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 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.     

Schistosoma mansoni: identification of immunoglobulins associated with the tegument of adult parasites from mice.

Immunocytochemical and immunodiffusion studies were conducted in an attempt to identify the immunoglobulins associated with the tegumental surfaces of Schistosoma mansoni. Peroxidase-labeled monospecific rabbit anti-mouse immunoglobulin class or subclass sera revealed the presence of mouse IgG₁, IgG₂, IgG₃, IgA, and IgM on the surface of adult parasites recovered from mice. These observations were confirmed by double gel diffusion of the various rabbit antisera against an eluate obtained from mouse worms.     

Characterization of immunoglobulin classes of human antibodies to cryptococcus neoformans

Immunoglobulin G (IgG), immunoglobulin M (IgM) and immunoglobulin A (IgA) levels were determined by radial immunodiffusion techniques in sera from 11 patients with cryptococcosis. Most specimens showed increased levels of IgM. Studies with fluorescein-labeled monospecific antihuman IgG and IgM, however, indicated that IgG was the immunoglobulin reactive in the indirect fluorescent antibody (IFA) test. In addition, cross-reacting sera from mycotic infections other than cryptococcosis were also shown to contain IFA antibodies of the IgG class. Sera treated with 2-mercaptoethanol continued to react in both the IFA test and the tube agglutination test. No correlation could be established between IgG and IgM concentrations and serological reactivity in the sera evaluated in this study.     

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.     

Comparison of two methods for measuring human serum immunoglobulins (laser-nephelometry and radial immunodiffusion)

The AA. have tested 50 serum samples for immunoglobulins (IgG, IgA, IgM) with two different methods: laser-nephelometry (LN) and radial immunodiffusion (RID). Mean values of IgG and IgA are almost the same in the two tested methods and there is a good correlation between LN and RID (IgG: r = 0,98; IgA: = 0,96). Also IgM have showed a good correlation (r = 0,987) but mean values obtained with LN are just a few lower than those obtained with RID. Regression lines, calculated for all the Ig, confirm these conclusions. The AA. conclude affirming that the obtained difference for IgM is due to the different standards used for LN and RID determinations.     

Changes in serum immunoglobulins in subjects with acute myocardial infarct and essential hypertension

20 (12 men and 8 women) acute myocardial infarction (AMI) patients and 17 (14 men and 3 women) patients with arterial hypertension (II degrees stage according to OMS) in comparison to controls age and sex matched, were studied, serum IgA, IgG, IgM were evaluated with radial immunodiffusion and serum IgE with RIA. Ho significant changes ef immunoglobulins were observed between hypertensive patients and controls; whereas a significant increase of IgM, IgG and IgE, with out changes of IgA, were shown in AMI patients. Serum Ig and IgM were significantly augmented in AMI patients in comparison to hypertensive patients.     

Comparability of some serum protein determinations by radial immunodiffusion, laser nephelometric and turbidimetric assays employing Q-antisera SEVAC

Quantitation of IgG, IgA and IgM immunoglobulins and C3, C4 components in human serum samples by the radial immunodiffusion technique and by the nephelometric and turbidimetric assays was compared using the linear regression analysis method. Comparisons of the two methods run in polyethylene glycol showed close agreement between methods and a relatively high degree of correlation between the parameters studied. Compared to the radial immunodiffusion technique, nephelometry and turbidimetry gave good correlation between parameters, but the agreement between tests was worse, especially in the case of C3 component determinations in fresh samples of patients' sera. All tests were carried out using the Q-antisera and Control human serum preparations SEVAC.     

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.     

Serum and secretory immunoglobulins in allergic diseases

A total of 158 patients with pollinosis, bronchial asthma, urticaria and Quincke's edema were examined. The immunoglobulin and C3 levels in sera and the immunoglobulin and albumin levels in saliva were determined by the method of single radial immunodiffusion with the corresponding monospecific antisera. In all the groups of patients subjected to examination the presence of polyclonal hypergammaglobulinemia was detected, which was manifested by a rise in the levels of IgG, IgA and especially IgM; the level of IgD was low. A decrease in the level of C3 was detected in pollinosis patients in the absence of the exacerbation of the disease. No circulating immune complexes were detected. An essential increase in the level of IgG in saliva was revealed, which was due to the local synthesis of this immunoglobulin. In winter the level of salivary IgA in pollinosis patients was found to be essentially below normal, but at the period of exacerbation it increased twofold, probably in response to local stimulation with antigen-allergen. Patients with bronchial asthma and pollinosis were found to have a high level of free secretory component (SC); in pollinosis the level of free SC sharply increased during the stage of exacerbation, which was due to the increase of its synthesis and secretion by the epithelial cells of the mucous membranes. The importance of these data for the pathogenesis of allergic diseases are discussed.     

An immunohistochemical study of cells with surface and cytoplasmic immunoglobulins in situ in Peyer's patches and lamina propria of rat small intestine

The distribution of cells with surface and cytoplasmic immunoglobulins was studied in Peyer's patches (PP) and intestine of rats, using both frozen and paraffin sections, with a two-step peroxidase technique. Anti IgM, IgG, IgA and IgE sera were used. Surface staining was found within PP with all antisera used. Although the villi contained predominantly IgA plasma cells (PC), IgG PC and a few IgM and IgE PC were also found. Within PP, however, no IgA PC were found but IgM and IgG PC were present in all stages of development, mainly in the dome. PC of all types, but mostly IgA cells, were present in and around high endothelial venules (HEV). The results suggest that IgM and IgG PC precursors can develop to PC within PP whereas IgA precursors do not. PC appear to home to the gut preferentially via HEV.     

