Humoral response to selected antigens of Yersinia enterocolitica and Yersinia pseudotuberculosis in the course of yersiniosis in humans. I. Occurrence of antibodies to Yersinia Yop proteins by Western-blot

The results obtained with the use of the western-blotting showed that antibodies for released proteins YopD (33-36 kDa) were the most frequently detected antibodies in serum samples from patients suspected for yersiniosis. Reactions between serum samples studied and the YopD protein were very intense, suggesting that protein is the strongest immunogen among the utilised, released proteins Yop of Yersinia. Antibodies IgM were more often diagnosed in patients with abdominal pain in the contrary to antibodies IgA which were characteristic to patients with reactive arthritis. Detailed analysis of the results of western-blotting on serum samples obtained several times from individuals with yersinosis during the course of infection in this investigation have showed also that antibodies of the IgA class hold longer in serum of individuals with arthritis compared with individuals with yersinosis not complicated by arthritis. In joint-fluid samples obtained from patients with arthritis antibodies for particular released proteins Yop were detected in the same class of immunoglobulins like in serum samples obtained from those individuals.     

Preparation and immunochemical properties of monoclonal antibodies to potato ring rot pathogen Corynebacterium sepedonicum

Five stable hybridoma lines producing monoclonal antibodies to Corynebacterium sepedonicum were obtained. The specificity of monoclonal antibodies obtained was characterized. Interactions of the antibodies with native cells and antigenic preparations from bacterial cell extracts were studied. The epitope specificity of these antibodies to their recognized antigens and the use of the antibodies in advanced immunodiagnostic assays are discussed.     

Monoclonal antibodies against human beta-glucocerebrosidase

Monoclonal antibodies were obtained against the membrane-bound lysosomal enzyme beta-glucocerebrosidase (acid beta-glucosidase), which is deficient in Gaucher's disease. BALB/c mice were immunized with homogeneous enzyme protein extracted from a sodium dodecyl sulphate/polyacrylamide gel. The mice were subsequently hyperimmunized with partially purified enzyme prior to fusion of spleen cells with myeloma cells. After fusion, 32 primary hybrid cell populations were obtained which continued to produce antibodies against beta-glucocerebrosidase after prolonged time of culture. All antibodies reacted with both native and denatured enzyme. Four primary cell populations were subcloned and the antibodies produced were characterized. The antibodies were all four of the IgG1 subclass. Three of these antibodies bind to protein A whereas one does not. The results of binding assays indicated that three of the antibodies react with the same antigenic domain (epitope 1), but the fourth with a different one (epitope 2). Probably two antigenic determinants are present in epitope 1 since one of the antibodies with specificity for epitope 1 is inactivated after iodination by the chloramine-T procedure whereas a second one is not.     

Production and immunochemical characterization of monoclonal antibodies toCorynebacterium sepedonicum, the causative agent of potato ring rot

Five stable hybridoma lines producing monoclonal antibodies toCorynebacterium sepedonicum were obtained. The specificity of monoclonal antibodies obtained was characterized. Interactions of the antibodies with native cells and antigenic preparations from bacterial cell extracts were studied. The epitope specificity of these antibodies to their recognized antigens and the use of the antibodies in advanced immunodiagnostic assays are discussed.     

Heterogeneity of monoclonal antibody affinity

In experiments with Pasteurella pestis monovalent capsular antigen and hybridoma monoclonal antibodies obtained after cloning and recloning the heterogeneity of the active centers of antibodies with respect to their affinity was revealed. In ascitic fluids obtained from the animals inoculated with different hybridoma clones 5 groups of antibodies, differing in their affinity, were determined in each fluid sample.     

Serological investigations in retrospective diagnosis of malaria.

Sera were obtained in 415 known cases of malaria (88 residents, 327 immigrants) at different times after diagnosis. Three antigens were used in the indirect fluorscence antibody test to detect antibodies to either Plasmodium falciparum or P vivax. Results in residents and immigrants were analysed separately. Most residents had detectable antibodies within one week after an attack, which began to wane after a month. The strongest reactions were obtained in cases of falciparum malaria with the homologous antigen and in cases of vivax malaria with P fieldi. The overall pattern of results was the same in the immigrants but the proportions positive for malaria antibodies, mean titres, persistence of antibodies, and the cross-reaction were usually greater. Testing for malaria antibodies is probably of value in the retrospective differential diagnosis of malaria in patients who have not been exposed to malaria before but must be interpreted with caution in others.     

