Immunoelectrophoretic Studies Research Articles

Immunological Identification of Site of Vitellin Synthesis in Freshwater Prawn Macrobrachium malcolmsonii

Shanju, S. and Geraldine, P. 2009. Immunological identification of site of vitellin synthesis in freshwater prawn Macrobrachium malcolmsonii. J. Appl. Anim. Res., 36: 141–146. The site of vitellin synthesis in the freshwater prawn, Macrobrachium malcolmsonii, was examined immunologically using the antibodies raised against the vitellin purified from mature ovaries of M. malcolmsonii. In double diffusion test, single precipitin lines were found between center well containing immune serum and peripheral wells containing purified vitellin, ovarian homogenate, female hepatopancreas and female hemolymph. The precipitin lines were continuous suggesting that immune serum precipitated the proteins of complete identitiy. Immunoelectrophoretic and immunoblotting studies reveal positive reaction in ovary, egg, female hemolymph and female hepatopancreas but not in male hemolymph, male hepatpancreas, gill and muscle. These results suggest hepatopancreas as possible site of yolk protein synthesis in freshwater prawn M. malcolmsonii.

Immunoelectrophoretic study on common antigens of São Lourenço da Mata and Belo Horizonte strains of Schistosoma mansoni adult worms and Biomphalaria snails.

Immunoelectrophoretic studies on common antigens were carried out by using rabbits sera immunized against São Lourenço da Mata and Belo Horizonte strains of Schistosoma mansoni adult worms and antigens of Biomphalaria glabrata pigmented (Jaboatão--PE); B. glabrata albino (Belo Horizonte--MG) and B. straminea (São Lourenço da Mata, PE). Furthermore, the reverse approach was proceeded, namely, sera anti Biomphalaria snails produced in rabbits were tested against both strains of Schistosoma adult worm antigens. The analysis of the common antigens between the SLM strains of S. mansoni adult worm and B. glabrata pigmented showed 8 to 9 precipitin bands, 3 bands with B. glabrata albino and only 1 band with B. straminea crude extracts. On the other hand, the BH strain of S. mansoni adult worm antisera produced 6 to 7 bands with B. glabrata pigmented, 5 bands with B. glabrata albino and 1 band with B. straminea antigenic extract. Biomphalaria snails crude extracts were fractionated by Sephadex G-100 column and three fractions were collected from each snail strain. The fractions were tested with anti SLM and BH strains of S. mansoni adult worm sera by immunoelectrophoresis. The common antigens fractionated from Biomphalaria snails crude extracts and those found for both strains of S. mansoni adult worm mostly existed in the first fraction and they were estimated to have molecular weight over 158,000 daltons. In our laboratory, it was found a relationship between the antigenic similarities and experimental infection rates of S. mansoni towards Biomphalaria snails so that more bands were seen with increasing infection rates of S. mansoni.

Inhibition of cell proliferation with an HLA‐A‐specific monoclonal antibody

A new mouse IgG2A monoclonal antibody--JD 12--is described, which was selected for its ability to inhibit peripheral blood mononuclear cell (PBMC) proliferation. Serologic, immunoprecipitation and immunoelectrophoretic studies showed JD12 to be monomorphically reactive with HLA-A molecules with an affinity of 10(9) M-1. JD12 was capable of inhibiting growth in resting or stimulated PBMC with an ID50 of 300 ng/ml in a time-dependent and dose-dependent manner without cytotoxicity. IgG, F(ab)2 and F(ab) were active. Growth stimulation by phorbol ester was not inhibited, suggesting that JD12 action occurred prior to protein kinase C activation. DNA and RNA synthesis were both slowed with a resulting G0/G1 block. Analysis of separated PBMC components showed that adherent cells displayed increased activity (clumping and RNA synthesis) upon JD12 binding. In addition, the inhibitory activity of JD12 could be transferred to new cultures by cell-free, IgG-free supernatant from JD12-treated PBMC, suggesting that the antiproliferative effects could be mediated, at least in part, in an indirect manner through an inhibitory factor. Therefore, potent, noncytotoxic inhibition of cell proliferation, DNA and RNA synthesis via MHC molecules can be mediated specifically through effects initiated by binding to HLA-A molecules alone.

