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.     

The respective advantages of complement fixation and ELISA for routine diagnosis of cytomegalovirus infection

The results of CF and ELISA tests for cytomegalovirus performed on 270 sera of hospitalized patients show a positive correlation. As a general rule, ELISA is more sensitive than CF, except for a few sera collected from patients with immunological disorders. When two sequential sera are available, the CF remains a reliable and inexpensive method. But when only one serum can be obtained, the probability of an active CMV infection can be estimated on the IgM/IgG ratio. In 26% of the patients, this ratio was greater than or equal to 1. The ELISA is twice as expensive as the CF test. To reduce its cost, a simple method for preparing ELISA antigen from commercially-obtained CF antigens is described.     

Monoclonal antibodies to parasite antigens found in the serum of Dirofilaria immitis-infected dogs

We recently reported that parasite antigens are detectable in the serum of Dirofilaria immitis-infected dogs by counterimmunoelectrophoresis (CIE). Hybridoma cell lines that produce monoclonal antibodies specific for these antigens were obtained by immunizing mice with a partially purified antigen preparation, fusing spleen cells with SP-2 myeloma cells, and screening cell culture supernatants for antibody by ELISA and CIE inhibition. Antibodies specific for two epitopes shared by the two major circulating parasite antigens were identified. Immunoperoxidase studies showed that the epitopes recognized by the monoclonals were widely distributed in D. immitis, but the female uterus and eggs were particularly strongly labeled. A monoclonal antibody-based ELISA was developed to measure parasite antigens in dog sera. Parasite antigens were detected in 45 of 46 sera from infected dogs but were absent in sera from uninfected dogs and sera from dogs infected with Dipetalonema reconditum. Serum antigen content was significantly correlated with the number of female worms recovered from infected dogs (r = 0.82, p less than 0.001). Antigenemia was first detected 6 mo after infection, and antigen levels remained fairly stable between 9 and 21 mo after infection. Parasite antigen detection with this monoclonal antibody-based ELISA appears to be superior to previously described diagnostic methods for canine dirofilariasis in terms of sensitivity, specificity, and relation to infection intensity.     

Isolation, purification and characterization of surface antigens of the bovine filarial parasite Setaria digitata for the immunodiagnosis of bancroftian filariasis

The surface antigens of the bovine filarial parasite Setaria digitata were isolated by EDTA extraction and purified by affinity chromatography using sepharose bound human filarial (Wuchereria bancrofti) antibodies obtained from chronic human filarial sera. The purified and crude antigens were used in enzyme-linked immunosorbent assay (ELISA) for the detection of serum antibodies in bancroftian filariasis. The purified antigen showed sensitive and specific reactions in ELISA for the detection of antibodies in filarial sera and showed least cross reactivity with other parasitic infections. The crude and purified antigens showed about 18 and 6 peptide bands respectively in SDS-PAGE and about 11 and 6 antigenic bands respectively in enzyme-linked immunoelectrotransfer blot (EITB). The purified antigen was observed to be glycoprotein in nature. It was possible to identify the stage-specific infection in human filariasis by using the crude and purified antigens in EITB.     

Evaluation of purified H and M antigens of histoplasmin as reagents in the complement fixation test.

Complement-fixation (CF) tests were performed with purified H and M antigens, histoplasmin, and Histoplasma capsulatum whole cell yeast phase antigen using sera of 126 patients with proven or suspected histoplasmosis. Specific titers for either H or for M antibody were obtained with the individual purified antigens; the highest titers were comparable to those obtained with histoplasmin. However, in sera containing only anti-M antibody, the titers obtained with the purified M antigen were 2 to 16 times those obtained with the histoplasmin or yeast phase antigens. The CF test for either H or M antibody was 4 to 32 times as reactive as the agar-gel microimmunodiffusion test; in general precipitin lines were obtained with either H or M antigens from sera with CF titers greater than or equal to 8. With sera containing H antibody, there was an excellent correlation between the CF titers obtained with purified M antigen and histoplasmin. The correlations of CF titers with H antigen and either histoplasmin or yeast phase antigen were very low.     