Mechanism of action of DNA-hydrolyzing antibodies to DNA from blood of patients with systemic lupus erythematosus

Four fractions of IgG antibodies to native DNA (nDNA) were obtained from blood of patients with systemic lupus erythematosus (SLE). These antibodies displayed a thermostable DNA-hydrolyzing activity and were different in affinity for DNA-cellulose and sorption on DEAE-cellulose. DNA-hydrolyzing antibodies to nDNA are metal-dependent endonucleases, cause mainly single-strand breaks in DNA, and are active over a wide range of pH. By atomic-force microscopy, three-dimensional images of DNA complexes with DNA-hydrolyzing antibodies to nDNA were obtained with nanometer resolution, and a nonprocessive action mechanism was shown for the DNase activity of antibodies to nDNA.     

Anti-idiotypic antibodies to HLA-DR4 and DR2

The aim of our study was to determine whether antibodies recognizing epitopes of HLA-DR antigens (idiotypic antibodies or Ab1) induce the production of anti-idiotypic antibodies (Ab2). We tested the capacity of the F(ab')2 fragment obtained from two sera, one with no anti-HLA antibodies (serum ES) and one depleted by absorption of anti-HLA lymphocytotoxins (serum FH), to block the anti-DR antibodies reacting with the HLA-DR antigens of the immunizing donor. The F(ab')2 fragment obtained from serum ES inhibited the anti-DR2 activity of an earlier post-delivery bleeding obtained from the same woman. The anti-idiotypic antibodies contained by this serum also inhibited the anti-DR2 activity of a reference anti-DR2 antiserum 8W907 and of an anti-MT1 antiserum 8W1231. Similarly, the F(ab')2 fragment obtained from serum FH, after absorption of her anti-DR4 antibody, inhibited the anti-DR4 activity of autologous and homologous antisera. These data suggest that sera of parous women contain anti-idiotypic antibodies directed against regulatory idiotypes of anti-DR antibodies.     

Selection of monoclonal antibodies for immunoaffinity chromatography: model studies with antibodies against soy bean trypsin inhibitor

To facilitate selection of monoclonal antibodies for immunoaffinity chromatography, an ELISA screening procedure was developed. The assay is based on the avidin-biotin system and provides a profile of the monoclonal antibody which is based on the binding characteristics of the antigen binding site when exposed to different elution reagents. The elution profiles of 5 monoclonal antibodies to soy bean trypsin inhibitor (SBTI) were determined and for 2 of the antibodies the results obtained in the ELISA were verified using column experiments. The affinity constants were determined for the same 5 monoclonal antibodies and no correlation was seen with the ease of elution. The elution profiles presented here are easily obtained and the results indicate that a general screening procedure for suitable combinations of antibodies and elution conditions can be carried out using an elution ELISA assay when modified as described herein.     

An ABA-binding protein with nucleic acid-binding property

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The absence of specific interactions of Sertoli-cell-secreted proteins with antibodies directed against H-Y antigen

Radiolabeled proteins secreted into the medium by rat Sertoli cells in primary culture have been examined for specific interactions with polyclonal and monoclonal antibodies directed against serologically detectable H-Y antigen(s). None of the proteins secreted by Sertoli cells reacted specifically with H-Y antibodies, as determined with immunoprecipitation procedures and immunoabsorbent affinity chromatography, followed by SDS gel electrophoresis. Radioactivity profiles of proteins obtained after reaction with H-Y antibodies were similar to those observed after treatment with nonimmune sera or with irrelevant antibodies. We obtained comparable findings with proteins secreted by the mouse cell line TM4, which is of presumptive Sertoli cell origin, and with proteins present in ram rete testis fluid. These and other findings presented do not support the contention that Sertoli cells secrete a protein having the properties of serologically detectable H-Y antigen as previously described.     

A Study on the Comparative Stability of Insoluble Complexes of Glucose Oxidase Obtained with Concanavalin A and Specific Polyclonal Antibodies

Summary The formation of insoluble complexes of glycoenzymes with lectins and antibodies is one of the simplest methods of enzyme immobilization. Insoluble complexes of glucose oxidase were simply obtained by mixing the enzyme with concanavalin A or a specific polyclonal antibodies solution. The concanavalin A and immunocomplexes of glucose oxidase retained more than 80% of the original enzyme activity. Expression of very high enzyme activity in insoluble complexes suggested that these aggregates were quite porous and easily accessible to substrates. Insoluble complexes of glucose oxidase showed very high stability against denaturation induced by pH, temperature, urea and water-miscible organic solvents. Complexes of glucose oxidase obtained with concanavalin A and glycosyl-specific antiglucose oxidase polyclonal antibodies were quite comparable in stability while complexes prepared using polyclonal antibodies raised against the native glucose oxidase were slightly less stable. The complexes of glucose oxidase obtained with glycosyl-specific antiglucose oxidase polyclonal antibodies showed very high stability against inactivation mediated by exposure to water-miscible organic solvents. Insoluble complexes of glucose oxidase were cross-linked with glutaraldehyde to maintain their integrity in the presence of substrates. The cross-linking of complexes resulted in a slight decrease in enzyme activity but showed a pronounced enhancement in stability against various forms of denaturation.     

