Composition of immunoglobulins in brucellosis patients using DEAE-cellulose column chromatography.

The composition of immunoglobulins in patients with brucellosis was studied. The method of ion-exchange chromatography on DEAE-cellulose columns was used to define more precisely the physico-chemical character of cysteine-resistant antibodies. The study of IgM, IgA and IgG fractions obtained from the patients sera showed the IgG fraction to possess the greatest serological activity in the agglutination reaction, in the passive haemagglutination reaction and in Coomb's test. Specific antibodies in the remaining 2 fractions (IgA and IgM) were found only in single patients in low titres, mainly in Coomb's test (incomplete antibodies). The study of IgM, IgA and IgG serum fractions before and after cysteine treatment revealed cysteine-resistant antibodies to be usually IgG globulins. The presence of specific IgG antibodies in the sera of patients was found to correlate with active clinical manifestations of brucellosis.     

The development of an immunoenzyme method for determining human secretory IgA

The results of the work on the development of an enzyme immunoassay (EIA) system for the determination of secretory IgA (S-IgA) are presented. A first, S-IgA was isolated from human colostrum and used as the basis for obtaining biologically active immunosorbent; then antibodies to S-IgA were isolated and the specific conjugate was obtained. The determination of S-IgA was carried out by the method of sandwich EIA. The newly developed EIA system permitted the determination of S-IgA only, giving no positive reactions with serum immunoglobulins. The data thus obtained make it possible to regard this assay system as specific, sensitive and suitable for further trials.     

Fractionation of immunoglobulins by liquid-liquid partition chromatography in aqueous two-phase systems

In this paper we show that although immunoglobulins are easily precipitated in solutions containing polyethylene glycol (PEG), especially at pH's where the conformation of the proteins should be close to native, human and rabbit IgG can be solubilized in aqueous dextran/PEG two-phase systems containing glycine and sodium chloride at pH 7.0 and that human IgA and IgM can be solubilized in such systems if the pH is increased to 9.0. Liquid-liquid partition chromatography (LLPC) on Li-ParGel was used to separate immunoglobulins into subfractions. Human IgG, IgM, and IgA all gave three peaks in the system used. These results indicate the possibility of separating different classes of immunoglobulins with this method. Specific IgG antibodies isolated from a rabbit antiserum against human serum proteins gave only two peaks in the LLPC system while the total IgG population gave three, as did human IgG. Thus, partitioning of immunoglobulins seems to be related to antibody activity.     

The application of immunoperoxidase test for the detection of antibodies various classes (IgA, IgG and IgM) coating bacteria in urine

For the detection of bacteria coated with immunoglobulins in urine the monoclonal antibodies against human IgA, IgG and IgM conjugated with peroxidase were used. For comparison, the immunofluorescence technique was also employed. The results obtained by two methods revealed that immunofluorescence were less sensitive. It was found that bacteria were predominantly coated with IgA (41,9 +/- 22,4%) and IgG (34,1 +/- 15,3 %) immunoglobulins. The IgM antibodies were found rarely (12,8 +/- 8%).     

Monoclonal antibodies to Chlamydia psittaci: their isolation, characteristics and use]

A panel of 22 hybridomas producing monoclonal antibodies (McAb) to C. psittaci was obtained. 15 hybridomas produced IgG1 antibodies, 4 hybridomas produced IgM antibodies and 3 hybridomas produced IgG2b, IgG3 or IgA antibodies. IgG1 antibodies and 2 IgM antibodies did not bind complement in the complement fixation test. All McAb were reactive in the enzyme immunoassay and the indirect immunofluorescence test and did not precipitate specific antigens. Peroxidase conjugates on the basis of McAb effectively detected Chlamydia antigen, prepared from the crude suspension of chick embryo yolk sack infected with different strains of C. psittaci and C. trachomatis, in different modifications of EIA.     