Rapid antibody quantification and generation of whole proteome antibody response profiles using LIPS (luciferase immunoprecipitation systems)

The application of LIPS to the rapid quantification of antibody responses to infectious agents is described. Chimeric genes encoding pathogen antigens fused to Renilla luciferase are expressed in mammalian cells; crude extracts are prepared and, without purification, employed in immunoprecipitation assays to quantify pathogen-specific antibodies. In cross-sectional and longitudinal studies, antibody levels to the MSG-14 antigen of Pneumocystis jirovecii measured by this assay correlated well with levels previously obtained with an optimized ELISA. We also correctly predicted Hepatitis B (HBV), Hepatitis C (HCV), and HIV infection status in all but 2 of 99 assays analyzing 33 patient sera. We then used 15 HIV-encoded proteins comprising the whole HIV proteome to generate antibody response profiles for these 33 sera. Each HIV antigen was recognized by antibodies in serum from at least one HIV-infected individual. Data generated with these simple, quantitative antibody-detection assays have both clinical and research applications.     

Antibodies to nontypeable Haemophilus influenzae in blood donors. A comparison between different assay methods

Abstract Antibodies to nontypeable Haemophilus influenzae were determined in sera from healthy blood donors with complement fixation (CF) test, diffusion-in-gel enzyme-linked immunosorbent assay (DIG-ELISA), and enzyme-linked immunosorbent assay (ELISA). In most sera, antibodies to H. influenzae could be detected with all three methods, but DIG-ELISA as well as ELISA demonstrated a greater sensitivity than the CF test. Furthermore, the ELISA methods allowed analysis of separate immunoglobulin classes, which is of great advantage when sera from infected patients are to be analysed.     

Diagnosis of murine infections in relation to test methods employed

Comparative investigations of Sendai virus, pneumonia virus of mice (PVM), mouse encephalomyelitis virus (mouse polio), minute virus of mice (MVM), and reovirus type 3 (Reo 3) infected murine colonies revealed a 30% higher incidence of positive sera when enzyme-linked immunosorbent assay (ELISA) was employed instead of hemagglutination inhibition (HI) tests. Equivalent sensitivity as in the ELISA was obtained when the same sera were investigated by indirect immunofluorescence (IF) tests. The virus purification techniques described resulted in highly suitable antigens for all indirect ELISA established. Since IIF requires no purified antigens, this test is recommended as an alternative to ELISA as well as to HI and complement fixation (CF) tests for laboratories lacking the necessary equipment for high speed centrifugation. A high incidence of false positive HI reactions was found particularly in Reo 3 routine serology. An updated survey of seromonitoring showed that European murine colonies appeared to be infected far less with Reo 3 if ELISA or IIF tests were employed. During 1982-1984, only 13% of the mouse colonies screened possessed Reo 3 positive sera whereas no natural Reo 3 infection was found in rat colonies. Mouse hepatitis virus (MHV) and the coronaviruses of rats exhibited the highest incidence in murine colonies. A total of 60% of mouse and 41% of rat colonies were found to be infected by these viruses. In comparison with earlier serological surveys, the relative incidence of other murine infections was similar. Antibodies against Bacillus piliformis (Tyzzer's disease) were detected by the IIF test in 41% of the rat colonies screened.     

基因重组戊肝病毒多价复合抗原的表达及初步应用

目的：利用基因工程技术体外表达高效HEV多价复合抗原,建立一套敏感和特异的抗HEV检测体系,方法：将前期构建的基因重组戊肝病毒多价复合抗原表达质粒转化大肠埃希菌JM109。筛选鉴定阳性克隆并进行诱导表达,表达产物经分子筛层析纯化后,利用western blot作抗原特异性鉴定,以此抗原建立ELISA检测卫生部生物药品检定所HEV血清参比品及临床血清标本,同时以Genelab公司抗HEVELISA试剂盒作为对照进行对比研究。结果：重组质粒转化大肠埃希菌JM109株后的阳性克隆,经诱导在SDS-PAGE中出现一条分子量大小约为31.5kD的蛋白条带,该蛋白纯化后经Western Blotting检测证实为HEV特异性抗原,以此抗原包被酶联板建立ELISA检测53份国家卫生部标准HEV参比血清,灵敏度达100%（38/38）,特异度达93.3%(14/15)。用此方法检测68份临床标本,并与Gene4labELISA试剂盒结果比较,两者阴阳性相符的有64份,总符合率为94.1%。结论：本实验克隆表达的基因重组戊肝病毒多价复合抗原具有良好抗原性,以此抗原建立的抗HEVELISA具有灵敏度高,特异性强的特点,可望为戊型肝炎的血清学诊断和流行病学调查提供一种更为有效的检测手段。     

