The results of using direct solid-phase immunoenzyme analysis for assessing the specific activity of rabies vaccines

The effectiveness of the solid-phase enzyme immunoassay (EIA) in the determination of the specific activity of rabies vaccines is evaluated in comparison with that of the protective test in mice. Inactivated tissue-culture, concentrated tissue-culture and purified cerebral tissue vaccines for human use were studied. The methods for performing two EIA variants and evaluating the results are described. The average level of correlation between the results of EIA and the protective test for vaccines of different groups was revealed (0.546), the highest correlation index being obtained for tissue-culture vaccines: 0.753. On the basis of the data obtained in this study the expediency of using EIA for the determination of the specific activity of rabies vaccines has been substantiated.     

Methods of enhancing the specificity of immunoenzyme test systems for the determination of human IgE

The sensitivity and specificity of an enzyme immunoassay (EIA) system for the determination of total IgE in humans increases if polystyrene plates are sensitized with swine gamma-globulin and the conjugate is prepared with the use of sheep gamma-globulin obtained from the commercial preparation of sheep antiserum to human IgE. Such system becomes even more specific and sensitive if sheep antibodies to human IgE, purified by affinity chromatography, are used both for the sensitization of polystyrene plates and for the preparation of the conjugate. This conjugate is necessary for the development of EIA systems intended for the determination of specific human IgE-antibodies to various allergens.     

Relationship between canine mucosal and serum immunoglobulin A (IgA) concentrations: serum IgA does not assess duodenal secretory IgA

A double-sandwich enzyme immunoassay method was developed for determination of serum immunoglobulin A (S-IgA) and mucosal secretory immunoglobulin A (sIgA) in duodenal brush samples obtained via endoscopy and the relationship between enteric mucosal sIgA, salivary sIgA and S-IgA in dogs was examined. Twenty healthy dogs underwent routine endoscopy. A brush sample from the duodenal mucosa was obtained and washed in PBS, with a serum sample being taken concurrently. A saliva sample was collected from twelve of these dogs. S-IgA and sIgA with total protein concentrations in the duodenal washings and saliva samples were determined. A significant negative correlation (r = -0.64, P = 0.0059) was found between duodenal sIgA/protein ratios and S-IgA concentrations. Saliva sIgA/protein ratios did not correlate with sIgA/protein ratios of duodenal samples. The method described here allows for direct assessment of duodenal IgA; therefore indirect measures based on serum IgA or salivary IgA can be avoided. In addition, these indirect measures appear to be poor indicators of duodenal sIgA competence in dogs.     

Secretory immunoglobulin A: from mucosal protection to vaccine development

Immune responses taking place in mucosal tissues are typified by secretory immunoglobulin A (S-IgA) molecules, which are assembled from proteins expressed in two cell lineages. The heavy and light chains as well as the J chain are produced in plasma cells, whereas the secretory component (SC) is associated to the immunoglobulin complex during transcytosis across the epithelial layer. S-IgA antibodies represent the predominant immunoglobulin class in external secretions, and the best defined entity providing specific immune protection for mucosal surfaces by blocking attachment of bacteria and viruses. S-IgA constitutes greater than 80% of all antibodies produced in mucosa-associated lymphoid tissues in humans. The existence of a common mucosal immune system permits immunization on one mucosal surface to induce secretion of antigen-specific S-IgA at distant sites. In addition, S-IgA antibodies not only function in external secretions, but also exert their antimicrobial properties within the epithelial cell during transport across the epithelium. Passive mucosal delivery of monoclonal IgA molecules neutralizes pathogens responsible for gastrointestinal and respiratory infections. Mucosal and systemic immunity can be achieved by orally administered recombinant S-IgA molecules carrying a protective bacterial epitope within the SC polypeptide primary sequence.     

