The characteristics of detecting the tick-borne encephalitis virus antigen in the ELISA and indirect hemagglutination reaction by means of scanning electron microscopy

The surface of polystyrene plates was studied at different stages of the enzyme immunoassay (EIA) and the passive hemagglutination (PHA) test by the method of scanning electron microscopy in the detection of tick-borne encephalitis (TBE) virus antigen. The study revealed that in the process of EIA larger antigens were washed away from the plate surface. The objects detected on the polystyrene surface were identified as conglomerations of the virions of TBE virus, but whole virions were shown to play no decisive role in EIA. The conclusion was made that, due to some specific features of this method, EIA was more sensitive in reaction with small antigens (individual glycoproteids, their small complexes). And, respectively, the PHA test was more sensitive in reaction with large antigenic complexes (whole virions, their conglomerations, immune complexes).     

Adhesion in meningococcus

The adhesive activity of 113 meningococcal strains with different invasive properties and 10 Neisseria nonpathogenic strains have been studied. Adhesive properties have been revealed in 80-82% of these strains. Meningococcal strains isolated from the nasopharynx of carriers possess high hemagglutinating activity. The percentage of cells with pili in these strain was also higher than that in meningococcal strains isolated from the liquor of patients. This shows the presence of direct correlation between the number of pili in the cells (according to the data of electron microscopy) and their activity in the hemagglutination test, which makes it possible to use this test for the determination of the adhesive capacity of meningococci, associated with the presence of pili.     

Heterogeneity of antibody response to Salmonella lipopolysaccharide measured by passive hemagglutination and hemolysis in mice

The complement-requiring passive hemolysis test with Salmonella typhimurium lipopolysaccharide-coated sheep erythrocytes is more sensitive for antibodies directed against the lipopolysaccharide than is the passive hemagglutination test. The hemagglutinating and hemolyzing antibodies produced in Swiss mice by hyperimmunization, either with or without Freund's adjuvant, were distributed in both the light and heavy fractions isolated by sucrose density gradient fractionation and gel filtration. IgM fractions, whether tested by hemagglutination or hemolysis, were sensitive to 2-mercaptoethanol (0.15 m). On the other hand, IgG hemolytic antibodies were more sensitive to 2-mercaptoethanol than were IgG hemagglutinating antibodies. The resistance of IgG hemagglutinating activity amounted to about 72 to 95% of the total IgG recovered, whereas the resistant portion of the IgG hemolytic activity was approximately 40 to 53%. It is suggested that, although mercaptoethanol sensitivity is not a definitive test for IgM antibody, its use in connection with the hemagglutination test gives at least an approximation of the IgG antibody, whereas the hemolysis test gives a better approximation of maximal measurable antibody against Salmonella lipopolysaccharides.     

Interrelation between the blood neutrophil damage test and the passive hemagglutination reaction in detecting antibodies

Statistical analysis of the results of examinations of vaccinees against plague has revealed that the values of the neutrophil damage index (NDI) and antibody titers as determined in the passive hemagglutination (PHA) test qualitatively (but not quantitatively) correlate. The statistical series for the PHA test and NDI belong to different variations, i. e. they describe different functions of antibodies with respect to the same antigen. Besides, the determination of NDI permits the detection of antibodies in 95% of cases with probability equal to 0.06, while the PHA test determines antibodies in 67% of the vaccinees with probability equal to 0.30. The determination of NDI, used as an alternative qualitative method for antibody detection, is particularly effective in the evaluation of faintly immunogenic antigens, as well as at the early and late periods of immune state.     

Serologic diagnosis of intestinal yersiniosis. The design of a stable erythrocyte preparation for the indirect hemagglutination reaction

Nine types of erythrocyte diagnostica of serovars O3 and O9, differing in the methods of obtaining sensitins and the physical state of erythrocytes, were put on trial. The preparations were used for the titration of hyperimmune sera and blood sera obtained from about 500 healthy persons, 300 patients with Yersinia enteric infection and 300 patients with other diseases. Freeze-dried diagnostica, when compared with liquid ones, were found to be less sensitive, but more stable and specific. Sensitins isolated by the methods of Westphal ad Boivin showed the highest degree of specificity. The authors believe freeze-dried sheep red blood with activated Boivin's antigen adsorbed onto them to be the optimal preparation for use in the passive hemagglutination (PHA) test. The preparation was found to retain its serological activity for as long as 2-3 years. The titer 1:160 (1:200) in the PHA test is recommended as the minimal diagnostic indicator. Erythrocyte diagnosticum is more sensitive, specific and stable than bacterial one. Since 1984 dried Yersinia erythrocyte diagnostica (serovars O3 and O9) have gone into quantity production at the Leningrad Research Institute for Vaccines and Sera.     