Immunodiagnosis of nocardiosis.

A specific immunodominant 54-kDa antigen was purified from a culture filtrate of Nocardia asteroides by immunoaffinity chromatography. The chromatography column was prepared with immunoglobulin G obtained from sera from patients with lepromatous leprosy. Unbound solutes consisted of specific, partially purified N. asteroides antigens, primarily a 54-kDa band, accompanied by two others of 31 and 62 kDa. The Western blot (immunoblot) technique was applied to detecting the immunologic response to nocardiae in the serum of nocardiosis patients. Each of the serum samples from immunosuppressed or immunocompetent patients infected with N. asteroides reacted with the 54-kDa band, and two reacted with the 31- and 62-kDa bands. There was no reaction to either the 54- or the 31-kDa antigen with all serum samples obtained from patients with tuberculosis, except for one, with all serum samples obtained from patients with leprosy, or with all sera obtained from healthy controls. The partially purified 54-kDa antigen, specific for N. asteroides, was used as the immunogen to generate monoclonal antibodies (mAbs) and two mAbs were selected. As determined by Western blot, both mAbs reacted with the 54-kDa band. Using indirect immunofluorescence or enzyme immunoassay with whole N. asteroides micro-organisms, the mAbs did not react with N. asteroides cells. No cross-reactivity with mycobacterial antigens, either culture-filtrate antigens or tuberculin, was exhibited with any of the two mAbs. These mAbs are candidates to be used for the development of a sensitive and specific diagnostic test for nocardiosis.     

Evaluation of a monoclonal antibody affinity purified antigen for zymodeme specific serological diagnosis of Trypanosoma cruzi infection

Theoretically, serological assays with affinity purified marker antigens can allow strain-specific diagnosis even when parasites cannot be retrieved from an infected host. A Trypanosoma cruzi antigen was purified by affinity chromatography using a zymodeme (Z) 2 specific monoclonal antibody (2E2C11). An indirect enzyme-linked immunosorbent assay (ELISA) based on the purified antigen could discriminate between sera from rabbits immunized with T. cruzi zymodeme clones but could not discriminate between sera from mice infected with different zymodemes.     

Trichinella spiralis: recognition of muscle larva antigens during experimental infection of swine and its potential use in diagnosis

Longitudinal studies with Trichinella spiralis experimentally infected pigs were carried out to identify muscle larva antigens recognized during infection. This was approached using Western blot analysis and ELISA assays. Immunoblots of sera from experimentally infected pigs using total parasite extracts revealed five principal parasite antigens throughout infection. A similar pattern of antigen recognition was given by sera from backyard pigs in areas of Mexico, some of them endemic for Trichinella. Four of the five antigens recognized (MW 47, 52, 67, and 72 kDa) corresponded to surface/stichosomal antigens purified by monoclonal antibody NIM-M1. In addition, Western blots of excretions-secretions of muscle larva contained three (MW 52, 67, and 72 kDa) of the four surface/stichosomal components recognized by NIM-M1. Affinity-purified surface/stichosomal components, total soluble extracts, and excretory-secretory antigens of muscle larva were then evaluated in ELISA for detection of T. spiralis infections in experimentally infected, noninfected control, and 295 backyard pigs. These assays showed that purified surface/stichosomal components and excretory-secretory antigens increased the specificity of ELISA. These results suggest that muscle larva components purified by monoclonal antibody NIM-M1 are the major antigens recognized during infection of pigs with T. spiralis and therefore potentially useful for diagnosis of swine trichinellosis.     

