Influenza A Neuraminidase Antibody Assay with Sensitized Erythrocytes

Erythrocytes sensitized with purified neuraminidase (Hong Kong) antigens were used for assay of influenza A neuraminidase antibodies. The neuraminidase indirect hemagglutination test was equal to the neuraminidase hemagglutination-inhibition (enhancement) test and appeared to be better than the neuraminidase inhibition test for detection of fourfold or greater antibody rises in paired sera from influenza patients or vaccinees. It was better than both tests for detection of neuraminidase antibody. The neuraminidase indirect hemagglutination test is simple to perform and has the advantage of direct antigen-antibody assay.     

The characteristics of the antibody response of animals immunized with components of the surface structures of Francisella tularensis

Antibody formation in animals immunized with one of the components of F. tularensis surface structures was studied. The time course of antibody formation in 20 hamadryas baboons was studied in the passive hemagglutination (PHA) test, microagglutination (MA) test, and indirect enzyme immunoassay, used for the determination of IgG, IgA and IgM antibodies. The character of antibody response in the animals immunized with components of F. tularensis surface structures (S-complex) and with live tularemia vaccine was compared. The study revealed that immunization with the S-complex induced the formation of antibodies detected by all three methods. Antibody formation to the S-complex was found to be dose-dependent. With the increase of the injected dose of the S-complex, antibody titers determined in the PHA test decreased and those determined in the MA test increased, which was seemingly due to the induction of antibodies differing in their isotypes. After immunization with the S-complex the levels of IgG antibodies were lower and the levels of IgM antibodies by day 28 after immunization higher than after the injection of live tularemia vaccine.     

Erythrocyte diagnostic kits for detection of Pseudomonas aeruginosa exotoxin A

The use of formulated chick red blood cells loaded with IgG preparations and affinity-purified antibodies, in comparison with initial immune serum to P. aeruginosa exotoxin A (ETA), has been shown to increase the sensitivity of antibody erythrocyte diagnosticum (AbED) 17-fold and to ensure the detection of ETA at a concentration of 1.2 mg of protein per ml. The passive hemagglutination (PHA) test with AbED has proved to be a more sensitive method for the detection of ETA than the antibody neutralization test with the use of antigenic erythrocyte diagnosticum, the latex agglutination test, the coagglutination test and the enzyme immunoassay. The PHA test has permitted the detection of ETA in the culture fluid of 80% of P. aeruginosa cultures under study.     

A comparative evaluation of the sensitivity of different methods for the serological diagnosis of leptospirosis

In this work the comparative evaluation of the sensitivity and serological specificity of the microcapsular agglutination (MCA) test, the passive hemagglutination (PHA) test and the microagglutination (MA) test are presented. In the MCA test leptospiral antigens, adsorbed on synthetic carrier capsules produced by Japan Lyophilization Laboratory, were used and the PHA test was made with the use of polyvalent erythrocyte diagnosticum. The study of blood serum samples from 46 leptospirosis patients revealed that the values of antibody titers in the PHA and MCA tests were 5.5-8.1 times higher than the traditional MA test. In the MCA and PHA tests antileptospiral antibodies could be detected as early as on days 1-3 of the disease when the results of the MA test were negative or very low. The maximum values of antibody titers in the MCA and PHA tests were detected on days 11-15 of the disease and in the MA test, on days 21-25. The MCA and PHA tests are genus-specific and permit the detection of antileptospiral antibodies irrespective of the serogroup of the infective agent. In the study of the blood sera of 40 patients with diseases of nonleptospiral etiology the MCA and MA tests yielded false positive results in 7.5% and the PHA test, in 12.5% of cases in titers below the diagnostic level. These data are indicative of high sensitivity and specificity of the serological tests used in this study.     

Detection of postvaccination mumps virus antibody by neutralization test, enzyme-linked immunosorbent assay and sensitive hemagglutination inhibition test