Shigella endotoxin protein--its electrophoretic and serological properties

The electrophoretic analysis of lipid A-associated protein (LAP), obtained from S. sonnei, in polyacrylamide gel in the presence of sodium dodecyl sulfate and urea has revealed the heterogeneity of the preparation; it has found to contain three main components with molecular weights of 43, 38 and 18 KD and some minor components with molecular weights of 49, 45 35, 30, 29, 27, 5, 21 and 14 KD. The electrophoretic mobility of the main protein components in the isolated preparation of LAP coincides with that of endotoxin components. The dissociation of proteins and lipopolysaccharide in the process of boiling the endotoxin in 2% sodium dodecyl sulfate is indicative of the noncovalent binding of these components. LAP contained in the endotoxin, in contrast to isolated LAP, is resistant to trypsin and proteinase K. The enzyme immunoassay (EIA) system with the use of LAP as a component of its solid phase has been developed, which makes it possible to carry out the quantitative determination of antibodies to this protein. The EIA system shows high sensitivity in the determination of anti-LAP IgG antibodies: in hyperimmune rabbit sera their titer is 1:250,000-1:800,000. As shown by the method of competitive EIA, the antigenic affinity of LAP of different origin corresponds to the degree of taxonomic propinquity of microorganisms: the maximal degree of cross reactions is observed between LAP obtained from S. sonnei, S. flexneri and Escherichia coli, while their affinity to Salmonella typhi is considerably less; remote microbial species (Bacterium bifidum and Sarcina marcescens) give practically no cross reactions.     

A comparative study of the sensitivity and specific activity of an immunoenzyme test system for determining class-M antibodies to the hepatitis A virus

The diagnostic value of the first experimental production batches of assay kit "DIAGN-A-HEP", produced at the Institute of Poliomyelitis and Viral Encephalitides (USSR Acad. Med. Sci.) and intended for the determination of IgM to hepatitis A virus (HAV) in the enzyme immunoassay (EIA), has been studied in comparison with that of the internationally known and widely approved commercial EIA system "HAVAB-MEIA" for the determination of antibodies to HAV (the product of Abbott, USA). The study has revealed that the EIA kit "DIAGN-A-HEP" is highly sensitive and specific, and the diagnostic value of this kit is not inferior to that of the commercial assay system "HAVAB-MEIA". On the basis of this study the use of the EIA kits "DIAGN-A-HEP" in medical practice has been allowed by the decree of the Ministry of Health of the USSR.     

Development of a sensitive enzymeimmunoassay (EIA) for FSH determination in bovine plasma.

A highly sensitive enzymeimmunoassay (EIA) procedure for FSH determination in bovine plasma on microtiterplates using the biotin-streptavidin amplification system and the second antibody coating was developed. Biotin was coupled to FSH and used to bridge between streptavidin-peroxidase and the immobilized antiserum in the competitive assay. The EIA was carried out directly in 50 microl of bovine plasma and compared with an established radioimmunoassay (RIA) employing 100 microl plasma. Same FSH standards and FSH specific antiserum were used in both procedures. FSH standards prepared in hormone free plasma were used. The sensitivity of the EIA procedure was 6.25 pg/well FSH which corresponded to 125 pg/ml plasma; the 50% relative binding sensitivity was seen at 200 pg/well. In comparison to RIA, the EIA was at least four times more sensitive besides requiring 6 times less FSH specific antiserum. Plasma volumes for the EIA ranging from 12.5 to 50 microl did not influence the shape of the standard curve even though a slight drop in the OD450 was seen with higher plasma volumes. When both EIA and RIA methods were used to measure FSH in cows, the levels were detectable only by the EIA procedure. The assay detects high and low plasma FSH levels within the physiological variation as well as changes in plasma FSH after stimulation with a GnRH analog. In conclusion, in addition to being non-radioactive and low cost in nature, the method offers several advantages over the conventional FSH RIA procedure; these are (a) higher sensitivity, (b) less labour and time saving, (c) more economical use of precious FSH antiserum and (d) long shelf-life of the biotinyl-FSH label (in contrast to the short half life of iodinated FSH in RIA).     