Properties of pili from Escherichia coli SS142 that mediate mannose-resistant adhesion to mammalian cells

We isolated pili from Escherichia coli SS142. These pili had a diameter of 6 nm and an average length of 400 nm. They were composed of subunits with a molecular weight of 18,000. Their amino acid composition was determined; methionine and proline were not detected. The isolated pili retained mannose-resistant hemagglutinating activity. Proteolytic digestion and glutaraldehyde fixation led to partial or complete loss of the hemagglutinating activity of the pili without causing any detectable damage to their supramolecular structure, which was only disintegrated by treatment with hot sodium dodecyl sulfate. The hemagglutinating activity of E. coli SS142 was inhibited by the glycoproteins fetuin and Tamm-Horsfall protein, as well as by the glycolipids phytyl lactoside, dansyl-sphingosine lactoside, and digalactosyl diglyceride. Isolated pili inhibited the adhesion of the homologous strain E. coli SS142 to Intestine 407 cell monolayers, but did not inhibit the adhesion of E. coli strain B-413, B-506, or 2699. This indicates that E. coli SS142 binds to a receptor different from those recognized by the other strains and that mannose-resistant adhesion to tissue culture cells can be classified into different subtypes.     

The use of immunoenzyme analysis and the vibriocidal antibody reaction in the serological examination of persons who have had cholera and of those in contact with them

The preparation of cholera toxin obtained from Vibrio cholerae strain 1310 has been used for producing solid-phase immunosorbent intended for the enzyme immunoassay (EIA). The use of EIA and the vibriocidal antibody test (VAT) in the serological study of former cholera patients and persons having contacts with them has made it possible to show the excess of the antitoxic activity of sera over their vibriocidal activity in all subjects covered by the dynamic study (from 5-14 days to 8-10 months). EIA and VAT can be used as auxiliary methods in epidemiological survey and analysis.     

Demonstration of feline leukemia virus antibody in cats by passive hemagglutination (38583).

Two FeLV fractions from Sephadex G-150 gel filtration were used to sensitize RBC for the PHA test. When the cells were coated with the fraction from the second peak consisting mostly of a mojor gs antigen of FeLV, no antibodies could be detected either in hyperimmunized cat sera or sera from leukemic cats, whereas antibodies were readily detectable in immunized rabbits. Using cells coated with the first peak eluants or Tween-ether disrupted FeLV, PHA antibodies were detected in cat sera. There existed, however, one significant exception that a cat with the diagnosis of mast cell leukemia showed antibody against the second peak fraction. Little or no antibodies could be detected in cat sera by CF or gel-diffusion. There was some correlation between hemagglutinating antibodies and conglutinating complement absorbing antibodies, but these antibodies did seem to differ from neutralizing antibody.     

The danger of hepatitis B virus infection from transfusions of plasma in which the HBs-antigen was detected retrospectively by highly sensitive methods

The prospective dynamic laboratory and clinical study of premature children was carried out: 55 children who received plasma transfusion during the first weeks of their life and were retrospectively (i. e. after plasma transfusion) found to have HBsAg detected by the passive hemagglutination (PHA) test, the enzyme immunoassay (EIA) and the radioimmunoassay (RIA) and 127 children who received the transfusion of plasma found to be HBsAg-negative according to the results of EIA and RIA. As revealed in this study, the risk of hepatitis B virus infection in such children after the transfusion of plasma containing HBsAg at low concentrations, determined only in the PHA test or in EIA and RIA, was, respectively, 7.5 and 6.3 times greater than in children receiving plasma found to be HBsAg-negative according to the results of EIA and RIA. The results of this investigation showed the tendency towards a decrease not only in the total contamination of plasma recipients with hepatitis B virus, but also in morbidity rate in icteric forms of acute posttransfusion hepatitis B, depending on the concentration of HBsAg in plasma used for transfusion.     

Properties of hemagglutination by Prevotella melaninogenica

Although Prevotella melaninogenica belongs to the commensal oral microbiota, some strains possess putative virulence factors. For example, we have previously described fimbriated, hemagglutinating strains of P. melaninogenica, isolated from patients with periodontal disease. The aim of this investigation was to compare some chemical and physical properties of hemagglutination (HA) of P. melaninogenica with those of other pigmented gram-negative anaerobes. HA of 13 P. melaninogenica strains proved to be considerably weaker than that of the major periodontal pathogen, Porphyromonas gingivalis. Vigorous shaking reduced HA of shaken cells but the shaken supernatant had the same hemagglutinating activity as non-shaken cells. The hemagglutinating agent on P. melaninogenica seemed to be a protein, which can be separated from the cell and binds to lactose-, galactose-, and raffinose-containing carbohydrates on the erythrocytes. Adherence to epithelial cells did not differ significantly between the hemagglutinating and non-hemagglutinating strains of P. melaninogenica. Although P. melaninogenica is able to agglutinate erythrocytes, this potential virulence factor is of a considerably lower magnitude than that of major periodontal pathogens.     