A group of 251 children aged 2-3 years given live attenuated mumps virus vaccine PAVIVAC of Czechoslovak production were tested for antiparotitis antibody levels in pre- and postvaccination sera by neutralization test (NT), enzyme-linked immunosorbent assay (ELISA) and sensitive hemagglutination inhibition test, enhanced by heterologous antibody to human immunoglobulin G (E-HIT). The prevaccination findings were as follows: positive ELISA IgG titres, neutralization antibodies and hemagglutination inhibition antibodies were present in, respectively, 35%, 25.9% and 27.9% of the sera. Postvaccination seroconversions were evaluated in 159 susceptible vaccinees whose prevaccination sera had been negative by all three tests. The lowest seroconversion was detected by NT (74.2%), seroconversions by ELISA and E-HIT were appreciably higher (82.4% and 86.8%, respectively). The seven children showing a seroconversion by E-HIT but not by ELISA had a 4 fold increase of anti-mumps ELISA IgG antibodies as well, but the rise of antibody titres was at a level falling in the range below the positivity criterion for ELISA. The statistically evaluated detection rate for antibodies was significantly higher (significance test "t") by ELISA as compared with neutralization test. However, antibody levels (geometric mean titres) were 8-10 times lower in postvaccination sera than in convalescent sera of 30 children with mumps in all three tests.     

Passive Hemagglutination Assays for the Detection of Antibodies to Herpes Viruses

A simple and effective method for the detection of antibodies to herpes simplex virus (HSV), human cytomegalovirus (HCMV) and varicella-zoster virus (VZV), has been established using the passive hemagglutination assay (PHA) in combination with viral specific glycoproteins. The results obtained with the PHA were compared with those from neutralization (NT) and complement fixation (CF) tests. The PHA test for each of the herpes viruses appears to compare favorably with the other assays tested. The specificity and sensitivity of HSV PHA to NT were 100%, whereas the specificity and sensitivity of HSV CF test to NT were 98% and 100%, respectively. For HCMV, the specificity and sensitivity of PHA to NT and PHA to CF were 100%. Similarly, the specificity and sensitivity of VZV PHA to NT were 100%. Because of the low sensitivity of the VZV CF, the sensitivity of CF to NT was 83%. Furthermore, the range of antibody titers and their absolute levels obtained in the PHAs were significantly greater than those in the NT and CF tests.     

Assessment of antibody responses to antigens of Mycobacterium tuberculosis and Cysticercus cellulosae in cerebrospinal fluid of chronic meningitis patients for definitive diagnosis as TBM/NCC by passive hemagglutination and immunoblot assays

Tanned sheep erythrocytes stabilized with pyruvic aldehyde and glutaraldehyde, called double-aldehyde-stabilized cells, were used to standardize passive hemagglutination assay (PHA) for detection of antibody responses to sonicate extract of Mycobacterium tuberculosis and Cysticercus cellulosae soluble antigens. PHA was performed in the following groups of cerebrospinal fluid (CSF) samples: group I - chronic infections of the central nervous system with the possible diagnosis of tuberculous meningitis (TBM), tuberculoma and neurocysticercosis (NCC) (n=88), and group II - controls which included (a) non-infectious non-neurological conditions (n=30), (b) infectious neurological conditions (n=21) and (c) non-infectious neurological conditions (n=133). PHA could detect anti-mycobacterial antibodies at the sensitivity level of 80.76% with a specificity of 92.4% and anti-cysticercal antibodies with a sensitivity of 100% and specificity of 92.94%. However, in 6.33% (i.e. 14/221) of group I and group II (c) CSFs both anti-mycobacterial and anti-cysticercal antibodies were detected. Immunoblot analysis of CSFs derived from TBM patients reacted predominantly to 120-kDa, 96-kDa, 65-kDa, 38-kDa, 26-kDa, 23-kDa, 19-kDa and 12-14-kDa and 4-6-kDa antigens of M. tuberculosis sonicate extract (MTSE), whilst CSFs of proven NCC reacted to >110-kDa, 96-kDa, 80-kDa, 66-68-kDa, 52-kDa and 26-28-kDa antigens of porcine whole cyst sonicate extract (PCSE). On immunoblot analysis, some of the CSFs of TBM patients were PHA positive for both MTSE and PCSE showed antibody reactivity to 70-kDa and 10-kDa antigens of C. cellulosae. Similarly CSF antibody of some Guillain Barre syndrome and myeloradiculopathy patients reacted with cysticercal antigens. But per se no cross-reactivity between MTSE and anti-cysticercal antibodies and vice-versa were observed. However, findings of this study should alert laboratory personnel especially in endemic areas to be extra careful in interpretation of antibody detection results.     