Protein kinase activity of immunoglobulins in human blood plasma

Protein kinase activity of the immunoglobulins (Ig) fractions from blood plasma of clinically healthy humans has been studied. IgA, IgG and IgM preparations have been obtained using column chromatography on sorbents with rabbit antibody to H-chains of human Ig. The level of 32P incorporation in casein in the presence of [gamma-32P]ATP was used to determine protein kinase activity of the Ig-fractions. The protein kinase activity of the preparation of IgA (but not IgG or IgM) was defined. The high-purified preparation of IgA for studing the protein kinase activity has been obtained. Three stages of purifications were used--the separation of plasma proteins by polyethylenglycol 6000, gel-filtration on the column with Toyopearl HW-60 Fine and affinity chromatography on the column containing rabbit antibody to H-chains of human IgA. It was revealed that the fraction of IgA possesses the casein phosphorylation activity. Heparin and trifluoperazine completely and partially inhibited protein kinase activity of IgA while spermidine did not render essential influence. On the basis of the obtained results the conclusion is made that the blood of clinical by healthy humans contains IgA possessing the protein kinase activity.     

Ordering of antibodies, that react with cardiac muscle fiber and interstitial connective tissue antigens, in different immunoglobulin classes

Sera of patients suffering from rheumatic diseases and myocarditis were examined on the sections of human and bovine myocardial tissue by indirect immunofluorescence with the use of pure IgG antibodies or monospecific sera against IgG, IgA and IgM. It was shown that antibodies reacting with different myofibers and interstitial connective tissue of the heart belong to the main immunoglobulin classes (IgG, IgA and IgM). There was a significant predominance of IgG antibodies as shown by the frequency of their detection and by the titer height. The predominance of antibodies to certain classes of immunoglobulins did not correlate with a specific disease entity. The frequency of detecting antibodies to a certain immunoglobulin class was in good agreement with the time of the disease onset. Moreover, the frequency of positive reactions due to IgG, IgA, and IgM antibodies correlated with the level of the appropriate immunoglobulins in the test sera.     

Specific immunoglobulin response in mice immunized with porins and challenged with Salmonella typhi

Specific antiporin antibody (IgG, IgM, and IgA) response was studied in control, infected, immunized-infected, and immunized mice. The activity of specific IgG immunoglobulins was found to be the highest in immunized and immunized-infected groups in which 87.5% protection had been observed by laboratory potency test in mice; the next-highest activities were those of IgM and IgA immunoglobulins. However, in the infected group the activity of specific IgM and IgG immunoglobulins was almost similar.     

Isotype specific immune responses in murine experimental toxocariasis

In this work, a murine experimental model of toxocariasis has been developed in BALB/c, C57BL/10 and C3H murine strains orally inoculated with 4,000 Toxocara canis embryonated eggs, in order to investigate the isotype-specific immune responses against excretory-secretory antigens from larvae. T. canis specific IgG+M, IgM, IgG, IgA, IgG1, IgG2a and IgG3 were tested by ELISA. The dynamics of the specific immunoglobulins (IgG+IgM) production showed a contrasting profile regarding the murine strain. Conversely to the results obtained with the IgM isotype, the IgG antibody class showed similar patterns to those obtained with IgG+IgM antibodies, only in the case of the BALB/c strain, being different and much higher than the obtained with IgG+IgM antibodies, when the C3H murine strain was used. The antibodies IgG+IgM tested in BALB/c and C57BL/10 were both of the IgM and IgG isotypes. Conversely, in the C3H strain only IgG specific antibody levels were detected. The IgG1 subclass responses showed a similar profile in the three murine strains studied, with high values in BALB/c, as in the case of the IgG responses.     

Immunomodulative properties of conjugates composed of detoxified lipopolysaccharide and capsular polysaccharide of <Emphasis Type="Italic">Vibrio cholerae</Emphasis> O135 bound to BSA-protein carrier

O135 serotype Vibrio cholerae isolated from Slovak river was used as a source of surface polysaccharide antigens. Following detoxification procedure, fractions of polysaccharides were separated by size exclusion chromatography. Two resultant fractions were the capsular polysaccharide (M _w ∼ 197,000 Da) and the lipopolysaccharide fragment (M _w ∼ 13,300 Da). These materials were used for preparation of four novel glycoconjugates. Two of them containing detoxified lipopolysaccharide as antigen were prepared by original chemical method using the new biocompatible polymer as carrier of antigen. Additionally, other two conjugates were prepared by direct linking of capsular and detoxified lipopolysaccharide antigens to the protein carrier using adipic acid dihydrazide spacer. The immunogenicities (induced IgM, IgG, IgA antibodies) of all conjugates were determined by enzyme-linked immunosorbent assay. Polymer containing conjugates elicited higher levels of specific anti-lipopolysaccharide IgM and IgG antibodies in comparison with other conjugates without polymer carrier. Enhanced IgM vibriocidal activity of mice antisera was also evident here.     