The thienopyridine PCR 4099 selectively inhibits ADP-induced platelet aggregation and fibrinogen binding without modifying the membrane glycoprotein IIb–IIIa complex in rat and in man

The thienopyridines, ticlopidine and PCR 4099, inhibit ex vivo aggregation in response to ADP and other agonists. It has been shown that ticlopidine induces a functional defect in the binding of fibrinogen to its platelet membrane receptor. We have studied the effects on platelet functions of PCR 4099 in rat and in man. The aim of the study was to check the possibility of a direct modification of the fibrinogen binding site on the GB IIb–IIIa complex. Washed platelet suspensions were used for aggregation and fibrinogen binding studies. Platelet lysates were submitted to SDS-polyacrylamide gel electrophoresis, crossed immunoelectrophoresis and immunoprecipitation. We found that administration of PCR 4099 inhibited selectively and irreversibly ADP-induced aggregation. Although the effect of ADP on aggregation was blocked, PCR 4099 did not modify ADP-induced shape change. Only the effects of low concentrations of thrombin on platelet aggregation were inhibited. Fibrinogen binding was dramatically inhibited in rat and in man when platelets were stimulated with ADP and low concentrations of thrombin. At high concentration of thrombin there still remained a part of fibrinogen binding inhibition although aggregation was not impaired. Electrophoretic and immunoelectrophoretic studies showed no difference before and after treatment by PCR 4099. In particular, the GP IIb–IIIa-complex was not dissociated, its electrophoretic mobility was not changed and three monoclonal anticomplex antibodies recognized it in the same manner before and after treatment. We conclude that PCR 4099 selectively inhibits the ADP aggregation pathway and that the inhibition of fibrinogen binding is probably not due to a direct modification of the GP IIb–IIIa complex.

Myopathy with respiratory failure and typical myofibrillar lesions

16 patients representing 7 different pedigrees exhibited an unusual, adult onset limb-girdle myopathy with typical clinical hallmarks. In a majority of cases there was evidence of an autosomal dominant inheritance. A prominent early finding in all cases was respiratory muscle weakness, and in many of these an acute respiratory incapacity was the reason for the first neurological examination. Neck flexor and sometimes foot extensor weakness were other early symptoms. The clinical picture seems to be at variance with that of the more well known hereditary myopathies. Electrophysiological analysis confirmed a myopathy and serum muscle enzyme concentrations were normal or slightly elevated. Muscle biopsy findings revealed myofibrillar changes which, at the light microscopy level, included plaques that stained strongly with rhodamine-conjugated phalloidin, a specific marker for F-actin. At the ultrastructural level, these plaques were observed to be composed of moderately dense, thin filaments and were related to splitting of Z-discs or formed extensions from Z-discs. We believe that the muscle biopsy changes revealed by cytochemical and ultrastructural observations indicate defective myofibrillogenesis, and the possibility of defective actin polymerization is discussed. A conclusive answer requires further immunocytochemical and immunoelectrophoretic studies and possibly the application of molecular genetics.

Pigeon breeders' disease: quantitative immunoelectrophoretic studies of pigeon bloom antigen.

This study employed quantitative immunoelectrophoretic techniques, on sera from confirmed cases of pigeon breeders' disease (PBD), to investigate the antigenicity of a pigeon bloom extract, implicated as a sensitizing agent in this disease. On crossed immunoelectrophoresis the maximum number of antigenic components identified was 29 for the bloom compared to 10 for pigeon serum. A major component was shown to be closely related to pigeon IgA, and demonstrated partial crossreactivity to the pigeon IgG. This component also showed identity with the major component of a pigeon droppings extract, considered to be derived from intestinal IgA. Only trace amounts of serum albumin were detected and most other bloom components were not serum-related. Although greater overall antigenic similarity was found to pigeon droppings extract, at least three of the bloom components appeared to be specific. The bloom extract also contained a low amount of an alpha-techoic acid-like component, causing some non-specific reactivity. Pigeon feather dust or 'bloom', like pigeon droppings, is therefore a potent source of antigens associated with PDB--pigeon IgA being a major component of both antigens.

