The potency determination of human varicella-zoster immunoglobulin by enzyme-linked immunosorbent assay, complement-fixation test and indirect fluorescent antibody tests

Traditionally, plasma for the production of the human varicella-zoster immunoglobulin (VZIG) has been selected on the basis of the complement-fixing antibody (CFA) titre. Since immune individuals may lack CFA to varicella-zoster virus (VZV), non-CFA may be of importance in protection. In a search for a simple and reliable method for potency determination, 24 VZIG preparations were quantified by enzyme-linked immunosorbent assay (ELISA), the complement-fixation test (CFT), the indirect fluorescent antibody test to acetone-fixed (IF) and viable (FAMA) VZV-infected cells, respectively. The antibody titres obtained by the various methods were compared. Arranged in order of decreasing agreement, the correlation coefficients (r) of the regression equations between the variables were 0.62 for CFT and FAMA, 0.50 for CFT and ELISA and 0.26 for CFT and IF in a log2 plot. There was complete agreement between the titres obtained by the commercially available Enzygnost Varicella/Zoster kits (Behring Institute, Marburg, F.R. Germany) and the ELISA microtitre plates produced at our institute (r = 1). The regression equation lines for ELISA/CFT and FAMA/CFT titres tended to be parallel to each other, while the line for IF/CFT titres had a less steep slope. Similar titration curves were obtained for VZIGs fractionated by two different methods. Furthermore, the titration curves of serum pools from varicella and zoster convalescents, respectively, had a similar shape below delta OD = 0.4. Generally, a steeper slope was observed above delta OD = 0.4. As antibody detectable by ELISA seems to correlate with protection and the method is sensitive, specific, reproduceable, simple to carry out and easily automated, it may be suitable for the potency determination of VZIGs.     

Enzyme linked immunosorbent assay (ELISA) for determination of IgG antibodies to human cytomegalovirus

A solid-phase enzyme linked immunosorbent assay (ELISA) for determination of IgG antibodies to cytomegalovirus (CMV) is described. The assay used purified CMV and extracts of CMV infected cells as antigen. Antigens were desiccated onto the bottom surface of polystyrene microcuvettes. The antibodies bound to the antigens were assayed by anti-IgG-alkaline phosphate conjugate followed by addition of the enzyme substrate. Titration curves have been obtained from the sera of 35 blood donors and of 23 patients. Comparison of results obtained by ELISA with those obtained by complement fixation (CF) shows that there is agreement between the tests. Both purified CMV and extracts of CMV infected cells were found to be suitable antigens. Purified CMV was of value particularly in those sera which show high reactivity against control antigen. The ELISA technique described is approximately 412 to 548 times more sensitive than the CF test when purified CMV or extracts of CMV infected cells, respectively, are used as antigens. No significant heterotypic rise to CMV was observed by ELISA in three sets of sera with seroconversion to herpes simplex virus. The ELISA technique gives objective results, is easily performed, and may be adaptable as a routine test both for serological diagnosis of CMV infection and for screening of the general population.     

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.     

Evaluation of the enzyme-linked immunosorbent assay for the serodiagnosis of tularemia

The usefulness of the ELISA using as antigen prepared in our laboratory supernatant obtained after centrifugation of sonicated F. tularensis cell suspension was compared with the tube agglutination test with commercial available antigen. Paired serum specimens obtained from 6 patients with ulceroglandular syndrome of tularemia were tested in both tests. The cut-off limit of serum antibodies was set at mean antibody titre determined in the sera of 115 blood donors exceeded by three standard deviations. Antibodies to F. tularensis in diagnostically significant titre were detected in all 12 serum samples by both tests. However the titres obtained in ELISA were several times higher than in tube agglutination test. In the second serum sample the level of IgA and IgM was lower but the level of IgG higher than in the first sample. We could not observe any difference in the level of antibodies between paired serum specimens in tube agglutination test.     

Attempts to demonstrate specific antibody responses in the vitreous body of the eye

Postmortem serum and vitreous humor specimens obtained from 31 autopsied human bodies were assayed for specific antibody responses to adenoviruses, RS virus and Mycoplasma pneumoniae using the complement-fixation (CF) test and the ELISA procedure (in 23 of the bodies examined). The antibody responses as measured by the CF test were negative in all vitreous body samples tested, with the ELISA five specimens gave a positive reaction at a titre 1 : 40 and one at 1 : 80. These positive antibody titres turned out to invariably coincide with the high-titre antibody levels in the serum. Implicitly, at high-titre levels in the serum these antibodies tend to penetrate in the vitreous body of the eye.     