Determination of Shigella antigens in the feces of people, immunized with enteral vaccine prepared from S. flexneri antigen

The dynamic determination of the presence of the specific antigen and its activity in the excreta of humans subjected to enteral immunization with vaccine prepared from S. flexneri antigen was made in the agglutination test and neutralization test with the use of, respectively, antibody and antigenic erythrocyte diagnosticums. In the feces and urine of the vaccinees antibodies occurred less commonly and, as a rule, they were less active than those detected in dysentery patients at the corresponding time from the beginning of the disease. The occurrence of Shigella antigens in the feces of the vaccinees was greater than in their urine at the corresponding time. Similarities and differences in the dynamics of the isolation of Shigella antigen from dysentery patients and from the vaccinees receiving enteral vaccine prepared from S. flexneri antigens were established.     

The role and place of serologic research in the system of epidemiologic surveillance of meningococcal infection

The results of serological surveys, carried out with a view to the detection of capsular meningococcal polysaccharides in the passive hemagglutination (PHA) test and covering 1,289 patients with systemic meningococcal infection, 221 patients with bacteriologically confirmed meningococcal nasopharyngitis, 2,820 persons in organized groups of children and adults with different epidemiological situation (including 650 carriers) and 4,050 residents of 8 cities with different morbidity levels, were analyzed. The patients, the carriers and the members of organized groups underwent multiple surveys. As shown in this study, the confirmation of the meningococcal etiology of the systemic forms of meningococcal infection (meningitis, meningococcemia) in all cases with the exception of children under 1 year of age, as well as the determination of the groups of meningococci, could be achieved in the PHA test twice as frequently, i.e. in 60-70% of cases, as with the use of the bacteriological method. The same regularities were observed in nasopharyngitis. The conclusion was made on the possibility of using the PHA test for more exact determination of the group of meningococci, prevailing at the initial stage of diseases, this determination playing an essential role in the epidemiological surveillance. The data obtained as the result of serological surveys in organized groups may lead to conclusions on the circulation of meningococci and their group composition, thus showing the necessity of profound epidemiological study with ensuring epidemic-control measures. The selective serological study of some hundreds of donor sera is not expedient in big cities, as it provides no data on the circulation of meningococci there.(ABSTRACT TRUNCATED AT 250 WORDS)     

Adenovirus antibody measured by the passive hemagglutination test

Lefkowitz, Stanley S. (Variety Children's Research Foundation, Miami, Fla.), Julia A. Williams, Bernard E. Howard, and M. Michael Sigel. Adenovirus antibody measured by the passive hemagglutination test. J. Bacteriol. 91:205-212. 1966.-Rabbits immunized intravenously with adenovirus type 5 antigen were tested for antibody titers by use of the passive hemagglutination test (PHA). Primary and secondary responses were studied, and the class of antibody was determined by means of density gradient centrifugation and reduction with 2-mercaptoethanol (ME). It was found that the PHA was 10 to 100 times more sensitive than complement-fixation and neutralization tests for the detection of antibodies to adenovirus. The immunological response to primary immunization was dependent on the dose of antigen, with antibody appearing in as early as 3 days. After secondary stimulation with the same antigen, there was a rapid response which appeared to be less dose-dependent. It was found that a heavy 19S antibody sensitive to ME was produced early and was followed by a lighter, presumably 7S, ME-resistant antibody. Upon secondary stimulation, both 7S and 19S antibody increased to levels greater than those of the primary injection.     

Antibodies to Clostridium botulinum toxins in free-living birds and mammals.

Naturally-occuring antibodies against Clostridium botulinum toxins were found in Cathartes aura (turkey vultures), Canis latrans (coyotes) and Corvus brachyrhynchos (crows) by the passive hemagglutination (PHA) test and verified by the serum neutralization (SN) test. The prevalence of IHA antibodies was 18 of 20 vultures (90%), 5 of 12 crows (42%) and 25 to 110 coyotes (23%). Vultures and coyotes were seropositive by the PHA test against A, B, C, D, and F toxins. The highest antibody titer 1:8192 was in vulture serum against type C. In descending order, the highest antibody levels were against type C, D, F, E, A and B toxins.     