Isolation of immunoglobulin G by affinity chromatography using an IgG Fc receptor protein from Streptococcus dysgalactiae coupled to a solid phase

A culture of Streptococcus dysgalactiae (C 26) was shown to bind only to 125I-IgG, whereas another S. dysgalactiae culture (C 12) bound both 125I-IgG and 125I-albumin. The IgG- binding proteins could be readily solubilized by lysozyme treatment of the bacteria and isolated by affinity chromatography on IgG Sepharose. The purified IgG-binding protein from S. dysgalactiae C 26, which lacked simultaneous albumin binding activity, precipitated with IgG preparations from man, cow, horse, pig and mouse but not with chicken IgG. This IgG-binding protein was coupled to CNBr-activated Sepharose and subsequently used for the purification of IgG from both bovine and human serum. SDS-PAGE and immunoelectrophoretic studies confirmed the purity of the eluted proteins.

Binding of Immunoglobulin G and Albumin to Streptococcus dysgalactiae

All 24 cultures of Streptococcus dysgalactiae investigated bound 125I-IgG, 13 cultures additionally interacted with 125I-albumin. Inhibition experiments with unlabelled IgG and albumin preparations from humans and various animal species indicated the specificity of the binding sites which showed characteristics of IgG Fc-receptors of type III and albumin-receptors of type c. IgG and albumin-binding proteins could be removed from the streptococcal surface by solubilization and subsequently isolated by affinity chromatography. The isolated binding proteins of S. dysgalactiae strains C 12 and C 8 obtained from IgG and albumin sepharose precipitated with IgG in immunodiffusion reactions, and in immunoelectrophoretic studies, and they reacted, after transfer onto nitrocellulose, with 125I-IgG or 125I-albumin and vice versa. Antisera produced against IgG-binding proteins of S. dysgalactiae C 12 inhibited binding of 125I-IgG and 125I-albumin. Solubilization of binding proteins by trypsinization yielded low molecular weight fragments with 125I-IgG but not with 125I-albumin binding activities. IgG-binding proteins isolated from S. dysgalactiae C 26 reacted with 125I-IgG but not with 125I-albumin, indicating the presence of 2 groups of type III Fc-receptors among S. dysgalactiae strains.

Effects of aflatoxin on the immune system of the chick

A decreased antibody response to injected sheep red blood cells and weight losses to the extent of 25-38% were observed in the bursa Fabricii and thymus in chicks fed an aflatoxin diet (10% w/w) for 3 weeks. The immune organs did not show any significant histopathological changes. The antibody titre decreased from day 6 of treatment onward until the 15th day, when the antibody titre had disappeared completely in the test chicks. The body weight gain was also less in test chicks (9.7-37.3%) given test feed or test feed along with Salmonella pullorum in water (10(8) cfu ml 1) in comparison to chicks given toxin-free diet and water after 3 weeks of feeding trials. The disappearing component in the test chicks' liver homogenate was found to be antigenic by immunoelectrophoretic studies.

Aggregation to Thrombin and Collagen of Platelets from a Glanzmann Thrombasthenic Patient Lacking Glycoproteins IIb and IIIa

The aim of this study was to investigate the platelets of a Glanzmann thrombasthenic patient, which in citrated PRP failed to respond to various agonists, but aggregated and secreted to high concentrations of thrombin (0.36, 0.72 and 1 U/ml) and collagen (4, 10 and 20 micrograms/ml) when washed and resuspended in a Tyrode-albumin solution (containing 2 mM Ca2+). Aggregation of the patient platelets was not affected by anti-IIb/IIIa monoclonal antibody (P18) which strongly inhibits thrombin or collagen induced aggregation of normal platelets. Washed platelets of this patient did not aggregate to ADP (10-100 microM) in the presence of added fibrinogen (2 mg/ml) nor bind 125I-labelled fibrinogen (40 to 320 micrograms/ml) when thrombin-stimulated. Different anti-IIb/IIIa monoclonal antibodies (P2, P18) when used in binding or crossed immunoelectrophoretic studies showed a complete absence of the IIb-IIIa glycoprotein complex on the patient platelets. Moreover, glycoproteins IIb or IIIa were absent on silver-stained two-dimensional (non-reduced/reduced) polyacrylamide gel separations of the patient platelets and were not detected by Western blots used in combination with anti-PLA1 (antigen present on IIIa), anti-Leka (antigen present on IIb). This study shows that platelets lacking glycoproteins IIb or IIIa can aggregate in response to high concentrations of collagen or thrombin when resuspended in the presence of physiological concentrations of calcium. Results obtained in this study could indicate the existence of other mechanisms (other than the IIb-IIIa glycoprotein complex) involving glycolipids, heparans, proteoglycans, and/or unknown membrane glycoproteins to mediate platelet aggregation of stimulated thrombasthenic platelets.