Jatobal virus antigenic characterization by ELISA and neutralization test using EIA as indicator, on tissue culture

A virus antigenic characterization methodology using an indirect method of antibody detection ELISA with virus-infected cultured cells as antigen and a micro virus neutralisation test using EIA (NT-EIA) as an aid to reading were used for antigenic characterization of Jatobal (BeAn 423380). Jatobal virus was characterized as a Bunyaviridae, Bunyavirus genus, Simbu serogroup virus. ELISA using infected cultured cells as antigen is a sensitive and reliable method for identification of viruses and has many advantages over conventional antibody capture ELISA's and other tests: it eliminates solid phase coating with virus and laborious antigen preparation; it permits screening of large numbers of virus antisera faster and more easily than by CF, HAI, or plaque reduction NT. ELISA and NT using EIA as an aid to reading can be applicable to viruses which do not produce cytopathogenic effect. Both techniques are applicable to identification of viruses which grow in mosquito cells.     

Determination of antibodies to pertussis toxin in working reference preparations of anti-pertussis sera from various national control laboratories.

Working reference preparations of anti-pertussis sera from various National Control Laboratories were assayed for anti-PT antibodies by standardized ELISA and toxin neutralization (Nt) test. Both the ELISA and Nt tests gave highly reproducible results for various preparations when these preparations were assayed repeatedly on different days. Various working reference preparations were assigned units against the proposed International standard for anti-pertussis serum (JNIH-10) assuming its unitage of 250. Assigning unitage to various preparations would help in comparing results of ELISA and Nt tests for anti-PT antibodies reported in various studies.     

Problems posed by the preparation of specific anti-cytomegalovirus immunoglobulins

The authors describe the preparation of a first batch of intravenous cytomegalovirus (CMV) immune globulin at the Nancy Regional blood transfusion centre. Immune plasmas were selected from 3 640 healthy volunteer blood donors on the basis of CF antibody titers to CMV (Kolmer's method modified) of, at least, 1:8; plasmas from approximately 10% of the donors were therefore selected. The 68 liters of pooled immune plasma had à CF antibody titer of 1:16 (CMV antibody titers of 1: 10 000 and 1: 640 when tested in the ELISA assay and passive hemagglutination assay respectively). Intravenous immune globulin was produced from pooled plasma by Cohn fractionation and treatment with pepsin at pH 4; 4.8 liters of immune globulin were prepared and divided in 96 doses of 50 ml each. The final product was found to have a CMV antibody titer of 1: 32 (CF) 1: 50 000 (ELISA) or 1: 2 560 (passive hemagglutination). Recent reports on the preparation of CMV immune globulin are briefly reviewed.     

Use of the indirect hemagglutination reaction for detecting antibodies to Pseudomonas pyocyanea in acute suppurative destructive pneumonia]

No less than 4-fold increase in the antibody titres to Pseudomonas aeruginosa during the infectious process served as laboratory confirmation of its participation in the infectious process. In 49 of 91 patients hemagglutining titre exceeded the diagnostic one (1:640). In 9 patients (chiefly in infants) hemagglutinin titre remained low, but there was a rise of antibody level to Pseudomonas aeruginosa during the disease. High hemagglutinin titres were noted in 12 patients on admission to the clinic, with reduction of the antibody titres at the late periods of the disease. Antibody titres remained unchanged during the disease in 15 patients. In 3 cases the indirect hemagglutination test was assesed as negative. In the rest of the patients hemagglutinin titres varied within the range of the diagnostic titre. Thus, the indirect hemagglutination test with erythrocytic diagnostic agent permitting to determine antibodies to Pseudomonas aeruginosa could be used at the clinic in combination with bacteriological and other investigations for establishing etiology of the destructive process.     

Indirect microhemagglutination test for varicella--zoster antibody determination.

An indirect microhemagglutination assay (IHA) was devised because of a need to provide an alternative test to complement fixation (CF) for varicella-zoster (V-Z) antibody determination. Human erythrocytes were sequentially treated with 2% glutaraldehyde, 0.04% tannic acid, and 2% pyruvic aldehyde then exposed to sonicated V-Z infected cells. This same tanning procedure was suitable for herpes simplex and Epstein-Barr virus antigen attachment but unsatisfactory for several non-herpes-group viruses. V-Z antibody titres determined by IHA were generally 2 to 6 times higher than CF titres. Cross-reaction with herpes simplex antibody was minimal.     

