Gel Diffusion Technique Research Articles

APPLICATION OF A RADIAL DIFFUSION ASSAY FOR THE MEASUREMENT OF β-GLUCANASE IN MALT

An assay for the routine determination of β-glucanase by a gel diffusion technique has been established. The method has many features which make it preferable to the viscometric procedure currently employed.

Teichoic acid antibodies in osteomyelitis and septic arthritis caused by Staphylococcus aureus.

Counterimmunoelectrophoresis and gel-diffusion techniques were used to study the development of teichoic acid antibodies in eighteen patients with osteomyelitis and seven patients with septic arthritis caused by Staphylococcus aureus. Nine of eleven patients with acute osteomyelitis and three of seven with chronic osteomyelitis had a positive response to tests for teichoic acid antibody. However, only two of seven patients with septic arthritis generated a positive teichoic-acid antibody response. In two patients the test was extremely valuable in the diagnosis and management of osteomyelitis. Antibody detection appears to be a sensitive test for detecting staphylococcal osteomyelitis, especially the acute variety. It does not appear to be a reliable test for septic arthritis. It is also useful for the detection of antibody in patients who had received prior antibiotic therapy, yet have persistent foci of infection. The presence of the teichoic acid antibody, as well as its titer, is of diagnostic value in patients with serious infections caused by Staphylococcus aureus. The detection and quantification of teichoic acid antibodies is of great value for the early diagnosis of patients with acute osteomyelitis caused by Staphylococcus aureus and for assessing the clinical response of such patients.

Degradation of amyloid by a serum component and inhibition of degradation.

ADA of human serum was demonstrated and investigated with an agar gel diffusion technique using amyloid-impregnated agar plates. Sera of 20 healthy adults, 40 patients with AA-amyloidosis, and 86 nonamyloidotic patients were tested. The presence of an ADF, showing enzymatic properties and strongly bound to albumin, was demonstrated in normals and amyloidotic and nonamyloidotic patients. ADA in the serum of amyloidotic and cirrhotic patients was markedly decreased due to the presence of an inhibitor of ADF. ADA of amyloidotic sera was restored to normal by EDTA, citric acid, and ascorbic acid. The ADA of 16 FMF patients and four of 34 patients with rheumatoid arthritis without amyloidosis was intermediate between normal and amyloidotic values, indicating the presence of lADF at low concentrations in these patients. These findings suggest that amyloid is a normal protein metabolite, possibly with a high metabolic turnover. Accumulation of amyloid may be caused by decrease of the ADA of the serum by its inhibitor, rather than by accelerated production.

Precipitating antibodies against excretory/secretory antigens of Fasciola hepatica in sheep serum

Gel diffusion techniques were used to study antigen-antibody reactions in precipitates forming around juvenile, semi-mature and adult Fasciola hepatica cultured in serum from infected sheep. The number of reactions was analysed in both primary and challenge infections. The number of antigens shared by the various developmental stages of the fluke was also examined. At least 2 antigens were involved in precipitate formation around juvenile flukes. These antigens were also produced by the later stages of development. Two additional antigens were produced by semi-mature flukes and these, together with two others, were produced by adult flukes. Challenge infections had little effect on the number of antigens or antibodies in the precipitate.

Microplate enzyme-linked immunosorbent assay for bovine leukemia virus antibody.

A microplate enzyme-linked immunosorbent assay method was developed for the measurement of bovine immunoglobulin G antibody specific to the envelope antigen (glycoprotein 60) of bovine leukemia virus. The test was then performed on 440 serum samples from dairy cows belonging to herds in which bovine leukemia was suspected or which were leukemia free, and the results were compared with those obtained with the gel-diffusion technique.

Study of guinea pig and human antibodies to toluene diisocyanate.

Antibodies to toluene diisocyanate (TDI) have previously been reported in both sensitized guinea pigs and industrial workers. Detection of antibodies to TDI is difficult because the ability of TDI to self-polymerize and cross-link carrier proteins causes problems in preparing satisfactory hapten-protein conjugate antigens. Use of monoisocyanate analogues of TDI for antigen preparation eliminates the problem. This study compared 3 tolyl monoisocyanate isomers with TDI for haptenic ability to detect antibodies to TDI in sensitized guinea pigs and humans. Using the gel-diffusion technique, antibodies from guinea pigs reacted with ortho-, meta- and para-tolyl monoisocyanate haptens and with a TDI hapten. In the guinea pigs, reactions appeared to be strongest with the TDI hapten and weakest with meta-tolyl isocyanate hapten. On the other hand, antibodies in TDI-sensitized workers, as determined by radioallergosorbent testing, reacted comparably with each of the haptens.