Application of sensitive enzymeimmunoassays for oxytocin and prolactin determination in blood plasma of yaks (Poephagus grunniens L.) during milk let down and cyclicity

Highly sensitive and specific enzymeimmunoassays for oxytocin and prolactin determination in yak plasma using the biotin-streptavidin amplification system and the second antibody coating technique were validated and applied for determining their profiles during milk let down and cyclicity in yaks. Oxytocin EIA was conducted taking duplicate 200 microl of unknown plasma samples and standards per well. The lowest detection limit was 0.2 pg/well, which corresponded to 1pg/ml plasma. Prolactin EIA was carried out directly in 50 microl of yak plasma. The sensitivity of EIA procedure was 5 pg/well prolactin, which corresponded to 0.1 ng/ml plasma. Mean plasma prolactin concentrations although high at estrus were not statistically different (P > 0.05) from the hormone concentrations on other days. Mean plasma prolactin concentrations during non-breeding season were significantly higher (P < 0.001) than that recorded in breeding season. Oxytocin and prolactin profiles were also obtained in two yaks before, during and after milking. A sharp release of oxytocin and prolactin shortly after udder stimulation was observed. High levels of oxytocin and prolactin were maintained during milking, falling sharply thereafter.     

Use of recombinant HBcore antigen for development of the diagnostic test-system

The plasmid construction expressing recombinant HBc antigen (HbcAg) in Escherichia coli cells under the control of the PL promoter of phage I, was obtained. The specific activity of the antigen thus obtained was controlled by the enzyme immunoassay (EIA) method and compared with the reference system "AxSYM CORE assay" ("Abbott", USA) with four panels of sera (altogether 111 samples). The coincidence of the results of the compared test system with the reference was 96.4%, which made it possible to recommend this genetic construction of recombinant HBcAg for the production of EIA systems.     

Enzyme immunoassay for the measurement of 17alpha-estradiol 17-N-acetylglucosaminide in rabbit urine.

17alpha-estradiol 17-N-acetylglucosaminide (17alphaE2 17NAG) is an estrogen metabolite hitherto obtained only in rabbits. To gain insight into this unique conjugate, an enzyme immunoassay (EIA) was established by using antiserum elicited against 3-[3-(1-carboxypropyl)] ether of 17alphaE2 17NAG-bovine serum albumin conjugate; horseradish peroxidase, as a label; and 3,3',5,5'-tetramethylbenzidine, as a chromogen. The method proved to be specific, and the detection range of the assay was 0.20-10.00 ng/ml. A proposed double conjugate, 3-glucuronide of 17alphaE2 17NAG, was synthesized to validate the EIA. The EIA was applied to the determination of the urinary level of 17alphaE2 17NAG in male and female (pregnant and non-pregnant) rabbits with and without beta-glucuronidase-sulfatase preparation from Helix pomatia. The results showed that 17alphaE2 17NAG was mainly excreted as a double conjugate (17alphaE2 17NAG 3-glucuronide and/or 3-sulfate) and that its level varies during pregnancy.     

The development and use of an immunoenzyme test system for detecting and the species identification of the monkey pox virus]

An enzyme immunoassay (EIA) system for the species-specific diagnosis of monkeypox, based on the use of monoclonal antibodies (McAb) to monkeypox virus, has been developed. Immunoglobulins, isolated from McAb-containing cultural and immune ascitic fluids, have been conjugated with horse-radish peroxidase and used as detector antibodies. For immunosorption, rabbit polyclonal antibodies to the vaccine virus have been used. The specificity and sensitivity of the EIA system thus obtained have been tested on animals and humans having monkeypox and confirmed by traditional diagnostic methods (the isolation of the virus on chick embryo chorioallantoic membranes and in cell culture).     

The production of specific immunoenzyme conjugates to human immunoglobulins by the glutaraldehyde method]

The results of studies aimed at obtaining class-specific conjugates to human immunoglobulins to be used in the enzyme immunoassay (EIA) are presented. At the first stage of the studies purified IgA, IgM and IgG preparations were obtained. These preparations were used for obtaining immunologically active immunosorbents on the basis of bromocyanic Sepharose. Specific antibodies to human IgA, IgM and IgG were isolated from animal sera by the method of affinity chromatography. These antibodies were conjugated with peroxidase by the glutaraldehyde method. The specific activity of the conjugates were determined in EIA. The results thus obtained revealed that all preparations exhibited high specific activity and gave no cross reactions with immunoglobulins of other classes.     