Interaction of specific and innate factors of immunity: IgA enhances the antimicrobial effect of the lactoperoxidase system against Streptococcus mutans

The antimicrobial effect of the lactoperoxidase (LPO) system (enzyme with the thiocyanate ion and hydrogen peroxide) on Streptococcus mutans NCTC 10449 (serotype c) was significantly enhanced when the system was combined with secretory IgA. Similar enhancement was observed with LPO-myeloma IgA1 or IgA2 combinations. This enhancement of the antimicrobial efficiency was not dependent on the presence of specific antibodies to S. mutans in the IgA preparation, but seemed to require binding between LPO and immunoglobulin. However, neither human polyclonal nor myeloma IgG or IgM nor rabbit IgG enhanced the antibacterial activity of the LPO system. None of the immunoglobulins, when added alone, produced antimicrobial effects. LPO was shown to bind to colostral secretory IgA, myeloma IgA1, IgA2, and to a lesser degree to monoclonal and polyclonal IgG and monoclonal IgM. This binding had a stabilizing effect on the enzyme activity. Our results suggest that IgA significantly enhances the antibacterial efficiency of one of the innate immune factors--the LPO system.     

Study of the antigenic relationship between animal and human immunoglobulins using antisera to human immunoglobulins]

Cross-reactivity of monospecific antisera to human immunoglobulins with animal sera of 10 species was studied by immunoelectrophoresis and radial immunodiffusion. Antisera to IgG were shown to reveal IgG of all the species studied, antisera to IgM and especially to IgA cross reacted less extensively. The greatest number of cross reactions were given by the antisera obtained as a result of hyperimmunization. Hyperimmune monospecific antisera to human IgG, IgA, and IgM can be used for the identification of animal immunoglobulins during their isolation from the sera and for their quantitation by radial immunodiffusion.     

Isolation of monospecific antisera to guinea pig immunoglobulins

The results of the production and analysis of monospecific rabbit antisera to guinea pig IgG1, IgG2, IgA and IgM are presented. Isolated immunoglobulins of different isotypes, as well as immune precipitates obtained by immunoelectrophoresis, were used for immunization. After adsorption antisera of each type there formed one precipitation line with guinea pig serum in immunoelectrophoresis, thus indicating that they contained antibodies to immunoglobulins of the definite isotype.     

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.     

Bound class M, A and G immunoglobulins in the thymus of myasthenia gravis patients]

Deposits of granular material containing IgM, IgA, and IgG were revealed in the thymus of patients with myasthenia by direct immunofluorescence. Treatment of the thymus sections by unlabeled preparations against individual classes of human immunoglobulins inhibited the reaction of the granular material with the homologous labeled preparations. Disappearance of fluorescence of these deposits was also seen in treatment of the sections with glycine-HCl-buffer, pH 2.8. These data permitted a suggestion that granular material represented immune complexes where IgM, IgA, and IgG served as antibody, and thymus tissue components--as an antigen. The presence of bound immunoglobulin in the thymus indicated that an autoimmune process directed against the tissues of this organ occurred in myasthenia.     

Naturally occurring catalytic antibodies: evidence for preferred development of the catalytic function in IgA class antibodies

IgG class antibodies express catalytic activities rarely and at very low levels. Here, we studied polyclonal IgA and IgG preparations from healthy human sera and saliva for the ability to hydrolyze model peptidyl-aminomethylcoumarin (peptide-AMC) substrates. These substrates permit objective evaluation of the catalytic potential of the antibody classes with minimal effects of noncovalent interactions occurring at sites remote from the reaction center. The IgA preparations hydrolyzed Glu-Ala-Arg-AMC at rates 3-orders of magnitude greater than IgG preparations from the same individuals. The cleavage occurred preferentially on the C terminal side of a basic residue. The activity was confirmed using monoclonal IgAs isolated from patients with multiple myeloma. Active site-directed inhibitors of serine proteases inhibited the catalytic activity and were bound irreversibly by the IgA, suggesting the involvement of a serine protease-like mechanism similar to that utilized by previously described IgM antibodies. These observations suggest that mechanisms underlying B cell clonal selection favor the retention and improvement of catalytic activity in the IgA, but not the IgG compartment of the immune response.     