Evaluation of commercial imported fire ant extracts by crossed immunoelectrophoresis and radioallergosorbent test

Imported fire ant whole-body extracts (IFAWBE) from three major U.S. commercial suppliers and a reference IFAWBE and imported fire ant venom (IFAV) preparation were compared by crossed immunoelectrophoresis and RAST-inhibition testing. Results of crossed immunoelectrophoretic studies showed major differences in antigen content between commercial preparations. IFAWBE and IFAV RAST-inhibition testing showed that no commercial extract had equivalent inhibitory activity to the reference IFAWBE. Reference IFAWBE reached 50% inhibition of IFAWBE and IFAV RAST at 52 and 492 μg/ml, respectively. IFAV gave 50% inhibition of IFAWBE and IFAV RAST at 5.9 and 58 μg/ml, respectively. Of the commercial extracts, only those from one supplier were able to reach 50% inhibition of the reference IFAWBE RAST, and no commercial extract from any supplier was able to reach 50% inhibition of IFAV RAST. Analysis of inhibition slopes and intercepts confirmed that there were both qualitative and quantitative differences among the commercial extracts and the reference IFAWBE and IFAV. Our findings suggest possible reasons why some individuals fail to respond to testing or immunotherapy with IFAWBE and show the urgent need for standardization of allergenic extracts for diagnosis and treatment of imported fire ant sensitivity.

Extraction of serologic and delayed hypersensitivity antigens from spherules of Coccidioides immitis.

We have used an aqueous toluene extraction procedure to obtain antigens from mature spherules of Coccidioides immitis. This extract contained many antigens as determined by immunoblotting and two-dimensional immunoelectrophoretic studies. These included antigens with specificity for tube precipitin-type antibodies having molecular weights greater than or equal to 100 KDa. The extract also displayed lymphocyte-transforming activity when tested on human peripheral blood mononuclear leukocytes from donors who react to coccidioidal skin tests but elicited no such stimulation of cells from persons whose coccidioidal skin tests were nonreactive. At high concentrations of the extract, lymphocyte transformation did not occur, a finding that could not be explained by nonspecific toxicity. When gel filtration was employed to separate antigens by size, tube precipitin-like activity and specific coccidioidal delayed-type hypersensitivity displayed overlap, although only the latter activity was apparent in lower molecular weight pools.

Humoral and cellular response to infection with Echinostoma revolutum in the golden hamster, Mesocricetus auratus.

Laboratory hamsters (Mesocricetus auratus) were infected with Echinostoma revolutum (Trematoda). Immunoelectrophoretic studies of hamster serum showed no demonstrable antibody response to E. revolutum. Histopathologic examination of intestinal tissue of infected hamsters showed erosion of intestinal villi and lymphocytic infiltration as the primary host response. Spleens from infected hamsters were hyperplastic during the first 3 weeks of infection and atrophic from 4 to 8 weeks postinfection. Hamsters were unable to acquire a resistance to E. revolutum infection. Lack of resistance was demonstrated in hamsters where the parasite infection was no longer detected based on the absence of eggs in the faeces; these hamsters were then reinfected. Hamsters treated with the anthelmintic oxyclozanide were also reinfected with E. revolutum.