Detection of the antibody to lymphocytic choriomeningitis virus in sera of laboratory rodents infected with viruses of laboratory and newly isolated strains by ELISA using purified recombinant nucleoprotein

An enzyme-linked immunosorbent assay (ELISA) was developed to detect the antibody against lymphocytic choriomeningitis virus (LCMV) in sera of laboratory animals. In this ELISA system, LCMV-nucleoprotein (NP) expressed by recombinant baculovirus and purified with high molar urea was used as the antigen. Sera from laboratory animals experimentally infected with the Armstrong strain or the newly isolated M1 strain of LCMV were examined to detect anti-LCMV antibody by the ELISA system, and the reactivity was compared with that of IFA test. Regardless of LCMV strain, all the sera of adult mice infected with LCMV were positive with very high optical density (OD). Also, the sera from mice neonatally infected with LCMV M1 strain were positive with slightly lower OD than adult mice. In contrast, all the sera of uninfected mice were negative to LCMV-NP antigen. Similarly, anti-LCMV antibodies were detected in all the sera of hamsters, mastomyses, and gerbils infected with the LCMV Armstrong strain. The results of the ELISA were in complete agreement with those of IFA, and indicate the high sensitivity and specificity of the ELISA system in the detection of anti-LCMV antibody. Because this ELISA system does not require handling infectious LCMV in the course of the antigen preparation and serological assay, there is no risk of contamination in the laboratory or nearby animal facility. In addition, by using negative control antigen in parallel with positive antigen in ELISA, we can exactly check the LCMV contamination in laboratory animals.     

Autodisplay of 60-kDa Ro/SS-A antigen and development of a surface display enzyme-linked immunosorbent assay for systemic lupus erythematosus patient sera screening

Enzyme-linked immunosorbent assay (ELISA) is a common tool to test human sera on an antibody reaction against a specific antigen. The 60-kDa Ro/SS-A antigen for autoantibodies can be found in sera from systemic lupus erythematosus (SLE) patients. As in the case of 60-kDa Ro/SS-A, antigens used in ELISAs are recombinantly expressed in Escherichia coli and time-consuming purification steps are needed to get the proteins. To avoid these disadvantages, 60-kDa Ro/SS-A was expressed on the surface of E. coli using autodisplay, an efficient surface display system. Cells displaying 60-kDa Ro/SS-A on the surface were applied as an antigen source instead of the purified antigen. In total, 39 patients and 30 control sera were screened on a 60-kDa Ro/SS-A antibody reaction. To eliminate antibodies against native E. coli, human sera were preabsorbed with E. coli cells prior to the assay. The new ELISA protocol (surface display ELISA [SD-ELISA]) using E. coli with autodisplayed 60-kDa Ro/SS-A showed a sensitivity of 86.67% and a specificity of 83.33% by a cutoff value of 0.28. Our results show that autodisplay provides simple, rapid, and cheap access to human antigens for an ELISA to screen human sera against specific antibody reactions.     

Development of an indirect sandwich ELISA for detection of urinary antigen,using Legionella pneumophila PAL protein

Legionella pneumophila peptidoglycan-associated lipoprotein (PAL) protein is an extremely conserved antigen among Legionella species. In this study, rabbit and rat anti-PAL immunoglobulin G antibodies were produced by immunization with purified, recombinant PAL (r-PAL) protein of L. pneumophila serogroup 1 and used as capture and detection antibodies in the PAL antigen-based enzyme-linked immunosorbent assay (ELISA) to detect urinary PAL antigen. Urine samples were obtained from rats experimentally infected with L. pneumophila serogroup 1. The PAL antigen was measured in urine samples of 40 infected and 40 uninfected rats. After choosing the cut-off value of 0.192, the sensitivity and specificity of the PAL antigen-based ELISA were 87.5 and 97.5 %, respectively. The results obtained by PAL antigen base ELISA were compared with those obtained by Biotest. The PAL antigen was detected efficiently by both of the assays and all of the control human urine samples were negative by the ELISA test. The PAL antigen-based ELISA assay was relatively simple to perform, precise, highly sensitive and specific, and reproducible. Based on our data the PAL antigen-based ELISA described here is the first indirect sandwich ELISA for urinary antigen detection which could easily be applied for diagnosis of Legionnaires disease.     

