Immunological diagnosis of cytomegalovirus infections. Application to blood transfusion centers

The Blood Transfusion Centers (B.T.C.) are mainly concerned with the selection of CMV infection free blood donors, the screening of the anti CMV antibody high titre plasma donors and the evaluation of specific anti CMV Immunoglobulin preparations. Various serological methods could be used but they are of different value depending the purposes of the B.T.C. The neutralization test (Nt), with the addition of complement is specific and detects the protecting AB against the glycoproteins of the viral envelope. The complement fixation test (CF) using extracts of CMV infected cells as antigen largely varies in its sensitivity according to the quality of the antigen. In any case, the CF test is not sensitive enough to detect a latent CMV infection in a certain percentage of the non immunosuppressed adults, but could be used for the selection of anti CMV antibody high titres carriers. Three sensitive methods: passive haemagglutination, indirect immunofluorescence and indirect ELISA tests, might be used for the detection of latent CMV infections. They detect various AB against various internal and external components of the CMV. They are submitted to various sources of errors. The sensitivity of the indirect IF test is mainly restricted by the quality of the antigen preparation, its specificity by the presence of anti cells antibodies in the sera, the Fc receptors in the antigens and the specificity of the conjugates. The indirect ELISA which is submitted to the same causes of errors is a highly sensitive test, easy to perform, reagents are available, and automatic processors have been developed. When compared with the previous techniques, the ELISA test is suitable for the screening of CMV free donors, when it is performed with an highly sensitive antigen. It could be also used for the screening of high antibody titre carriers: its correlation with the CF test is quite good (r = 0,82). When comparatively applied to the titration of Immunoglobulins preparations, made from plasmas or placentas, for either IM or IV administration, the Elisa test gives AB titres different from those obtained with Nt and indirect IF. The calibration of a standard Immunoglobulin reagent is urgently needed and double blind clinical assays of the protective effect of various preparations of specific anti CMV Immunoglobulins have to be promptly designed.     

Comparison of 4 serological tests--complement fixation, neutralization, fluorescent antibody to membrane antigen and immune adherence hemagglutination--for assay of antibody to varicella-zoster (V-Z) virus.

Four tests for antibody to varicella-zoster (V-Z) virus were compared; these were tests of complement fixation (CF), neutralization (NT), fluorescent antibody to membrane antigen (FAMA) and immune adherence hemagglutination (IAHA). Fifty-two sera from patients with varicella and zoster and from recipients of live varicella vaccine were examined by the 4 tests. The CF test was least sensitive, but the antibody titers by the NT, FAMA and IAHA tests were roughly comparable. The IAHA test was the simplest and fastest to perform, and appeared suitable for routine serological assay to V-Z virus. The correlation between the IAHA antibody titer and susceptibility of individuals to clinical varicella was investigated retrospectively using sera obtained during 2 outbreaks of varicella in an institution for children, where all the unvaccinated children had developed varicella symptoms. Most of the 25 pre-exposure sera from unvaccinated children examined by the IAHA test had tiers of less than 1:2. In contrast, all the 23 sera from vaccinated children who did not develop varicella had detectable antibody titers of 1:2 to 1:64. These results indicate that the IAHA titer reflects the susceptibility or resistance of individuals to clinical varicella.     

Cross-reactive antibodies to mycoplasmas found in human sera by the enzyme-linked immunosorbent assay (ELISA)

Antibodies against Mycoplasma pneumoniae in patients' sera with M. pneumoniae infection were measured by the complement fixation (CF) test and enzyme-linked immunosorbent assay (ELISA). Many patients' sera cross-reacted with heterologous mycoplasmal ELISA antigens such as M. hominis, M. hyorhinis, M. orale, M. pulmonis and M. salivarium. The sera with high CF (CF greater than or equal to 40) titers gave significantly higher ELISA values to M. hyorhinis (P less than 0.001) and M. pulmonis (P less than 0.001), which are not parasitic for humans, than those with low CF (CF less than 20) titer. Human normal immunoglobulin G (human normal IgG) containing 98% or more IgG, prepared from pooled plasma of at least 500 normal human donors, showed ELISA reactions with all mycoplasmal strains used. The nonspecific adsorption of human normal IgG on the surface of plate wells and on medium components which might contaminate mycoplasmal ELISA antigens could be disregarded. These results suggest that cross-reactive antibodies to mycoplasmas exist in human sera, and they affect the results of ELISA for serodiagnosis of M. pneumoniae infection.     