Specific antibodies in children excreting cytomegalovirus

Acute-and convalescent-phase sera from 22 children were examined by ELISA in comparison with a routine complement fixation (CF) test for detection of anti-CMV antibodies. All these subjects were excreting CMV from urine and/or saliva. The results showed that ELISA is more sensitive than CF test. Particularly ten children showed, by ELISA, anti-CMV antibody titers more agreeing with clinical-virological features. Generally, in other subjects the results of the two serological tests were similar. Three cases showed discordances both between the two methods and between serological data and clinical virological findings.     

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.     

Evaluation of the sensitivity of selected serological tests and activity of Coxiella burnetii antigens in the diagnosis of Q fever]

Sensitivity of three serological tests: indirect immunofluorescence assay (If), complement fixation test (CF), and microagglutination test (MA) was evaluated. Sera (118 samples) of humans suspected of C. burnetii infection were tested. Phase II antibodies were detected in 68.6% of sera and phase I antibodies--in 38.2% of sera. Among seropositive to phase II antigen--93.8% of sera reacted in IF, 62.9% in MA, and 32.1% in CF; among seropositive to phase I antigens--100% of samples reacted in IF, 2.6% in MA and 2.6% in CF. Calculated sensitivity of above tests was as followed: IF-93.8%, MA-67.1%, CF-34.2%. Some human sera (6.1%) reacted with hen egg antigens in CF. Reactivity of diagnostic antigens prepared from reference Henzerling strain and four others isolated in Poland with rabbit immune sera and sera of individuals suspected of C. burnetii infection in IF was compared. Generally, the immune sera reacted in highest titres with homologous antigens derived from homologous strains. Human sera showed differentiated activity to particular antigens. The titres of phase I antibodies fluctuated from 0 to 16 depending on the antigen applied. Because of that fact diagnostic antigens should be prepared from the mixture of reference strains and isolates from a region under study.     

The effect on kaolin adsorption of serum on the virus neutralization and enzyme-linked immunosorbent assays of antibody to bovine herpesvirus 1

Two procedures have been used for measuring antibody titres to bovine herpes virus 1 (BHV1): the serum neutralization (SN) test and enzyme-linked immunosorbent assay (ELISA). One hundred and thirty-two sera selected for their low SN titres were tested both unadsorbed and after adsorption with kaolin to determine the effect of kaolin on the titres. With ELISA, the titres of unadsorbed and kaolin adsorbed were not significantly different but with the SN test many treated sera, originally with weak positive titres, became negative after kaolin adsorption. Thus, if the ELISA results are specific for BHV1 antibody then the SN test findings suggest that treatment of sera with kaolin, rather than removing a viral inhibitor, removes a substance from the serum which potentiates SN antibody. This in turn indicates that low SN titres (reciprocal of titre less than or equal to 4, for instance) are probably specific for BHV1 SN antibody whether or not they are abolished by kaolin treatment of the serum.     

Experimental dicrocoeliasis: the humoral immune response of golden hamsters and rabbits to primary infection with Dicrocoelium dendriticum

The results presented deal with the humoral immune response of golden hamsters to primary experimental infection with D. dendriticum. The development of serum antibodies has been comparatively investigated with three hamster groups (n = 43) harbouring different burdens of adult flukes. The mean numbers of parasites were 11, 30, or 130 per animal. Serum antibody response was studied during an observation period of at least 331 and up to 496 days postinfection. For antibody detection the sensitivities of precipitation test (PTs) (double diffusion test, immuno- and counterimmunoelectrophoresis), of the indirect haemagglutination test (IHAT), the complement fixation test (CFT), and the enzyme linked immuno sorbent assay (ELISA) were compared using aqueous crude fluke antigen and crude egg antigen. CFT and ELISA were most sensitive for the early detection of initial response. Thereafter all the tests employed revealed increasing antibody titres, which in general remained at constant levels and persisted until the end of the observation period with the exception of CF-antibodies. In general fluke antigen was found to be more sensitive than egg antigen. However, in CFT this antigen occasionally has been associated with unspecific inhibition of haemolysis. Comparison of the results shows that ELISA using crude fluke antigen gave the most realistic picture of the actual fluke burden. Also preliminary results on the precipitin response of rabbits (n = 3) after primary experimental exposure to different numbers of metacercariae (500, 1,000, and 3,000 per animal respectively) are reported. Employing the above mentioned PTs a persisting antibody response could be demonstrated only after exposure to at least 3,000 infective larvae. The initial response was found on day 63, the observation period was 550 days.     