<Emphasis Type="Italic">Blastomyces Dermatitidis</Emphasis> Antigen Detection in Urine Specimens from Dogs with Blastomycosis Using a Competitive Binding Inhibition ELISA

A competitive binding inhibition enzyme linked immunosorbent assay (ELISA) was used to detect Blastomyces dermatitidis antigens in urine specimens from dogs with blastomycosis. Sera from rabbits immunized with B. dermatitidis killed whole yeast cells were used as the primary antibody in the competitive ELISA. This initial study was performed to determine if B. dermatitidis antigen detection was possible and to test the efficacy of the rabbit sera as a primary antibody. An indirect ELISA was also performed to compare antigen detection in urine to antibody detection in the sera of the infected dogs. The results indicate 100% (36/36 specimens) detection of both antigen and antibody. Cross reactivity with Histoplasma capsulatum, as well as non-specific binding with the normal urine specimens, was observed with the competitive binding inhibition ELISA.     

Serological Investigation of Corynebacterium Pseudotuberculosis Infection in Sheep–Correlation between the Hemolysis Inhibition Test and the ELISA Test

Corynebacterium pseudotuberculosis causes caseous lymphadenitis in sheep and goats. The animals may be infected without showing clinical symptoms. Several serotests have therefore been employed to detect infected animals. Shen et al (1982) performed an enzyme linked immunosorbent assay (ELISA) to detect antibodies against the organism using cell wall antigens. Maki et al (1985) found that the toxin of the bacterium was a better antigen for assessing infection in the ELISA test. They reported that the antitoxin ELISA appeared to be as sensitive as the antihemolysin inhibition test (Zaki 1968).     

The serodiagnosis of human hydatid disease: 2. Additional studies on selected sera using indirect haemagglutination (IHA), enzyme linked immunosorbent assay (ELISA) and defined antigen substrate spheres (DASS).

Sixty-one serum samples selected on the basis of reactivity in the complement fixation (CF) and latex agglutination (LA) test, were further examined for sensitivity and specificity by indirect haemagglutination (IHA), enzyme linked immunosorbent assay (ELISA) and defined antigen substrate spheres (DASS). Twenty sera from healthy Europeans and 48 samples from patients with either schistosomiasis or trichinosis were also tested. Comparable levels of sensitivity were found between the CF and LA positive sera and IHA, ELISA and DASS. Of the CF positive LA negative group of sera, many were positive by DASS but only a few reacted in IHA and ELISA. Some cross reactivity was also observed in the schistosomiasis sera tested by IHA and ELISA.     

Early diagnosis of Schistosoma mansoni in mice using assays directed against cercarial antigens isolated by hydrophobic chromatography

Two diagnostic assays are described for the early diagnosis of acute schistosomiasis, using a defined cercarial antigen preparation obtained by hydrophobic chromatography. Circulating IgM antibodies against this antigen fraction could be detected by ELISA as early as 1 wk after exposure in experimentally infected mice; IgM levels against other antigens and IgG levels against all the preparations examined were not significantly elevated until approximately 4-5 wk postinfection. Circulating antigen was detected as early as 3 days after exposure by a competitive inhibition ELISA using rabbit serum prepared against the cercarial antigen; antigen levels in the serum of mice with a 100-worm infection were found to exceed 100 ng/ml. Studies using sera from infected humans indicate that the assay can also recognize chronic S. mansoni, S. haematobium or S. japonicum infections. In a very limited field study, the specificity of the circulating antigen assay with regard to other helminthic infections was found to be 85%; sensitivity 100%. Preliminary characterization of the relevant antigen indicates that it is a relatively hydrophobic polypeptide with a molecular weight of approximately 41,000 daltons. The implications of these findings with regard to the treatment of travelers or the conduct of seroepidemiological studies in endemic areas are discussed.