured. RESULTS: The prevalence of seropositivity for H pylori antibody was high (> 80%), regardless of the extent of metaplasia. However, there was a negative association between the extent of metaplasia and the anti-H pylori titre: 75% of the subjects in the group without metaplasia had high (3+) antibody levels, as did 43% with moderate, and 37% with extensive metaplasia (absent v extensive). The inverse relation between the titre and the extent of metaplasia was evident when examined in those with normal PGI (> 30 ng/ml), whereas no such relation was apparent in subjects with low PGI (< or = 30 ng/ml). CONCLUSIONS: The anti-H pylori titre correlates inversely with the extent of intestinal metaplasia, particularly in subjects with less marked gastric atrophy.     

A fully human antibody neutralising biologically active human TGFbeta2 for use in therapy.

Phage display provides a methodology for obtaining fully human antibodies directed against human transforming growth factor-beta (TGFbeta) suitable for the treatment of fibrotic disorders. The strategy employed was to isolate a human single chain Fv (scFv) fragment that neutralises human TGFbeta2 from a phage display repertoire, convert it into a human IgG4 and then determine its TGFbeta binding and neutralisation properties and its physical characteristics. Several scFv fragments binding to TGFbeta2 were isolated by panning of an antibody phage display repertoire, and subsequent chain shuffling of the selected V(H) domains with a library of V(L) domains. The three most potent neutralising antibodies were chosen for conversion to IgG4 format. The IgG4 antibodies were ranked for their ability to neutralise TGFbeta2 and the most potent, 6B1 IgG4, was chosen for further characterisation. 6B1 IgG4 has a high affinity for TGFbeta2 with a dissociation constant of 0.89 nM as determined using the BIAcore biosensor and only 9% cross-reactivity with TGFbeta3 (dissociation constant, 10 nM). There was no detectable binding to TGFbeta1. 6B1 IgG4 strongly neutralises (IC50 = 2 nM) the anti-proliferative effect of TGFbeta2 in bioassays using TF1 human erythroleukaemia cells. Similarly, there was strong inhibition of binding of TGFbeta2 to cell surface receptors in a radioreceptor assay using A549 cells. 6B1 IgG4 shows no detectable cross-reactivity with related or unrelated antigens by immunocytochemistry or ELISA. The 6B1 V(L) domain has entirely germline framework regions and the V(H) domain has only three non-germline framework amino acids. This, together with its fully human nature, should minimise any potential immunogenicity of 6B1 IgG4 when used in therapy of fibrotic diseases mediated by TGFbeta2.     

Fibrillar anti-cellular antibody associated with mumps and measles infection

Fibrillar anti-cellular IgM antibody was found in sera of 8/10 children with acute mumps infection and in 8/12 children with acute measles infection. Absorption experiments showed that the antibody was against cellular components in cells of human origin and was unrelated to either viral antibody or to RF. Similar antibody was previously found in patients with MS.