Stability of Five Antibiotics

The stability of methicillin, amikacin, erythromycin lactobionate, vancomycin, and ticarcillin in plastic intravenous containers of sodium chloride injection 0.9%, USP and dextrose 5% injection, USP was studied. The study was conducted under aseptic conditions for a period of 24 hours. The samples, drawn at various times, were assayed by a modified agar gel diffusion technique. The study solutions were stored at room temperature. This study revealed that amikacin, methicillin, vancomycin, and ticarcillin are stable for 24 hours in the test solutions. Erythromycin lactobionate is stable for 24 hours in normal saline, but the addition of sodium bicarbonate 4% (Neut®) was necessary to assure stability in dextrose 5 %.

Fixation and Inactivation of Staphylococcal Leukocidin by Phosphatidylcholine and Ganglioside G M1 in Rabbit Polymorphonuclear Leukocytes

Staphylococcal leukocidin is resolved by chromatography on carboxymethyl cellulose columns into two components, which are designated F (fast) and S (slow). Fixation and inactivation of both components were studied as follows. (i) Leukocidin activity was confined to the first 10 min of intoxication, and the maximal effect resulted from treating 10(6) rabbit peripheral polymorphonuclear leukocytes per 20 mul with 0.5 ng of each component of leukocidin. The S component was more responsible for the interaction with the leukocytes than the F component. (ii) The F component was inactivated by phosphatidylcholine at concentrations which corresponded to molar proportions of 1:1 and bound to [(14)C]phosphatidylcholine at equimolar proportions. (iii) The S component was inactivated by ganglioside G(M1) at 1:1 molar proportions, but not by any of the related glycolipids. Ganglioside G(M1) also was precipitated with the S component by a gel diffusion technique. Subunit B of cholera toxin competitively inhibited the binding of the S component to rabbit leukocyte membranes. This indicates that ganglioside G(M1) may resemble or be part of the receptor site for the S component.

Serological differentiation between infected and vaccinated cattle by using purified soluble antigens from Brucella abortus in a hemagglutination system.

Serological tests used in current brucellosis eradication schemes, such as bacterial tube agglutination, do not readily distinguish between infected animals and those immunized with strain 19 or 45/20 Brucella abortus vaccines. In this study, sera from naturally infected cattle were used to identify serologically important antigens in extracts of virulent B. abortus by gel diffusion techniques. Antisera from rabbits hyperimmunized with selected precipitation lines were used for purification by affinity chromatography of two precipitating and one non-precipitating antigen from crude bacterial extracts. A passive hemagglutination test using these antigens was developed. A number of characterized bovine sera were screened by passive hemagglutination and conventional bacterial tube agglutination test. A considerable improvement in discrimination between sera from infected and vaccinated cattle was obtained with the hemagglutination test compared with bacterial tube agglutination.

Comparison of three methods for the estimation of plasma antithrombin.

Plasma antithrombin levels were measured by clotting, immunological, and amidolytic methods on two groups of subjects: 20 normal individuals and nine patients studied serially post-operatively (hip replacement). The postoperative patients were observed for the emergence of deep-vein thrombosis using 125I-fibrinogen uptake measurements (FUT). The three methods gave similar ranges for the normal subjects, were reproducible (cv less than 5%), and detected early postoperative reduction of antithrombin levels. All three methods failed to show any significant differences in preoperative antithrombin levels between the positive and negative FUT groups. Correlation studies were performed on the pooled data from the normal and postoperative group (range 60-130% of normal; 100 samples). The best correlation (r = 0.75; P less than 0.01) was achieved with the chromogenic kit assay method versus the Mancini immunoassay technique. The thrombin agarose (total antithrombin) gel diffusion technique correlated less well with the chromogenic (r = 0.65; P less than 0.01) and Mancini immunoassay (r = 0.45; P less than 0.01) methods. It is concluded that the chromogenic kit method gives a rapid, reproducible, and specific measurement of antithrombin III. The thrombin agarose diffusion method, though not specific for antithrombin III, is a cheap and simple method to perform. The potential of the three methods for detecting the prethrombotic stage and early thrombosis is discussed.