The effects of strain heterology on the epidemiology of equine influenza in a vaccinated population

We assess the effects of strain heterology (strains that are immunologically similar but not identical) on equine influenza in a vaccinated population. Using data relating to individual animals, for both homologous and heterologous vaccinees, we estimate distributions for the latent and infectious periods, quantify the risk of becoming infected in terms of the quantity of cross-reactive antibodies to a key surface protein of the virus (haemagglutinin) and estimate the probability of excreting virus (i.e. becoming infectious) given that infection has occurred. The data suggest that the infectious period, the risk of becoming infected (for a given vaccine-induced level of cross-reactive antibodies) and the probability of excreting virus are increased for heterologously vaccinated animals when compared with homologously vaccinated animals. The data are used to parameterize a modified susceptible, exposed, infectious and recovered/resistant (SEIR) model, which shows that these relatively small differences combine to have a large effect at the population level, where populations of heterologous vaccinees face a significantly increased risk of an epidemic occurring.     

Etiology of acute and chronic bronchopulmonary diseases in children. II. A study of the specific immune response by counterimmunoelectrophoresis and the complement fixation and passive hemagglutination reactions

Specific immune response to Streptococcus pneumoniae and Haemophilus influenzae has been studied in 158 children with acute pneumonia and pleuritis and 128 children with chronic pneumonia by countercurrent immunoelectrophoresis (CIE) and in the complement fixation (CFT) and passive hemagglutination (PHA) tests. The use of CIE leads to the detection of antibodies to H. influenzae in 23.7% of children with acute pneumonia and in 46.9% of children with chronic pneumonia. In the CFT antibodies to H. influenzae are also more often detected in children with chronic pneumonia (48%) than in those with acute respiratory infections (12.2%). In the PHA test high titers of antibodies to type b H. influenzae capsular polysaccharide occur in 11.9% of children with acute pneumonia and in 8.2% of children with chronic pneumonia.     

Epidemiological and immunological monitoring of tetanus in Georgia (Republic)

During the period of 1961-1990 the regular immunological and epidemiological monitoring of tetanus was carried out. At the prevaccination period (1937-1960) 140 cases of tetanus were registered, 77 patients died (morbidity rate being 0.97-0.11 per 100,000 of the population with mortality rate equal to 55.6%). At the period of mass vaccinal prophylaxis (1960-1990) 56 cases were registered and 27 patients died (the corresponding values: 0.09-0.11 with 48.2% mortality). In 1981-1990 altogether 53,213 vaccinees were examined. 4,864 vaccinees (8.9%) proved to be seronegative and 43,615 vaccinees (81.9%) were found to have protective antibody titers. 4,220 pregnant women were examined; 476 women (11.2%) proved to be seronegative and 3,576 women (84.3%) were found to have protective antibody titers. Timely specific prophylaxis proved to be the only way for decreasing tetanus morbidity.     

Serology of Rubella—Comparison of Fluorescent Antibody,Complement Fixation and Neutralization Tests for Diagnosis of Current Infections and Determination of Sero-immunity

Neutralization, complement fixation (CF) and indirect fluorescent antibody (FA) assays for rubella virus were compared for sensitivity in the serologic diagnosis of infection, for demonstrating antibody in the sera of infants with suspected rubella syndrome, and in the detection of antibody elicited by past infection (determination of immunity status). The combination of CF and FA tests was shown to be the most useful for serologic diagnosis of infection, largely eliminating the need for the slower and more cumbersome interference neutralization test.Neutralizing antibodies were found to appear rapidly in the course of infection, antibodies demonstrable by immunofluorescent staining appeared slightly later, and CF antibodies were rarely demonstrable in sera collected earlier than 14 days after onset of illness. Antibodies detected by all three techniques showed good correlation in infants with clinical evidence of rubella syndrome and corresponding maternal sera. The indirect FA technique compared favorably with the neutralization test for the detection of antibody elicited by past infection (determination of immunity status) and offered distinct advantages in ease of technical performance and more rapid results. In both current and past infections, FA titers tended to be higher than neutralizing antibody titers.     

Infectious Virion Capture by HIV-1 gp120-Specific IgG from RV144 Vaccinees

The detailed examination of the antibody repertoire from RV144 provides a unique template for understanding potentially protective antibody functions. Some potential immune correlates of protection were untested in the correlates analyses due to inherent assay limitations, as well as the need to keep the correlates analysis focused on a limited number of endpoints to achieve statistical power. In an RV144 pilot study, we determined that RV144 vaccination elicited antibodies that could bind infectious virions (including the vaccine strains HIV-1 CM244 and HIV-1 MN and an HIV-1 strain expressing transmitted/founder Env, B.WITO.c). Among vaccinees with the highest IgG binding antibody profile, the majority (78%) captured the infectious vaccine strain virus (CM244), while a smaller proportion of vaccinees (26%) captured HIV-1 transmitted/founder Env virus. We demonstrated that vaccine-elicited HIV-1 gp120 antibodies of multiple specificities (V3, V2, conformational C1, and gp120 conformational) mediated capture of infectious virions. Although capture of infectious HIV-1 correlated with other humoral immune responses, the extent of variation between these humoral responses and virion capture indicates that virion capture antibodies occupy unique immunological space.     