Serum Auto Antibodies and Clinical/Pathological Features in German Shepherd Dogs with a Lupuslike Syndrome

This study presents 8 dogs of German Shepherd breed (6 males, 2 females, 2–5 years of age at onset of the disease) with a lupus like syndrome characterized by febrile polyarthritis, wasting, nephropathy, cutaneous lesions and high positive titres of ANA (antinuclear antibodies) of speckled type. The serum autoantibodies were further characterized by double immunodiffusion against ENA (extractable nuclear antigen), ELISA for Histone antibodies (Histon fraction H-24A and H-3S), indirect IF on rat-liver sections, non treated and RNase/DNase digested sections for DNP/RNP antibodies, and smears of a nemoflagellate C. luciliae for antibodies vs doubbel strained DNA, (dsDNA). Thus, the high ANA titres in these dogs represent varying types of autoantibodies against nucleoproteins of both DNA and RNA nature, associated histone antigens and non-histone antibodies (RNA and Sm) as well. Rheumatoid Factor titres in serum from these dogs were low or negative. Immunoglobulin deposits at dermo-epidermal junctions were demonstrated in some of the dogs with hyperkeratotic skin lesions. High concentration of serum-IgG was a constant finding in combination with anemia and in most cases leukopenia probably related to the chronic inflammatory process in these animals. Autoimmune hemolytic anemia (AIHA) or thrombocytopenia was not detected in these dogs.     

Persistence of influenza serum antibodies in humans following immunization with a bivalent A/Victoria and A/New Jersey vaccine.

The persistence of serum antibodies 1 year after immunization with a bivalent vaccine containing recombinant viruses that were antigenically identical with A/Victoria/3/75 (H3N2) and A/New Jersey/8/76 (Hsw1N1) viruses was measured in 128 persons aged 18 to 65 years. Serum samples were tested with the hemagglutination inhibition assay against the two vaccine antigens and against A/Texas/1/77 (H3N2) and A/USSR/90/77 (H1N1) viruses. Prior to vaccination 56% and 79% of the participants had been found to be seronegative to A/Victoria and A/New Jersey antigens respectively; the geometric mean antibody titres were low (1:5 to 1:11) except in persons aged 51 to 65 years, whose mean titre of antibody to the A/New Jersey antigen was 1:23, and persons aged 26 to 35 years, whose mean titre of antibody to the A/USSR antigen was 1:25. By 3 weeks after vaccination 85% of the seronegative persons had a fourfold or greater rise in titres of antibodies to the viruses in the vaccine, and 70% had a fourfold increase in titre of antibody to the A/Texas antigen. Of the persons aged 26 to 35 years (seronegative and seropositive) 68% had a fourfold or greater increase in titre of antibody to the A/USSR antigen. There was no change in the mean titres of 19 unvaccinated control subjects during the observation period. At 6 and 12 months after vaccination the titres of antibodies to the A/Victoria and A/New Jersey antigens had declined moderately in all age groups from those observed 3 weeks after vaccination. The rate of decline was similar for the various antibodies except that to the A/USSR antigen in persons 26 to 35 years of age, in whom the decline was much slower.     

The serodiagnosis of human hydatid disease: 1. The routine use of latex-agglutination and complement-fixation in diagnosis

The combined use of complement fixation (CF) and latex agglutination (LA) tests is reported on sera from 6328 patients with suspected hydatid disease; 191 were confirmed positive at operation ('known positives'). Results by LA are related to CF titres. Both tests were negative in 90% of specimens. Nine patients were subsequently found infected of whom 3 became positive in tests after operation. Of sera positive in both tests, 75% were from 'known positives'. The remainder were almost certainly from infected patients. Half the patients whose sera were LA positive/CF less than or equal to 1/4 were follow-up 'known positives' in whom CF titres had waned; 2 were early infections. Only 3% of the cases with an LA negative/CF titre of greater than or equal to 1/16 were 'known positives' and 6% where the CF titre was 1/8. The remaining CF results in the group were false positives and accounted for 1.2% of all sera tested. Findings show that a CF titre greater than or equal to 1/8 with positive LA indicates past or present infection; a negative CF test with positive LA usually indicates past infection; rarely, infection is present when a CF titre is greater than or equal to 1/8 and LA is negative. A rising CF titre and positive LA indicates current infection; reliable prognosis following treatment is given by CF.     

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。