Use of monoclonal antibodies in the assay of hepatitis B core antigen and antibody

Hybridoma cells secreting antibody against hepatitis B core antigen (HBc Ag) were prepared. BALB/c mice were immunized with 0.2 ml of purified HBc Ag, and their spleen cells were fused with mouse myeloma (P3U1) cells by means of polyethylene glycol 1000. Activities of antibodies against HBc Ag (anti-HBc) were tested by the immune adherence hemagglutination (IAHA) and reverse passive hemagglutination inhibition (RPHI) techniques. Hybridoma cells found to contain antibodies accounted for 26.5% by IAHA and 52.1% by RPHI, respectively. Among 32 monoclonal anti-HBc antibodies, 18 were found to be positive by both IAHA and RPHI, and the remaining 14 positive by RPHI only. After cloning, they were injected intraperitoneally into ascitic mice. The highest anti-HBc activity with an IAHA titer of 1:4 X 10(6) and with an RPHI titer of 1:1 X 10(5) was detected in this ascitic fluid. Enzyme immunoassay (EIA) and RPHI with monoclonal antibody containing the highest anti-HBc activity were developed. All the sera in which anti-HBc was detected by IAHA and RPHI with polyclonal antibody were positive in EIA. RPHI titers obtained with monoclonal antibody were in good agreement with usual IAHA and RPHI titers obtained with polyclonal antibody. These results indicate that monoclonal antibody can be used in the HBc Ag and anti-HBc assay system.     

用巨细胞病毒抗原致敏的冻干血球作间接血凝试验

本文报告用巨细胞病毒(CMV)抗原致敏的冻干绵羊红细胞(简称冻干血球)做间接血凝试验。用5％正常兔血清和10％蔗糖磷酸盐缓冲液作保护剂,将巨细胞病毒抗原致敏的绵羊红细胞进行真空冷冻干燥,制成了冻干血球。经反复检测发现,用含百万分之一Tween20、2％正常兔血清的磷酸缓冲液稀释冻干血球,可消除冻干过程中产生的非特异性凝集反应。所建立的方法重复性较好,特异性与ELISA相同,敏感性比CF高。     

Three-Day Assay for Human Cytomegalovirus Applicable to Serum Neutralization Tests

A fluorescing cell assay (FCA) technique utilizing the indirect fluorescent-antibody method to measure human cytomegalovirus (CMV)-infected cells has been applied to the rapid determination of CMV-neutralizing antibody. Human sera with complement fixation titers to CMV of 1/32 or greater and fluorescein-conjugated rabbit anti-human globulin are the primary and secondary reagents in the fluorescent-antibody test. FCA measured in 3 days the same number of infectious units measured by plaque assay in 2 weeks. FCA and plaque assay yielded identical neutralizing antibody titers to CMV in 20 human sera.     

Plague in free-ranging mammals in western North Dakota.

From July through October of 1996, 48 blood samples were collected from coyotes (Canis latrans), badgers (Taxidea taxus), and raccoons (Procyon lotor) in western North Dakota (USA) for the purposes of determining antibody titers to the plague bacterium, Yersniia pestis. The passive hemagglutination paper-strip blood-sampling technique was utilized with hemagglutination inhibition controls. Two positive samples were obtained from McKenzie county, one from a coyote with a titer of 1:64 and one from a badger with a titer of 1:256. Considering coyote and badger population dynamics, this study documents plague in western North Dakota.     