Natural and immune human antibodies reactive with antigens of virulent Neisseria gonorrhoeae: immunoglobulins G, M, And A

Natural and immune human antibodies reactive with heat-labile and heat-stable antigens of virulent Neisseria gonorrhoeae were studied by use of an indirect fluorescent-antibody (IFA) procedure. The immunoglobulin class of the reactive antibodies was identified by using fluorescein-conjugated antisera specific for human IgG, IgA, or IgM in the IFA procedure. The effects of heat and mercaptoethanol on IFA reactivities were also studied. It appeared that antibodies of the IgG, IgM, and IgA classes present in the sera of both infected persons (immune antibodies) and normal persons with no history of gonococcal infection (natural antibodies) react with heat-stable somatic antigens. Immune IgG antibodies, however, were distinguishable from natural IgG antibodies by their ability to recognize heat-labile surface antigens. The distinction between natural and immune IgM antibodies was less obvious. IgM antibodies from both infected and normal persons appeared to react with heat-labile antigens. Some, but not all, infected persons had immune IgA antibodies to heat-labile as well as to heat-stable antigens. Treatment of sera with mercaptoethanol had no effect on IgG antibodies. The IFA activity of IgM antibodies was decreased, but not abolished. The effects of mercaptoethanol on IgA antibodies were variable. Some sera showed a decrease in IgA titer, and others showed an increase in IgA activity to certain antigens. Immune IgG antibodies were more resistant to heating than were natural IgG antibodies. Natural and immune IgM antibodies appeared equally sensitive to heating. IgA activity, on the other hand, was increased by heating sera at 60 C, but was decreased at higher temperatures. Thus, it appears that natural and immune human IgG antibodies to N. gonorrhoeae may be distinguished by their interactions with heat-labile antigens and by their resistance to heating.     

Normal human sera contain antibodies directed at Fab of IgA

Serum samples from 26 normal volunteers were evaluated by isotype-specific ELISA for the presence of IgG and IgM antibodies directed at IgA. Although there were wide variations in antibody levels, anti-IgA antibodies of both isotypes were found in all individuals tested. The anti-IgA activity was detected against a variety of polymeric and monomeric IgA1 and IgA2 myeloma proteins containing both kappa and lambda light chains. By using Fab and Fc fragments generated by incubation of an IgA1 myeloma protein with IgA1 protease, it was shown that the anti-IgA activity was specific for the Fab portion of the IgA molecule. It was also demonstrated that the serum of two individuals contained both IgG and IgM activity directed at autologous affinity-purified IgA. IgM antibody levels against both whole IgA and Fab of IgA were significantly higher than IgG antibody levels. Cells producing anti-IgA antibodies of both isotypes were detected in lipopolysaccharide-stimulated human spleen.     

Heterogeneity of humoral immune components in human cysticercosis

Twelve Taenia solium cysticerci, obtained from several human organs, were examined by immunofluorescence for IgG, IgM, IgA, IgE and C3b on their surfaces. Anti-cysticercus antibodies of the 4 classes of immunoglobulins were looked for in the cerebrospinal fluid of most neurologic patients, in the intraocular humors of a patient with eye cysticercosis, and in the serum of some other patients. The morphological appearance of the parasites as well as the clinical features of the patients were recorded. The distribution of components was heterogeneous among the different parasite surfaces. IgG was the most frequent, followed by IgA, IgM, C3b and IgE. No correlation was found between the presence of these molecules and signs of damage in the cysticerci, or with the classes of immunoglobulins found as anti-cysticercus antibodies. Possible explanations of these findings as well as the implications of heterogeneity in cysticercosis are discussed.     

Preparation of an IgM and IgA Enriched Fraction for Clinical Use

Fraction III obtained during large acale fractionation of human plasma served as starting material for obtaining an immunoglobulin fraction enriched in IgA and IgM. Caprylic acid was used for the precipitation of most proteins other than the immunoglobulins present in fraction III. The proteins remaining in the supernatant are then precipitated vith ethanol at pH 6. Prealbumin is the main contaminant of this immunoglobulin preparation which contains 20–25% IgM and 15–20% IgA. The residual IgG can be removed by adsorption of the other proteins on DEAE-cellulose at pH 5.7 (0.03 M acetate), elution being achieved at pH 5.7 (0.2 M acetate).