Determination of antibody affinity by ELISA. Theory

New approaches for the measurement of antibody affinity by ELISA are suggested and considered theoretically. It was shown that not only more precise and more convenient in comparison to that suggested earlier, but also more informative graphical representation of the experimental data in the appropriate coordinate could be used for evaluation of antibody affinity. The following cases were considered: (i) determination of antibody affinity for one kind of univalent antibodies, (ii) determination of antibody affinity for one kind of bivalent antibodies, (iii) determination of antibody affinity for two kinds of univalent antibodies, which are in a mixture, and (iv) determination of antibody affinity for two kinds of bivalent antibodies, which are in a mixture. Advantages and disadvantages of the different approaches are discussed.     

Rubella vaccination: persistence of antibodies for up to 16 years.

Sera from 123 volunteers vaccinated six to 16 years previously with one of four rubella vaccines (Cendehill, RA27/3, HPV77-DE5, and To-336) were tested for rubella antibodies by single radial haemolysis and radioimmunoassay. By radioimmunoassay 110 (89.4%) of the vaccinees had antibody concentrations greater than the minimum immune titre (that is, greater than 15,000 IU/1), 11 (8.9%) were seropositive but had concentrations less than or equal to 15,000 IU/1, and two (1.6%) were seronegative. Eight (6.5%) were seronegative by single radial haemolysis, of whom five had received Cendehill vaccine. Six to eight years after vaccination subjects who had received Cendehill vaccine had the lowest geometric mean titre of antibody by radioimmunoassay while the subjects who had received HPV77-DE5 vaccine had the highest. Although antibody concentrations less than or equal to 15,000 IU/1 were not detected among subjects given RA27/3 vaccine six to eight years previously, such low levels were detected in two (15.4%) vaccinated 11-16 years previously. These results emphasise the importance of long-term surveillance programmes so that vaccination policies may be reviewed.     

Quantitative and Qualitative Analyses of the Antibody Response of Adult Mice Tolerized with Deaggregated Bovine γ-Globulin

The antibody response of mice to bovine γ-globulin(BGG) was suppressed either specifically by an intravenous injection of deaggregated soluble BGG (sBGG) or nonspecifically by X-irradiation. Immunization with the subcutaneous injection of BGG in Freund's incomplete adjuvant was given to mice either various days after sBGG injection or immediately after X-irradiation. Antigen-elimination (AE) test and passive hemagglutination(PHA) test were employed for estimating the immune status. The AE test indicated that the induction of tolerance was accomplished in the first 2 days after sBGG injection and that the tolerant state was stable at least for about 30 days thereafter. The degree of suppression by 1000 μg of sBGG corresponded to that obtained by X-irradiation at the dose of 400 R or more, and 100 μg of sBGG was equivalent to 300 R X-irradiation. The PHA test indicated, however, that such a correspondence as mentioned above between the dose of tolerogen and that of X-irradiation was not so stable as was seen by the AE test. Thus, the PHA titers of tolerized animals tended to recover up to the level of untolerized animals during the period of time from 10 days to 20 days after the tolerogen injection. Such discrepancies between the features in the AE test and those in the PHA test seemed attributable to a low avidity antibody formation in the tolerized animals, as judged by the hemagglutination-dissociation test. Hemagglutination by means of the sera from tolerized animals was seen to be reversed by the addition of free antigen more easily than the hemagglutination achieved by the sera of control animals or X-irradiated animals. The relationship between PHA titers and AE capacities of antibodies was investigated by the passive immunization of normal mice previously given the antigen. The result showed that the PHA titer did not always correlate with the AE capacity.     

Determination of the concentration of active centers and the antibody heterogeneity index by using the precipitation in fluid reaction]

Equivalence zone in the precipitation test was characterized by the fact that the number of the antigen determinants was equal to the number of active antibodies centres, irrespective of the heterogeneity of the latter. This permits to assess the antibody concentration in the values of the antigen valency. The new method of determination of the antibody heterogeneity index is based on recording the titre of the antigen and antibodies in the equivalence zone.