Gel Double Immunodiffusion Research Articles

Efficacy of Counterimmunoelectrophoresis in the Detection of Fungal Antibodies in Allergic Bronchopulmonary Mycoses1

The allergic bronchopulmonary mycoses (ABPM) are characterized by dual antibody response which includes immediate Type I and delayed Type III Arthus reactions to fungal antigens. In such cases the demonstration of precipitins is of diagnostic importance. In the present study the efficacy of counterimmunoelectrophoresis (CIE) has been compared with that of conventional agar gel double immunodiffusion technique (DID). Cases of allergic bronchopulmonary aspergillosis (ABPA) and candidiasis (ABPC) were diagnosed in a survey among patients with pulmonary diseases on the basis of positive clinical, radiological, mycological and immunological findings. Precipitating fungal antibodies were detected by CIE and DID techniques in 77 and 59 cases of ABPA and ABPC respectively. The former technique revealed double or multiple bands as against a single band observed in double immunodiffusion in a great majority of these cases. With CIE it was further possible to demonstrate the presence of precipitins in about 65 per cent of suspected cases of ABPM found otherwise negative in DID tests. The CIE has proved to be a more efficacious serodiagnostic procedure in allergic bronchopulmonary mycoses in which antibody levels are known to vary a great deal.

Preparation of antibody (IgG)-ricin A-chain conjugate and its biologic activity.

As a model experiment for the preparation of cancer chemotherapeutic agents, a disulfide-linked conjugate of rabbit anti-mouse IgG (RaMIgG) and ricin A-cahin was synthesized. Activated sulfhydryl groups were first introduced into RaMIgG and then the modified RaMIgG was reacted with ricin A-chain. The fraction containing the conjugate was obtained from the reaction mixture by gel filtration through Sephadex G-150 and was characterized by sodium dodecyl sulfate-polyacrylamide disc gel electrophoresis and double immunodiffusion in agar. The inhibitory activity of A-chain against protein synthesis in a cell-free system and the binding activity of RaMIgG to mouse B lymphocytes were both fully retained. The purified conjugate selectively inhibited protein synthesis in mouse B lymphocytes possessing cell surface Ig, but not that in thymocytes. The cytotoxicity was inhibited by adding mouse IgG and an excess amount of RaMIgG to the cell culture. The effects of unlinked RaMIgG and A-chain on the cytotoxicity were negligible even when the two were used as a mixture.

Gel double immunodiffusion studies with six human rhinoviruses

Rabbits were immunized on six occasions during a seven month period with fluorocarbon and sucrose denstiy gradient purified preparations of human rhinovirus types 1A,2,3,4,9, OR 14. Sera collected 1 week after the final immunization formed 1 or 2 preciptin lines when reacted by immunodiffusion against fluorocarbon purified preparations of each homologous immunizing virus. Heterologous preciptin cross reactions were detected between: RV1A antigen and rabbit sera to RV2, RV9 and RV14; RV2 antigen and sera to RV1A; RV14 antigen and sera to RV3. The heterologous "group" or C-antigenic nature of the cross reactions was suggested by the merging of heterologous preciptin lines formed against rabbit sera with "group" antibody preciptin lines formed against human sera and by the location of heterologous reaction close to the antigen well. In addition, the presence of C-antigenic particles in the fluorocarbon treated RV2 preparation was suggested by the demonstration that a subpopulation of viral particles migrated to a pH of 4.4 by isoelectric focusing.

510 GALACTOCEREBROSIDASE(GC) CROSS REACTING MATERIAL(CRM) 5X0 IN GLOBOID CELL LEUKODYSTROPHY(GLD)

The primary genetic defect in GLD is a deficiency of GC activity. In order to clarify the nature of the mutation in this disease, we have purified GC from placenta and used antibodies prepared against this normal enzyme to look for CRM in tissues of patients with GLD. Antigenic identity was observed between GC of normal brain, liver and skin fibroblasts. The antibodies were monospecific to this enzyme, precipitated only β-galactosidase activities toward galactosylceramide and lactosylceramide (assay I)and did not react with either GM1 or neutral β-galactosidases. Preparations of 1 brain, 2 livers and 4 fibroblast lines from GLD patients were examined by double gel immunodiffusion against the anti-GC antibodies and found to be CRM positive. The patients' CRM was identical to the normal enzyme and demonstrated a similar electrophpretic mobility on immunoelectrophoresis. However, activity towards naphthol-AS-BI-β-galactoside could be demonstrated in the immune precipitates of normal controls and other sphingo-lipidoses but not in those of the GLD patients. A single radial immunodiffusion assay was developed for quantitation of the CRM in the various tissues independent of its enzymic activity. Quantities of CRM within the normal range were determined in the brain, liver, and skin fibroblasts of the GLD patients. The existence of normal amounts of CRM in the patient's tissues indicates a structural gene mutation producing a protein which has lost its enzymic activity but retained its antigenicity.