Pili of Vibrio cholerae O1 biotype E1 Tor: a comparative study on adhesive and non-adhesive strains

Pili were found on the cell surface of non-adhesive Vibrio cholerae O1 Biotype E1 Tor as well as the adhesive strain. Purified pili of the adhesive and non-adhesive strains were morphologically, electrophoretically, and immunologically, indistinguishable from each other. The molecular weights of both pilin (subunit protein of the pilus) were about 16,000 daltons as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. These 16 kDa pili are different from the pilus colonization factor, which is a 20.5 kDa protein, reported by Taylor et al. The 16 kDa pili of Vibrio cholerae O1 Biotype E1 Tor have hemagglutinating activity, but may have no role in colonization, because non-adhesive strains also have such pili.     

HBe antigen and its antibodies in HBsAg carriers in various regions of the USSR

A total of 300 blood serum samples, containing HBsAg and obtained from donors in three regions of the USSR (the RSFSR, the Uzbek SSR and the Moldavian SSR) differing in the level of HBsAg carriership, were studied for the presence of HBeAg and antibodies to this antigen in the passive hemagglutination (PHA) test and in the enzyme immunoassay (EIA). The occurrence of HBeAg was found to depend on the level of HBsAg carriership in the region. Thus, according to the EIA results, in Gorky, Kishinev and Tashkent HBeAg was detected, respectively, in 5.5%, 12.3% and 13.3% of serum samples, the level of HBsAg carriership in these cities being, according to the results of the PHA test, 1.4%, 5.0% and 9.0%. As shown by the results of EIA, the occurrence of HBeAg increased with the rise of the titer of HBsAg, while regarding the occurrence of antibodies to HBeAg the reverse relationship was observed.     

The use of microdot immunoenzyme analysis with visual detection for the determination of tularemia antibodies

The possibility of using the micropoint enzyme immunoassay (EIA) on a nitrocellulose membrane with the visual evaluation of results for the detection of tularemia IgG antibodies in hamadryas baboons at the postvaccinal period has been studied. The sensitivity of this assay has been compared with that of the passive hemagglutination (PHA) test, the microagglutination (MA) test and EIA with the spectrophotometric evaluation of results in plates. As shown in this study, EIA in the above-mentioned modification can be successfully used for the detection of tularemia antibodies in the blood serum. The sensitivity of micropoint EIA has proved to be not inferior to that of EIA in plates, while exceeding the sensitivity of the PHA test 10- to 20-fold and the sensitivity of the MA test 10- to 1,000-fold. This method is simple, reliable, highly sensitive, economic and requires no special equipment, which makes it highly promising for the diagnosis of tularemia and the evaluation of humoral immunity at the postvaccinal period.     

Serological reaction systems using enzyme-labelled immunospecific reagents

Different serological test systems, based on the use of enzyme-labeled immunospecific reagents and intended for testing the material under study for the presence of Yersinia pestis capsular antigen and antibodies to it, are described. Comparative data on the evaluation of their sensitivity to the antigen and antibodies to it in different schemes of enzyme immunoassays (EIA) are presented. As shown in this investigation, EIA systems for the detection of the antigen and antibodies to it can comprise, at the minimum, the following set of reagents: monoclonal antibodies to the capsular antigen, staphylococcal protein A, and the conjugates of the capsular antigen and monoclonal antibodies with horse-radish peroxidase. The authors have come to the conclusion that the use of the serological test systems can essentially increase the reliability of the assay of any individual sample by EIA techniques.     

Pili of Vibrio cholerae Widely Distributed in Serogroup O1 Strains

The distribution of Vibrio cholerae O1 pili consisting of 16 kDa subunit protein (16K-pili) was examined by Western blotting, using 211 strains from various origins and specific anti-16K-pili sera. The 16 kDa protein was detected in all 211 strains. The pili were purified from 3 El Tor and 3 classical strains, and characterized by hemagglutination and inhibition tests. All purified pili were hemagglutinative. However, the hemagglutinating activity of classical pili disappeared after exposure to 5 M urea and the agglutination induced by the classical pili was inhibited by D -mannose, alpha-methylmannoside, D -glucose and N-acetylglucosamine. On the contrary, El Tor pili were resistant to these sugars and urea.     