Antibodies to proteins from yolk of immunized hens.

Immunoglobulins IgY, extracted from yolks of hens immunized against several proteins and natural mixtures of proteins were examined by the Ouchterloney gel diffusion and Laurell immuno-electrophoresis techniques. The molecular weights of the proteins ranged from 8 x 10(6) to less than 2 x 10(4). The hens produced IgY antibodies readily when injected with the high molecular antigens > 150 000 but did not react as well to antigens of relatively low molecular weight < 30 000. Optimal precipitin reactions were obtained with low molecular antigens when the agarose gel contained 1.5 M NaCl. Salt concentration (0.15-1.5 M NaCl) has no effect on the precipitin reaction in gels on the systems of high molecular antigens and their IgY antibodies apart from a displacement of the precipitin line. From the relative positions of the precipitin lines to the wells containing the reactants it would appear that the IgY formed dimers when the concentration of NaCl is 1.6 M and remain as monomers in gels containing 0.15 M NaCl. An explanation of the salt effect is offered.

Preparation of conjugates using two tetanus toxin derived fragments: Their binding to gangliosides and isolated synaptic membranes and their immunological properties

B-II b fragment of tetanus toxin was coupled to I bc fragment which is the other part of the toxin molecule. The two fragments were conjugated with glutaraldehyde, by disulfide link formation or with m-maleimido-benzoyl- N-hydroxysuccinimide ester (MBS). The capacity of B-II b fragment to bind to gangliosides or crude synaptic membranes was retained after its coupling to I bc fragment using disulfide link formation or MBS. The immunological structure of the conjugates was analyzed by double gel diffusion techniques. The binding properties of B-II b fragment were lost in conjugates prepared using glutaraldehyde. B-II b fragment should be tried as a transport agent into the central nervous system for compounds without affinity for nervous tissue.

Influence of methyl methacrylate on quantitative gel diffusion assay of immunoglobulins.

Infection is not a common problem following implant surgery using the bone cement, polymethyl methacrylate. When infection occurs, its often disastrous results makes for major complications. Many methods have been used in an attempt to reduce bacterial contamination at the time of implant surgery, but little attention has been given to the problem of possible effects of implant materials on the immune mechanisms of the host. In the experiments reported here, the in vitro effect of methyl methacrylate monomer on human immunoglobulins was determined using a quantitative gel diffusion technique. When methyl methacrylate monomer was added to serum samples containing immunoglobulin, it caused no statistically significant change in the immunoglobulin concentration of immunoglobulin-G, immunoglobulin-A, or immunoglobulin-M. It is possible that methyl methacrylate could affect other types of antigen antibody reactions which may be important in resisting infection, but the evidence reported here indicates that in low concentrations, methyl methacrylate monomer does not affect the normal reactivity of immunoglobulins of the G, M, or A class.

Gel and Solution Growth of Large Crystals of Silver Iodide

After reviewing various methods for the preparation of AgI single crystals, this paper describes experiments based on the growth of such crystals from AgI-H I-H2O solution. Hexagonal pyramids of the β-phase have been produced by a gel diffusion technique, in which supersaturation is attained by slow water dilution. On the other side, hexagonal plates and pyramlds of the β-phase were grown by a convective circulation method in which supersaturation occurs by tcmperature Increase. Some characterIStic properties, e.g. purity, structure, refractive index, optical absorption, photoluminescence, crystallographic polarity and photographic sensitivity, have been measured and are discussed.