Brain isoenzyme of creatine kinase. I. Purification of rabbit enzyme and production of specific antibodies.

Creatine kinase (EC 2.7.3.2) of rabbit brain (BB) was purified to hemogeneity as judged by sedimentation velocity analysis in the ultracentrifuge. Nevertheless, the purified enzyme contained as many as seven extraneous protein species when examined by disc electrophoresis in polyacrylamide gel. The purified preparation also showed antigenic heterogeneity when it reacted in agar gel double immunodiffusion analysis with antibodies produced against it in the rooster. At least some of the extraneous proteins appeared to arise from BB during storage. Subsequently BB was purified to hemogeneity by disc electrophoresis in polyacrylamide gel. It was excised from the gel and injected repeatedly with Freund's complete adjuvant into roosters. A single immunoprecipitin line was noted in agar gel double immunodiffusion analysis when partially pure enzyme reacted with the antibodies raised to the electrophoretically pure protein. These antibodies inhibited the enzymatic activity of BB in both soluble and insoluble immune complexes. They did not, however, form immunoprecipitates with or inhibit rabbit muscle (MM) or rabbit hybrid (MB) creatine kinases. The antibodies were therefore specific for BB and did not react with MM or MB.

Identification of the 7S globulin with β-conglycinin in soybean seeds

The 7S globulin, a major ultracentrifugal component with the 11S globulin, was identical with β-conglycinin one of four antigenic components in the reserve proteins of soybean seeds ( Glycine max). Double gel immunodiffusion and immunoelectrophoresis in agar gel were used for their identification. In addition, some characteristic properties on ultracentrifugation and in carbohydrate content agreed well between the proteins. Their MWs were ca 180000.

La recherche de l'alpha 1 foetoproteine par immunoelectrophorèse à contre courant sur cellogel : Comparaison avec deux autres techniques immunochimiques

The authors compared the sensitivity of three immunochemical methods demonstrating the presence of alphafetoprotein in biological fluids; the study included 537 serums, ascitis fluids and tumor extracts. Double gel immunodiffusion is a reliable test in primary liver cancer diagnosis where high levels of alphafetoprotein are to be expected. On the contrary counterimmunoelectrophoresis on Cellogel is far better to detect values lower than 10 mg/l. With this quite sensitive method, alphafetoprotein was shown in primary liver cancer; the significance of its appearance in other liver diseases (secondary liver cancer, hepatitis, cirrhosis) is being discussed here.

Association-dissociation phenomena in glycinin

Ultraviolet difference spectra indicated that both tyrosine (287 mμ) and tryptophan (293 mμ) groups are exposed by treatment of glycinin with different concentrations of urea and with acid (pH 2.0). Glycinin solutions (0.34%) when heated at temperatures above 70 ° become increasingly turbid and the protein precipitates at 90 °. Glycinin appears to be stable to heating up to 50 ° and very little change is observed between 50 and 70 °. Heat, acid (pH 2.0), alkali (pH 11.0), and urea treatment cause dissociation of glycinin into subunits and subunit association products as examined by disc electrophoresis. Undissociated glycinin molecules can be detected after heat, acid, and alkali treatment by double gel immunodiffusion and immunoelectrophoresis. Urea (6 m) causes irreversible and complete dissociation of the protein into subunits. The subunits of glycinin, and reduced and carboxymethylated glycinin (RCM-glycinin) are soluble in the solvent system phenol-acetic acid-0.2 m mercaptoethanol, 2:1:1, w/v/v made 5 m in urea (PAMU). Disc electrophoresis using the PAMU solvent separates six major sub-units derived from glycinin, and five major subunits from RCM-glycinin. General considerations from existing data about the structure of glycinin are discussed.

Immunochemical properties of the 11S component of soybean proteins

Immunization of rabbits with soybean proteins in Freund's adjuvant results in the formation of specific antisera that can be used for the detection of these proteins by several immunochemical methods. The isolated 11S soybean protein was immunochemically homogeneous by double gel immunodiffusion, disc immunoelectrophoresis, and immunoelectrophoresis in agar. This protein retained its immunological properties after heating up to 80 °. Its diffusion coefficient was calculated by the double gel gradient diffusion technique. A water-insoluble polymer of the 11S soybean protein obtained by the reaction of the protein with ethyl chloroformate was used as an immunoadsorbent for the isolation of the specific anti-11S soybean protein antibody.