Immunogenic and antigenic characteristics of Salmonella heat-labile enterotoxin

The intramuscular immunization of rabbits with enterotoxin of S. weltevreden failed to provide protection against challenges with homologous Salmonella enterotoxin as well as heterologous enterotoxins (cholera toxin or E. coli LT). Similar results were obtained in rabbits immunized with cholera toxin, choleragenoid and E. coli LT. However, Salmonella antitoxin contained neutralizing antibodies against Salmonella enterotoxin (but not against cholera toxin) and thus was capable of neutralizing Salmonella enterotoxin when tested for skin permeability reaction. Immunodiffusion experiments showed that antitoxin prepared against the enterotoxin of one of the strains of S. weltevreden formed precipitin bands with enterotoxin preparations of 5 strains of S. weltevreden and 2 strains of S. anatum. However, Salmonella antitoxin failed to form precipitin bands with enterotoxins of other heterologous Salmonella species (S. dublin, S. enteritidis, S. hindmarsh and S. newport), cholera toxin and E. coli LT. The immunoelectrophoretic studies corroborated the results obtained by double immunodiffusion experiments. However, both Salmonella and cholera toxins migrated electrophoretically toward the cathode and resembled globulin in this respect. Salmonella enterotoxin, though immunogenic, yet proved unprotective through the parenteral route and appears to be antigenically distinct from cholera and E. coli enterotoxins.

Production, purification and partial characterization of a new adhesive factor (F42) produced by enterotoxigenic Escherichia coli isolated from pigs

Production of the F42 adhesive factor by porcine enterotoxigenic Escherichia coli (ETEC) grown on minimal solid medium was glucose-dependent. The addition of alanine and sodium acetate to this medium repressed the expression of this antigen whose production was also inhibited when the pH of the growing medium was lower than 7.4. The antigen was extracted from F42-positive ETEC grown in minimal liquid medium supplemented with 0.5% glucose. The cells were suspended in buffered 1 M NaCl and heated at 60°C. The supernatant was then treated with ammonium sulphate and the resulting precipitate treated with deoxycholate followed by chromatography of the deoxycholate-soluble material on Sepharose-4B. The molecular weight of F42 purified antigen was near 31,000 daltons and its pI 3.2, as determined by polyacrylamide gel electrophoresis and isoelectric focusing, respectively. Immunoelectrophoretic studies showed that the purified F42 antigen presented a slight anodic migration and was recognized only by its homologous antiserum.

Crossed immunoelectrophoretic studies of whole body extracts and venom from the imported fire ant Solenopsis invicta

Although allergic reactions occur after imported fire ant (IFA) sting, currently, only IFA whole body extract (IFAWBE) is available for diagnosis and immunotherapy of IFA-sensitive individuals. We report our crossed immunoelectrophoretic studies comparing antigenicity and allergenicity of Solenopsis invicta IFAWBE and IFA venom (IFAV). Rabbits were hyperimmunized with IFAWBE prepared from S. invicta or with IFAV obtained from S. invicta by an electric shock method. Crossed immunoelectrophoresis with anti-IFAWBE detected at least 29 precipitin lines in IFAWBE and three lines in IFAV, but none in a synthetic venom, transpiperidine. Anti-IFAV detected six precipitin lines in IFAV and five lines in IFAWBE, but no lines were detected with transpiperidine. Crossed-line immunoelectrophoresis confirmed the IFAV origin of at least three of the peaks in IFAWBE. Crossed radioimmunoelectrophoresis with 11 IFAWBE RAST-positive sera elicited radiostaining with five antigens in IFAWBE that were probably IFAV associated. One of these allergens was recognized by all sera; the other allergens were recognized by 8, 7, 5, and 4, respectively, of the 11 sera. Four of the antigens present in IFAV preparations had allergenicity. These findings indicate that IFA allergens probably originate in IFAV, but that transpiperidine, a major constituent of IFAV, does not appear to be immunogenic. The results suggest that IFAV may be a more appropriate reagent than IFAWBE for laboratory testing and for clinical diagnosis and immunotherapy of IFA-sensitive individuals.

Haemolytic activity in the serum of brown trout, Salmo trutta

Brown trout serum contains a natural, spontaneous, antibody‐independent lytic activity and a haemolysin antibody complement‐mediated lytic activity against unsensitized and trout antibody‐sensitized sheep erythrocytes, respectively. The use of various activators and inactivators of the mammalian complement system demonstrated that trout serum possesses complement or complement‐like components similar in activity to those present in the classical and alternative pathways found in mammals. A single injection of trout with sheep erythrocytes stimulated the production of antibody‐secreting cells in lymphoid organs and increased the levels of natural haemolysins. A second injection of sheep erythrocytes further raised the haemolysin values and antibody‐secreting cell counts. Serum complement from homologous or closely related fish species was more effective for use in the haemolysin and antibody‐secreting cell assays than that from heterologous sources, except guinea pig. Based on physico‐chemical properties, gel filtration and immunoelectrophoretic studies, natural and induced anti‐sheep erythrocyte haemolysins were found to be similar molecules and are possibly high molecular weight IgM antibodies.