Persistence of antibodies of the IgG class against cytomegaloviruses in blood donors

High titre plasmas gained from blood donors are the initial material used for producing human immunoglobulin containing cytomegalovirus antibodies (CMV-HIG). For this purpose the sera gained from 467 permanent donors at the District Institute for Blood and Transfusion Service in Berlin were investigated on their CMV antibody content of IgG class by means of an indirect immunofluorescence test (IFT). The infection rate of blood donors amounted to 62% (291/467). CMV-IgG titre greater than or equal to 1:40 was determined in 88 sera (18.8%) and greater than or equal to 1:160 in 14 sera (3%). Two CMV-HIG laboratory samples (charges 3113 and 3117) were produced from these plasmas. Particularly immunoglobulin fraction of charge 3117 (No. 3117 N II) revealed excellent antibody titres (IFT 1:640 [CMV-IgG] or 1:40 [CMV-IgM] respectively, neutralisation test 1:32). Checks made with the donors' CMV-IgG titres after repeated application of plasmapheresis resulted in maximal titres changes of two dilution stages in a period of 15 months. Thus, in producing CMV-HIG a sure, tested pool of donors can be resorted to.     

脱落酸人工抗原合成及多克隆抗体制备

目的:为了对植物细胞中的脱落酸(ABA)进行定量和定位分析,研究了脱落酸人工抗原的合成以及多克隆抗体的制备。方法:用重氮化法将ABA分别与牛血清白蛋白(BSA)、卵清白蛋白(OVA)联结,得到ABA的免疫抗原和包被抗原,并采用紫外全波长扫描和SDS-PAGE对合成的抗原进行了鉴定。以经过鉴定的抗原免疫白兔,制备出ABA的多克隆抗体;采用间接酶联免疫法(ELISA)对抗血清进行效价检测,通过离子交换层析法获得纯化的抗体。结果:ABA与BSA的平均偶联比为5.3∶1,抗血清效价为1∶16000。结论:人工抗原和多克隆抗体制备成功,为采用ELISA和免疫胶体金技术(ICG)检测ABA提供了有效工具。     

Recording and interpretation of the results obtained by using the ELISA Sevatest commercial immunoenzyme kit for detecting Toxoplasma gondii antibodies

The results of the repeated tests of 295 serum samples from patients, previously examined by means of enzyme immunoassay screening and traditional serological tests (complement fixation, indirect immunofluorescence, and indirect hemagglutination) and 115 serum samples from healthy donors, studied by enzyme immunoassay techniques with the use of the commercial kits Sevatest ELISA, have been analyzed. The methodological approach permitting the determination of the final titer of the serum under study, taken in a dilution of 1:800, by its optical density has been used. The mean geometric titer for the control group of donors, determined in the enzyme immunoassay, has been 1:800. This fact suggests that titers exceeding 1:1,600 should be considered diagnostically significant.     

流行性出血热免疫球蛋白的研制

本文首次报告采用纯化灭活流行性出血热（ＥＨＦ）Ⅰ型疫苗免疫健康献血员,采集ＥＨＦ抗体高滴度的血浆,用低温乙醇法及盐析法分离纯化三批ＥＨＦ免疫球蛋白。结果表明：（１）采用０,１,３,４（月）或０,１,４,５（月）免疫程序,疫苗剂量１～２ｍｌ（０．１５～０．３０ｍｇ蛋白）,受免献血员血清平均抗体滴度可达１∶４０６（ＥＬＩＳＡ）或１∶１１２（ＲＰＨＩ）。（２）通过生化检定,三批制品的电泳纯度为９７．０５％,９６．８４％,９９．２６％;ＩｇＧ单体和二聚体含量为８９．５５％,９１．３０％和９８．２１％。（３）用空斑抑制中和试验及免疫印染试验证明所纯化的免疫球蛋白具有抗ＥＨＦ病毒特异性。（４）三批ＥＨＦ免疫球蛋白的效价测定,结果为ＥＬＩＳＡ滴度≥１∶５１２,ＲＰＨＩ滴度≥１∶１０２４,ＰＲＮＴ（中和抗体）滴度１∶４０。按１６％蛋白计,ＥＨＦ免疫球蛋白的效价可达原料血浆的１０倍以上。（５）无菌、安全、毒性及热原质试验检定结果,全部通过《中国生物制品规程》要求。     