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.     

Development of a Soviet immunoenzyme test system for the quantitative determination of human immunoglobulin E

An EIA system for the quantitative determination of human IgE was developed. As the solid phase, polystyrene microplates sensitized with the gamma globulin fraction of sheep antiserum to human IgE were used in this system. Peroxidase conjugate with IgE was prepared with the use of periodate technique. The optimum parameters for the sorption of antibodies on polystyrene were determined: protein concentration, the pH of the buffer, temperature, the time of fixation, the working dilution of the conjugate. As the substrate of peroxidase activity, ortho-phenilenediamine solution with hydrogen peroxide was used. The result was evaluated by means of a photometer, model Titertek Multiskan MC (Flow Laboratories Ltd., Great Britain), at the wavelength 429 nm. The new EIA system proved to be specific and detected IgE in the sera under investigation at a concentration of 10(-9) g/ml. The system permitted the determination of the normal level of IgE in a group of healthy adults, this level being, on the average, 76 +/- 9 kU/1.     

Pili (fimbriae) of the causative agents of plague and pseudotuberculosis: their detection in the hemagglutination reaction, the passive hemagglutination reaction and in an immunoenzyme analysis system

Newly developed serological methods for the detection of pili in the passive hemagglutination (PHA) test with the use of immunoglobulin erythrocytic diagnosticum and in the enzyme immunoassay (EIA) with the use of specific immunoglobulins labeled with horse radish peroxidase have been found to exceed the method of detecting pili, based on the determination of their hemagglutinating activity, in sensitivity and specificity. Besides, the PHA test and EIA have proved to be capable of detecting low molecular fragments of pili, obtained by sonication and having lost their hemagglutinating activity.     

A simple and rapid method of determining the titer of herpes simplex virus antibodies in a study by immunoenzyme analysis of a single serum dilution

The result obtained in the study of the possibility of using the method for the determination of the titer of antibodies to herpes simplex virus by EIA techniques in a single dilution of the serum under test are presented. This method is based on the determination of the optical density of the serum titer (rcut) in different groups of sera with the use of the assay system, permitting the evaluation of the positive results obtained in the determination of their final dilution. The results obtained with the use of this method showed that error was 50% for high-titer sera, 60% for medium-titer sera and 30% for low-titer sera.     

Enzyme immunoassay of beta-hexosaminidase isoenzymes in human urine and renal cortex with monoclonal antibodies

Enzyme immunoassay (EIA) methods with monoclonal antibodies specific for N-acetyl-beta-D-glucosaminidase (NAG) isoenzymes A and B in human urine are presented. The proportion of NAG B obtained with the EIA methods was similar to that found with ion-exchange chromatography. In fresh human control urines, NAG B was found to be approximately 20% of the total NAG activity. A significant correlation was obtained between total NAG activity in human urine assayed with a conventional enzyme substrate method and the total NAG activity obtained as the sum of NAG A and NAG B analyzed with the EIA methods. Total NAG activity with the latter (EIA) methods showed about 30% higher values than found by the enzyme substrate method, which probably was due to inhibitors of NAG activity present in urine did not interfere with the EIA methods. The content of NAG A and NAG B in renal cortex was determined with the EIA methods. NAG B accounted for about 20% of the total NAG activity, which was similar to that found in fresh human urines.     