Allergy to rats: Quantitative immunoelectrophoretic studies of rat dust as a source of inhalant allergen

The antigenic composition of an extract of rat dust, as a source of aeroallergens for rat-sensitive individuals, has been investigated and compared to the antigenic composition of rat saliva and urine. Of four main antigenic peaks identified by crossed immunoelectrophoresis, one antigenic peak (Ag 4) was demonstrated to be antigenically closely related to and with similar molecular weight (approximately 22 kd) and isoelectric point values as urinary prealbumin, already recognized as a major rat allergen. Ag 4 was present in all dusts studied and was also identified as a minor component of saliva. However, no component with the same electrophoretic mobility or physicochemical characteristics of the α 2-euglobulin of male rat urine that shares partial identity with the prealbumin was detected, even in dust collected from a male rat room. A second high molecular weight (>200 kd) component, Ag 1, present in most of the dust extracts, could not be detected in either urine or saliva. Crossed immunoelectrophoresis and skin prick tests confirmed the allergenicity of both these antigens. Analysis of an air filter sample taken within a male rat room revealed significant amounts of the “prealbumin” component, and a monospecific antiserum to this component was used to quantitate levels in dusts collected from various locations. These findings suggest that a major inhalant allergen present in rat dust is closely related to urinary prealbumin but that this and other allergenic components may not be derived predominantly from rat urine or saliva but possibly from secretions originating from the skin of the animals and present in the fur.

Serotyping of aeromonas species using passive haemagglutination

Antisera were raised with heat-killed vaccines prepared from 25 Aeromonas hydrophila strains, 4 A. sobria strains and one A. caviae strain. Twentyseven of these 30 antisera gave high titers when tested in the microtiter tray agglutination (MTA) test with their homologous antigen heated at 100°C for 30 min. Three O antisera gave low titers in the MTA test but reacted to high titers in the haemagglutination (HA) test with conserved sheep red blood cells coated with alkali-treated heat extract. All 27 antisera showing high titers in the MTA test, showed even higher titers in the HA test. Therefore, the HA technique was employed to type 306 strains isolated from surface and drinking water, from food samples and from faeces of human patients with diarrhea. Of 155 A. hydrophila strains 21 (14%) could not be typed. For A. sobria and A. caviae, the percentage of untypable strains was 46% and 68% respetively. Many A. sobria and A. caviae strains reacted to titer in A. hydrophila O antisera. A limited number of strains reacted to titer in more than one specific O antiserum. Immunoelectrophoretic studies indicated that the O antigen is often represented by more than one precipitation line and that true K antigens occur in Aeromonas.

Immune response toTrichinellaepitopes: the antiphosphorylcholine plaque-forming cell response during the biological cycle

Phosphorylcholine (PC), an immunodominant component of the cell wall of certain bacteria, fungi and nematodes, is known to induce low anti-PC antibody levels during natural infection by Trichinella spiralis. This article reports a study in which spleen cells from BCF1 mice infected with Trichinella sp. larvae were found to produce large numbers of direct haemolytic plaques in response to PC conjugated to sheep red blood cells (SRBC) after muscle-encysted larvae had been killed by treatment with mebendazole. Inhibition of the response by PC-chloride, immunodiffusion and immunoelectrophoretic studies with the anti-PC IgA (TEPC-15) and anti-idiotype T15 serum assays showed the plaque-forming cell (PFC) response to be specific for PC. The absence of haemolytic plaques when unconjugated SRBC or TNP-SRBC were used as indicator cells ruled out involvement of a polyclonal response. Greatest anti-PC PFC response was found to be associated with a microsomal fraction designated FCp1, a particulate fraction behaving as a thymus-dependent antigen. The FCp1 fractions from all four strains of Trichinella employed induced anti-PC PFC responses when injected into mice. These results suggest that FCp1 is a suitable antigen for use in detailed studies of immune responses to Trichinella and related parasites.