The role of secretory antibodies in adenovirus infection

The protective role of secretory antibodies in the development of adenovirus infection has been established. More severe and prolonged course of adenovirus infection, accompanied by the involvement of the lower respiratory tract in the process, has been noted in patients with low titers of antibodies, or their absence, in secretions (O to 1:4). The presence of secretory antibodies in a titer of 1:8 considerably alleviated the course of the disease. The antibody titer 1:16 (according to the data of the passive hemagglutination test) has proved to be protective for children and adults in adenovirus infection. The results of the determination of antibodies in the secretions of the upper respiratory tract in the passive hemagglutination test may be used for the purposes of prognostication.     

The immunological diagnosis of peptostreptococcal infection

The work deals with different methods for the diagnosis of anaerobic streptococcal infection, experimentally tested and clinically approved in the examination of children with acute pneumonia. The passive hemagglutination test, the immune rosette formation test and the specific lymphocyte blastogenesis test were used for diagnosis. As sensitin and antigen, the preparation of the peptostreptococcal bacterial mass was used. The diagnostic titer of antibodies to anaerobic streptococci was 1:160. A twofold and greater increase in the number of antigen-dependent rosette-forming lymphocytes and in specific blast transformation in the presence of the diagnostic antibody titer confirms the etiological role of anaerobic peptostreptococci in the development of pneumonia in children.     

A trial application of the latex test for evaluation of antibody level in rabbit immune sera]

A latex test was elaborated which served for evaluation of quality of rabbit immune sera for antigen 0 of selected Gram-negative bacteria. Sensitivity and specificity of this test in comparison with passive hemagglutination and immunoenzymatic DOT-ELISA reactions was evaluated. These studies were performed on immune sera for antigen O of Salmonella groups B, C1, C2, D and E, Yersinia pseudotuberculosis and in antigen preparations for above listed microorganisms both in homologous and heterologous systems. It was found that sensitivity of the latex test is 9 to 160 times lower than that of passive hemagglutination and 7 to 307 lower than for DOT-ELISA. Sensitivity of the latex test and passive hemagglutination reaction was evaluated on the basis of results of cross reaction between studied antigens and unabsorbed rabbit sera, establishing so called sensitivity indexes, which were informing how many times heterologous titer is lower than homologous titer. So evaluated sensitivity of the latex test was close to sensitivity of the passive hemagglutination reaction. It was found that slide latex test is characterized by satisfactory sensitivity and good sensitiveness and may be used for evaluation of antibody level 0 antigens of Salmonella and Yersinia. The value of this test is characterized by high repeatability of results, as well as low work and time-consuming.     

Recombinant type 5 adenoviruses expressing bovine parainfluenza virus type 3 glycoproteins protect Sigmodon hispidus cotton rats from bovine parainfluenza virus type 3 infection.

Cotton rats were used to study the replication and pathogenesis of bovine parainfluenza virus type 3 (bPIV3) and to test the efficacy of the F and HN glycoproteins in modulating infection. In vitro cultures of cotton rat lung cells supported the growth of bPIV3 as shown by virus recovery, immunofluorescence, immunoprecipitation, and syncytium induction. Intranasal (i.n.) inoculation of cotton rats with 10(7) PFU resulted in peak recovery of virus after 2 days (8 x 10(4) PFU/g of lung tissue) and significant bronchiolitis with lymphocyte infiltration 5 to 7 days postinfection. Immunohistochemical staining of lungs and trachea demonstrated that virus antigen-positive cells increased in frequency over the course of infection to a maximum on day 5. Serum antibody responses were evaluated by enzyme-linked immunosorbent assays (ELISA), hemagglutination inhibition (HAI), and serum neutralization (SN). Following a single i.n. inoculation, serum antibody levels were 1/40,960, 1/32, and 1/80, as detected by ELISA, HAI, and SN, respectively. When an intramuscular inoculation of 10(7) PFU was administered 10 days prior to the i.n. inoculation, a secondary response which resulted in an ELISA titer of 1/163,000, an HAI titer of 1/640, and an SN titer of 1/512 was induced. IN inoculation of recombinant adenoviruses type 5 containing the bPIV3 F or HN protein or a combination of the two viruses protected cotton rats from bPIV3 challenge. Protection was evaluated serologically by ELISA, HAI, and SN titers, histopathology, immunohistochemistry, and virus recovery.     