Different reactivity of Z-DNA antibodies with human chromosomes modified by actinomycin D and 5-bromodeoxyuridine

Summary Antibodies against Z-DNA react with fixed metaphase chromosomes of man and other mammals. Indirect immunofluorescence staining shows that chromosomal segments corresponding to R- and T-bands preferentially fix Z-DNA antibodies. In this work Z-DNA antibodies were used as a probe for DNA conformation in euchromatin of fixed human chromosomes whose condensation or staining were modified by actinomycin D (AMD) and by 5-bromodeoxyuridine (BrdU). Treatments with AMD and BrdU were performed to induce a G-banding by modification of chromosomal segments corresponding to R- and T-bands. Long BrdU treatments were used to induce asymmetrical and partially undercondensed chromosomes by substitution of thymidine in one or both DNA strand. Our results show a clear difference of Z-DNA antibodies reactivity after AMD or BrdU treatment. The G-banding obtained after AMD treatment is not reversed by Z-DNA antibodies staining since these antibodies bind very weakly to the undercondensed R-bands. On the other hand, the G-banding obtained by BrdU is completely reversed giving typical R-banding, as on untreated chromosomes. For asymmetrical chromosomes an R-, T-banding pattern is always observed but there is a decrease of the fluorescence intensity proportional to the degree of BrdU incorporation. We conclude that AMD treatment greatly disturbs Z-DNA antibodies binding suggesting a change in DNA conformation, whereas BrdU treatments do not suppress but only weaken the specific binding of Z-DNA antibodies on R- and T-bands. The direct involvement of thymidine substitution in DNA sequences recognized by Z-DNA antibodies is discussed.     

Rapid single-tube method for small-scale affinity purification of polyclonal antibodies using HaloTag™ Technology

Even in this era of advanced biotechniques, specific antibodies against a protein still prove to be powerful tools to study proteins and their functions. The polyclonal antisera obtained from the immunized rabbits, however, are not always pure, high affinity, antigen-specific polyclonal antibodies. With our new rapid HaloTag-based procedure, specific antibodies are obtained in just two, short steps: (1) simultaneous purification and covalent coupling of the antigen to Sepharose resin via the HaloTag and HaloLink reaction, and (2) affinity column purification of the polyclonal serum (10 μl). The combined antigen purification and coupling step requires only 1 h of room-temperature incubation, plus successive washing steps. Because different regions of an antigen can elicit the production of low affinity antibodies with relatively high cross-reactivity, the best way to produce high affinity antibodies against a protein of interest is to survey all antigenic determinants of that protein and identify the epitopes that result in the production of antibodies with a high affinity and specificity for that protein. Because our HaloTag procedure is quite rapid and simple, potential epitopes can be assessed with relatively little effort for their ability to elicit the production of highly specific antibodies.     

Rapid single-tube method for small-scale affinity purification of polyclonal antibodies using HaloTag Technology

Even in this era of advanced biotechniques, specific antibodies against a protein still prove to be powerful tools to study proteins and their functions. The polyclonal antisera obtained from the immunized rabbits, however, are not always pure, high affinity, antigen-specific polyclonal antibodies. With our new rapid HaloTag-based procedure, specific antibodies are obtained in just two, short steps: (1) simultaneous purification and covalent coupling of the antigen to Sepharose resin via the HaloTag and HaloLink reaction, and (2) affinity column purification of the polyclonal serum (10 microl). The combined antigen purification and coupling step requires only 1 h of room-temperature incubation, plus successive washing steps. Because different regions of an antigen can elicit the production of low affinity antibodies with relatively high cross-reactivity, the best way to produce high affinity antibodies against a protein of interest is to survey all antigenic determinants of that protein and identify the epitopes that result in the production of antibodies with a high affinity and specificity for that protein. Because our HaloTag procedure is quite rapid and simple, potential epitopes can be assessed with relatively little effort for their ability to elicit the production of highly specific antibodies.     

Loss of rotational mobility of band 3 proteins in human erythrocyte membranes induced by antibodies to glycophorin A.