The characteristics of the serological diagnosis of meningococcal infection in children in the 1st years of life

The results of clinico-immunological examination of 181 children, aged 1 month to 6 years, with generalized forms of meningococcal infection are presented. In children under observation antimeningococcal antibodies to group-specific meningococci of the main groups A, B and C were determined over the course of the disease by passive hemagglutination (PHA) test and enzyme immunoassay (EIA). The level and frequency of seroconversion were found to depend on the patient's age and the severity of the clinical course of meningococcal infection. Antibody level was found to increase simultaneously with respect to several meningococcal polysaccharides: A, B in 18.5% and A, B, C in 3.3% of cases. In the clinical interpretation of data obtained in the PHA test and EIA not only the patient's age, the form and duration of meningococcal infection, but also serotherapy should be taken into consideration, as the latter may distort the serological results.     

Immunoenzyme method in the diagnosis of human brucellosis

The optimum conditions for the determination of specific antibodies in the sera of brucellosis patients by means of enzyme immunoassay (EIA) have been selected. The comparative study of the specificity and sensitivity of EIA and other serological tests has demonstrated that EIA has high diagnostic effectiveness in the diagnosis of acute and chronic brucellosis. The presence of direct correlation between the results of EIA and Coombs' test is observed, which is indicative of the capacity of EIA for detecting both complete and incomplete specific antibodies. It should be pointed out that in all cases the titer of specific antibodies in EIA has been found to be 5-16 times higher than in Coombs' test, the passive hemagglutination test, and agglutination test.     

The indirect hemagglutination reaction in the diagnosis of trichinosis

An indirect hemagglutination test (IHAT) for trichinosis using a Melcher's antigen was evaluated and compared with a precipitin test (PT) and a bentonite flocculation test (BFT). One hundred and forty eight serum samples from patients from the whole country confirmed or suspected to have trichinosis by clinical or epidemiological evidences were studied: 117 (79.1%) samples resulted positive by IHAT, 138 (93.2%) by PT and 65 (43.9%) by BFT. Sixty three serum samples from patients with strong clinical suspect of trichinosis presented the PT and the BFT positive and were compared with the IHAT for sensitivity study. IHAT was positive in 60 (95.2%) serum samples. In order to determine the specificity of IHAT 25 serum samples from healthy volunteers and 124 serum samples from individuals with other parasitoses, such as cysticercosis (48), hydatidosis (45) and fascioliasis (31) were studied. The specificity, using a titre > or = 1:16 as a possible diagnostic value was 96%. The use of IHAT with RP and BFT in the diagnosis of human trichinosis is discussed.     

Comparative evaluation of methods for detecting HBsAg in sera

The results of the analysis of 1209 serum samples, made with a view to detecting those containing HBsAg, are presented. This analysis was made by the radioimmunoassay (RIA) on a polyethylene film, by the standard RIA technique with the use of a diagnostic kit obtained from Abbott Laboratories (USA) and by the passive hemagglutination (PHA) test. The RIA film technique was found to have the sensitivity of about 2 ng/ml HBsAg, which is similar to the sensitivity of the kit from Abbott Laboratories and exceeds the sensitivity of the PHA test approximately 50-fold. The percentage of detected HBsAg-positive sera, yielded by analysis with the use of the RIA film technique and the standard RIA technique, is the same. The RIA technique make it possible to detect more positive sera than the PHA test by about 2.5%.     

Comparison of rotavirus strains by hemagglutination inhibition.

Rotaviruses have been shown to be of importance as aetiologic agents of gastroenteritis in infants and in domestic animals of several species. Hemagglutinins were prepared from two Canadian isolates of bovine rotavirus and from one isolate of a simian rotavirus. A United Kingdon isolate of bovine rotavirus was shown not to possess hemagglutinating activity, indicating a strain difference between a Canadian and United Kingdom bovine rotavirus. In hemagglutination-inhibition (HAI) tests a rabbit hyperimmune (two injections) serum, prepared to one of the bovine rotaviruses, was not helpful in distinguishing the two bovine viruses because of cross-reactions between the viruses. However, it was possible to distinguish the bovine viruses from the simian virus with this serum. When guinea pig immune sera were prepared to the four rotavirus strains and tested with the three hemagglutinins in the HAI test, antigenic differences between the four strains of rotavirus were demonstrated. Hyperimmune guinea pig serum prepared to a strain of human rotavirus did not inhibit any of three hemagglutinins indicating that the human strain is different from the three rotavirus strains which gave hemagglutinins.