A preclinical study comparing approaches for augmenting the immunogenicity of a heptavalent KLH-conjugate vaccine against epithelial cancers

Previously using a series of monovalent vaccines, we demonstrated that the optimal method for inducing an antibody response against cancer cell-surface antigens is covalent conjugation of the antigens to keyhole limpet hemocyanin (KLH) and the use of a saponin adjuvant. We have prepared a heptavalent-KLH conjugate vaccine containing the seven epithelial cancer antigens GM2, Globo H, Lewis(y), TF(c), Tn(c), STn(c), and glycosylated MUC1. In preparation for testing this vaccine in the clinic, we tested the impact on antibody induction of administering the individual conjugates plus adjuvant compared with a mixture of the seven conjugates plus adjuvant, and of several variables thought to augment immunogenicity. These include approaches for decreasing suppressor cell activity or increasing helper T-lymphocyte activity (low dose cyclophosphamide or anti-CTLA-4 MAb), different saponin adjuvants at various doses (QS-21 and GPI-0100), and different methods of formulation (lyophilization and use of polysorbate 80). We find that: (1). Immunization with the heptavalent-KLH conjugate plus GPI-0100 vaccine induces antibodies against the seven antigens of comparable titer to those induced by the individual-KLH conjugate vaccines, high titers of antibodies against Tn (median ELISA titer IgM/IgG 320/10240), STn (640/5120), TF (320/10240), MUC1 (80/20480), and globo H (640/40); while lower titers of antibodies against Lewis(y)()(160/0) and only occasional antibodies against GM2 are induced. (2). These antibodies reacted with the purified synthetic antigens by ELISA, and with naturally expressed antigens on the cancer cell surface by FACS. (3). None of the approaches for further altering the suppressor cell/helper T-cell balance nor changes to the standard formulation by lyophilization or use of polysorbate 80 had any impact on antibody titers. (4). An optimal dose of saponin adjuvant, QS-21 (50 microg) or GPI-0100 (1000 microg), is required for optimal antibody titers. This heptavalent vaccine is sufficiently optimized for testing in the clinic.     

Immunochemistry of Salmonella O-antigens: preparation of an octasaccharide-bovine serum albumin immunogen representative of Salmonella serogroup B O-antigen and characterization of the antibody response.

The O-antigenic polysaccharide of phenol-water extracted Salmonella typhimurium (O antigens 4, 12) lipopolysaccharide was enzymatically cleaved by phage P22 endorhamnosidase. An octasaccharide with the (formula: see text) structure Gal-Man-Rha-Gal-Man-Rha was isolated and shown to retain the O-antigen 4 specificity of the native polysaccharide. After oxidation of the terminal reducing rhamnose residue to the corresponding aldonic acid, the octasaccharide was covalently linked to bovine serum albumin (OLS-BSA) by use of a water-soluble carbodimide. The resulting conjugate showed O-antigen 4 specificity in enzyme-linked immunosorbent assay (ELISA) ans passive hemagglutination inhibition tests. Immunization of rabbits with the OLS-BSA conjugate gave rise to antibodies directed toward both the octasaccharide and the carrier protein. ELISA titration with synthetic disaccharide-protein conjugates as antigens revealed that the antibody titer against the mannose-rhamnose structure was higher than against the abequose-mannose structure. In rabbits immunized with heat-killed whole bacteria the titers against the two disaccharides were equal. The reason for this difference is not obvious. It is evident, however, that the OLS-BSA conjugate elicited in rabbits O-antibodies with the same specificity as whole bacteria.