The effect of antibodies to glycophorin A on the rotational diffusion of band 3 in human erythrocyte membranes was investigated by transient dichrosim. Three antibodies that recognize different epitopes on the exofacial domain of glycophorin A all strongly reduce the rotational mobility of band 3. The effect is at most only weakly dependent on the distance of the epitope from the membrane surface. The degree of immobilization obtained with two of the antibodies, BRIC14 and R18, is very similar to that produced by antibodies to band 3 itself. Similar results were obtained with membranes stripped of skeletal proteins. Fab fragments and an antibody to glycophorin C had no effect on band 3 rotational mobility. These results rule out a mechanism whereby band 3 rotational immobilization results from enhanced interactions with the membrane skeleton that are mediated by a conformational change in glycophorin A. Rather, they strongly indicate that the antibodies to glycophorin A cross-link existing band 3-glycophorin A complexes that have lifetimes that are long compared with the millisecond time scale of the transient dichroism measurements.     

Anti-idiotypic and natural catalytically active antibodies]

The principal existence of natural catalytic antibodies in the autoimmune sera is discussed. In the course of the autoimmune process, the induction of antiidiotypic antibodies against topoisomerase I has been shown in the sera of patients with scleroderma, systemic lupus erythematosus, and rheumatoid arthritis. The above antibodies were obtained in preparative amounts. Proceeding from the concept of the idiotypic network, the antibodies were suggested to be natural enzymes and their properties were studied. They appeared to be anti-DNA antibodies, competing with the native topoisomerase I for binding to anti-topoisomerase monoclonal antibodies and possessing highly specific DNA-binding activity (Kd is about 0.1 nM). The antiidiotypic antibodies specifically inhibit the topoisomerase-catalysed relaxation reaction and affect the formation of covalent DNA-protein complex. Possible involvement of antiidiotypic antibodies against topoisomerase in the catalysis of reactions of DNA transformation is analysed. Catalytic antibodies that are natural enzymes possessing DNA-nicking activity have been isolated from the blood sera of patients with different autoimmune pathologies.     

Effectiveness of methods of isolating specific class-G rabbit antibodies for the immunoenzyme determination of human placental alkaline phosphatase

Four different chromatographic methods of IgG isolation from rabbit antisera to placental alkaline phosphatase (HPAP) have been compared. The antibodies were obtained by ion-exchange chromatography and affinity chromatography on protein-A-sepharose, on the sepharose with immobilized antigen. IgG samples were characterized by the content of specific antibodies to HPAP and checked in enzyme immunoassay (EIA). IgG purified on immobilized antigen were found to be the optimal both from the point of view of the specific antibodies content and EIA sensitivity, but satisfactory results could be also obtained with ion-exchange and protein-A-chromatography purified IgG. The last two isolation methods are simpler and provide 3-10 ng/ml sensitivity of HPAP detection, which is lower, as compared with the test employing affinity antibodies (1 ng/ml), but allows the detection of HPAP in serum samples.     

Determination of antibodies to Streptococcus group A polysaccharide in human sera by an immunoenzyme method

Use was made of the ELISA to develop a highly sensitive quantitative method for detection of antibodies against Streptococcus group A polysaccharide (polysaccharide A) in human sera. The main advantage is that one can use only one optimal dilution of the sera together with the reference serum. Sera of 53 healthy volunteers and 77 patients with a history of Streptococcus group A infections were screened for the presence of polysaccharide A antibodies. Highly reproducible results were obtained in 97% of cases. The specificity of the method was shown with the polysaccharide A-induced inhibition of the reaction. Positive reactions obtained with the tested sera in gel immunodiffusion correlated with the data derived by the ELISA. Using the latter high level of specific antibodies was found in some of the sera that yielded negative reactions when tested by gel immunodiffusion. This may be associated with the presence of non-precipitating antibodies.     

Circulating cornea specific antibodies and keratoplasty

The presence of corneal antibodies in blood sera was investigated in 130 patients by immunoblotting analysis. Sera were obtained from patients before the first keratoplasty, repeated corneal transplantation or keratoplasty of the other eye. Baseline levels of antibodies in 15 healthy blood donors served to establish a positivity threshold. Patients were divided according to diagnosis and number of keratoplasties. Corneal antigens were prepared from donor corneas not suitable for surgery. Hyperimmune sera obtained by immunization of rabbits were used as positive controls. Significant increase in corneal antibodies specific to epithelial antigens was found in patients (62.5% positive) with keratitis of microbial origin. The presence of circulating antibodies to endothelial or stromal proteins was without significant changes. Depending on the number of keratoplasties no statistical difference in corneal antibodies production was observed. The group of patients with increased anti-corneal antibodies already before transplantation had to take immunosuppressive and anti-inflammatory therapy after keratoplasty.