Quantitation of serum immunoglobulins G, M, and A in the rhesus monkey (M. mulatta) using human monospecific antisera in the enzyme-linked immunosorbent assay: developmental aspects

Using human monospecific antisera, several parameters have been optimized for the micro-ELISA "sandwich" technique used in the quantitative measurement of total serum IgG, IgM and IgA levels in rhesus monkeys. Representation of the optical density as a four-parameter logistic function provided excellent fits of the data over a wide choice of dilutions of human antisera used for coating the ELISA plates and for the peroxidase-conjugated antisera used in the system. The micro-ELISA "sandwich" technique was shown to be specific, reliable, sensitive, and economical for use in the routine measurement of total serum IgG, IgM and IgA levels in the rhesus monkey.     

Studies on Immunoglobulins and Trypsin Inhibitor in Colostrum and Milk from Sows and in Serum of Their Piglets

The concentrations of IgG, IgM, IgA and the specific sow colostrum trypsin inhibitor (SCTI) were measured by radial immunodiffusion in colostrum and milk samples from sows and in serum samples from their offspring during the suckling period. A clear time dependence was found for all the measured variates in both whey and serum. Statistically significant positive correlations were found between, on the one hand, concentrations of IgG and IgA, but not IgM, in sera from 39 suckling piglets 1 and 3 days old, and, on the other hand, concentrations of the same immunoglobulins and of the trypsin inhibitor in maternal colostrum (n = 7). Multiple regression analyses showed that at day 1 and day 3 the levels of both IgG and IgA in serum samples from the suckling piglets were positively influenced by both the SCTI and the IgG or IgA contents in maternal colostrum.     

Quantitative determination of specific immunoglobulins in the serum of bacterial carriers of abdominal typhus]

As a result of determination of immunoglobulins of class A and M by the method of simple radial immunodiffusion with the use of monospecific antiglobulin sera it appeared that in the sera of carriers immunogolubins M were contained in much lesser quantity than in typhoid patients. The content of immunoglobulins A was somewhat greater in the blood sera of carriers.     

Antilipoprotein autoimmune hyperlipidemia. The Ig-Lp test

In the antilipoprotein type of autoimmune hyperlipidemia (AIH), the immunoglobulins (Ig) are bound to lipoproteins by their antibody site and circulate as immune Ig-Lp complexes. In the earlier studies, the specific antibody activities were demonstrated in vitro by specific but rather sophisticated methods which were not suitable for the screening of antilipoprotein AIH in large populations. In the Ig-Lp test described here, the immunoglobulins bound to the low density lipoproteins (Ig-Lp) are detected by floating the complexes at D 1.10 in the ultracentrifuge in a physiological saline sucrose density gradient; delipidating them by ether in the presence of 0.2 M urea, and assaying the protein by radial immunodiffusion and laser immunonephelometry with antisera specific for IgG, IgA, IgM, low density lipoproteins and albumin. Radial immunodioffusion and immunonephelometry gave similar results. This Ig-Lp test was positive in 5 myelomas associated with hyperlipidemia, which were previously classified as AIH with specific methods. And the test was specific for the Ig type of the monoclonal antibody involved in each case (3 IgA, 1 IgG and 1 IgM). It was negative in 6 normolipidemic myelomas and also in 40 sera from healthy blood donors and one normal serum taken 4 hours after a fat meal. Although the Ig-Lp test is not specific for antilipoprotein antibodies, the results of this study allow to use if for the screening for AIH.     

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.     

Concentrations of immunoglobulins and protein in piglet feces.

The concentrations of immunoglobulins and protein in piglet feces at one day to ten weeks of age were assayed by the single radial immunodiffusion and by the Lowry method, respectively. The concentrations of IgG, IgA, IgM and total immunoglobulin were found to be significantly higher at one day and two days of age than those at one week to ten weeks of age. The concentrations of IgA, IgM and total immunoglobulin at two days of age were the highest and those at one day of age were the next highest. However, in case of IgG, there seemed to be no such difference in concentrations. There was almost no significant difference in concentrations of IgG, IgA and total immunoglobulin in feces at one week to ten weeks of age. On the other hand, the concentration of IgM at eight weeks of age seemed to be the least from one day to ten weeks of age. The concentration of protein at two days of age was the highest and that at one day of age was the next highest. Those at one week to three weeks of age were the least among those at one day to ten weeks of age.     

Co-purification of soluble human galactosyltransferase and immunoglobulins

Preparations of human malignant effusion galactosyltransferase activity purified according to previously published techniques using enzyme-specific affinity chromatography consistently produced antibodies directed toward immunoglobulins with no detectable antigalactosyltransferase. Double immunodiffusion analysis of the antigen showed the presence of both IgG and IgA. Affinity chromatography with anti-human IgG-Sepharose and anti-human serum-Sepharose resulted in a 48,000-fold purification of galactosyltransferase activity with no detectable IgG by radioimmunoassay. Immunization of rabbits with this preparation produced antibodies directed against galactosyltransferase activity and minimal anti-Ig. The persistence of immunoglobulins during the purification of soluble galactosyltransferase activity through two enzyme-specific affinity chromatographic steps suggests an association of immunoglobulins with galactosyltransferase activity.     

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.     

Evolution of mouse intestinal and blood immunoglobulins after oral route vaccination]

The authors have studied the mice immunoglobulins level after vaccination by oral route with a killed-pathogenic strain of Salmonella typhimurium and an avirulent mutant of the same bacteria. The obtained results show an increase of the intestinal IgA and IgG1 levels and a slighter one of sera IgG between the 10 th and 30 th day following immunization. No correlation was observed concerning the IgM, IgG and IgA levels and the mice protection against a challenge of pathogenic bacteria.