Highly sensitive enzyme immunoassay for beta-nerve growth factor (NGF): a tool for measurement of NGF level in rat serum

A sensitive two-site enzyme immunoassay (EIA) system was established for mouse beta nerve growth factor (NGF) isolated from mouse submaxillary gland. Our EIA system is based on the sandwiching of antigen between anti-mouse beta NGF antibody IgG coated on a polystyrene plate and biotinylated anti-mouse beta NGF antibody IgG. The bound antibody complex was quantified with streptavidin linked-beta-D-galactosidase (beta-D-galactosidase, EC 3.2.1.23). With this system NGF concentrations as low as 0.02 pg/well (corresponding to 8 x 10(-19) mol) could be measured reproducibly. The sensitivity of this EIA system permitted the quantification of endogenous immunoreactive beta NGF in rat serum. The mean level in serum of male rats (153.2 pg/ml) was found to be almost the same as that of female rats (127.6 pg/ml).     

Cigarette smoke inhibits BAFF expression and mucosal immunoglobulin A responses in the lung during influenza virus infection

Background

It is incompletely understood how cigarette smoke (CS) exposure affects lung mucosal immune responses during viral respiratory infections. B cell activating factor belonging to the tumor necrosis factor family (BAFF) plays an important role in the induction of secretory immunoglobulin A (S-IgA) which is the main effector of the mucosal immune system. We therefore investigated the effects of CS exposure on BAFF expression and S-IgA responses in the lung during influenza virus infection.

Methods

Mice were exposed to CS and/or infected with influenza virus. Bronchoalveolar lavage fluid and lung compartments were analyzed for BAFF expression, influenza-specific S-IgA level and histological changes. Lung B cells were isolated and the activation-induced cytidine deaminase (Aicda) expression was determined. BEAS-2B cells were treated with CS extract (CSE), influenza virus, interferon beta or N-acetylcysteine and BAFF expression was measured.

Results

CS inhibited BAFF expression in the lung, particularly after long-term exposure. BAFF and S-IgA levels were increased during influenza virus infection. Three-month CS exposure prior to influenza virus infection resulted in reduced BAFF and S-IgA levels in the lung as well as augmented pulmonary inflammation on day 7 after infection. Prior CS exposure also caused decreased Aicda expression in lung B cells during infection. Neutralization of BAFF in the lung resulted in reduced S-IgA levels during influenza virus infection. CSE inhibited virus-mediated BAFF induction in a dose-dependent manner in BEAS-2B cells, while this inhibition of BAFF by CSE was prevented by pretreatment with the antioxidant N-acetylcysteine.

Conclusions

Our findings indicate that CS may hinder early mucosal IgA responses in the lung during influenza virus infection through oxidative inhibition of BAFF, which might contribute to the increased incidence and severity of viral infections in smokers.

Electronic supplementary material

The online version of this article (doi:10.1186/s12931-015-0201-y) contains supplementary material, which is available to authorized users.     

The characteristics of the O-specific humoral immune response of mice vaccinated with Salmonella choleraesuis antigens]

The work deals with the results of the comparative enzyme immunoassay (EIA) of serum samples taken from (CBA X C57BL/6) F1 mice immunized with O-specific polysaccharides, O-antigens (O-Ag) obtained by Boivin's method and antigenic preparations isolated with hydroxylamine (HA) from S. choleraesuis and S. typhimurium. O-Ag and lipopolysaccharide (LPS) of the corresponding bacterial species were used as antigens for the sensitization of polystyrene plates. The primary and secondary humoral immune response was studied by means of EIA. As revealed in this investigation, the immunization of mice with HA-isolated antigenic preparations and O-Ag, obtained from S. typhimurium, in a single injection (in doses of 1-100 micrograms) led to the development of weak specific immune response to O-Ag. Response to LPS was absent. After the second immunization of the animals pronounced immune response to O-Ag and LPS was observed. It developed as a response of both IgM and IgG type. The immunization of mice, made in a single injection, with HA-isolated antigenic preparations and O-Ag, obtained from S. choleraesuis, did not lead to the development of O-specific immune response. After the immunization of mice with these antigens in two injections sharply pronounced nonspecific activity of IgM and IgG serum antibodies with respect to O-Ag and LPS of homologous and heterologous bacterial species was noted in EIA. Neither S. typhimurium O-polysaccharide, nor S. choleraesuis O-polysaccharide did not induce O-specific immune response even after the second immunization.