Evaluation of the counter-immunoelectrophoresis (CIEP) and agar gel diffusion (AGD) techniques in the diagnosis of hydatidosis in Iran

Evaluation of the counter-immunoelectrophoresis (CIEP) and agar gel diffusion (AGD) techniques in the diagnosis of hydatidosis in Iran

Beetle Transmission of Cowpea Chlorotic Mottle Virus

HOBBS, H. A., and J. P. FULTON. 1979. Beetle transmission of cowpea, chlorotic mottle virus. Phytopathology 69:255-256. Cowpea chlorotic mottle virus was transmitted by the bean leaf beetle, from and transmitted to beans. Levels were much lower when the virus was Cerotoma trifurcata, and by the spotted cucumber beetle, Diabrotica either acquired from or transmitted to cowpeas. Lespedeza cuneata, which undecimpunctata howardii. Three strains of the virus were transmitted at may be an overwintering host, was a poor source of virus for vectors. low levels. Levels of transmission were highest when virus was acquired Several viruses in the bromovirus group are transmitted by antiserum, using the Ouchterlony gel diffusion technique. Virus in leaffeeding beetles. Cowpea chlorotic mottle virus (CCMV) was beetles that had fed on infected plants was tested by severing one transmitted by the chrysomelid species Cerotoma trifurcata metathoracic leg and collecting in a capillary tube the hemolymph (Forster) and Diabrotica undecimpunctata howardii (Barber) (6); that exuded. Hemolymph was diluted approximately 1:300 in 0.01 the bean yellow stipple strain (BYSV) of CCMV by C. ruficornis M phosphate buffer, pH 7.2, and inoculated to young Monarch Olivier and D. balteata LeConte (2,3); and broad bean mottle virus cowpea plants. (BBMV) by D. undecimpunctata howardii, Acalymma trivittatum (Mannerheim), and Colaspis brunnea (F.) (7). Levels of transmission were moderate to low in all cases. Because CCMV is

Staphylococcus aureus Bacteremia: Relationship between Formation of Antibodies to Teichoic Acid and Development of Metastatic Abscesses

Of 50 patients with bacteremia due to Staphylococcus aureus but without clinical evidence of endocarditis, 24 developed antibodies to the cell wall teichoic acid of S. aureus that were demonstrable by counterimmunoelectrophoresis. However, only 16 of the 24 patients developed titers of antibodies high enough for detection by passive gel diffusion. Eleven of the 16 patients developed evidence of complications due to metastatic infection. In contrast, of the 34 patients who were antibody-negative by gel diffusion, only one patient developed evidence of metastatic seeding. Thus, the development of antibodies to teichoic acid at a level detectable by the gel diffusion technique is regularly associated with complicated infections due to S. aureus that require more prolonged therapy, whereas bacteremic patients not developing such an antibody response rarely develop complications and may be treated with a two-week course of therapy.

The immune respone of plaice (Pleuronectes platessaL.) to the metacercariae ofCryptocotyle linguaandRhipidocoytle johnstonei

The humoral immune response of plaice to infestations of selected metacercariae was studied. Precipitating antibodies to both Cryptocotyle lingua and Rhipidocotyle johnstonei were demonstrated using agar gel diffusion techniques. Immunochemical investigations of these antibodies indicated they were macroglobulins, resembling immunoglobulin M (IgM) of mammals. The rate and magnitude of antibody production by the host was determined by the ambient temperature.

Immunochemical studies on human cancer. II. Detection of a urinary antigenic substance common to gastric cancer tissue.

Antibody reacting with urine from cancer patients was produced in rabbits immunized with urine from gastric cancer patients. Specimens of intact urine from the cancer patients revealed a direct precipitation reaction with the specific antiserum by the agar gel diffusion technique. The reaction of the specific antiserum with urine from cancer patients was inhibited by most concentrated urine specimens from the cancer patients. The reaction was also inhibited by D-glucosamine hydrochloride and D-galactosamine hydrochloride, i.e., by the same sugars as in the case of the reaction of anti-gastric cancer immune serum.

Purification and characterization of microsomal and lysosomal beta-glucuronidase from rat liver by use of immunoaffinity chromatography.

Rat liver beta-glucuronidase (EC 3.2.1.31), both from microsomal and lysosomal fractions, were purified about 9500-fold over the homogenate with high yield using affinity chromatography prepared by coupling purified specific immunoglobulin G against rat preputial gland beta-glucuronidase to Sepharose 2B and isoelectric focusing. The purified enzymes appeared homogeneous on electrophoresis in polyacrylamide gel and had a molecular weight of approximately 310000. In dodecylsulfate polyacrylamide gel electrophoresis, the microsomal beta-glucuronidase showed a single band corresponding to a molecular weight of 79000, while the lysosomal beta-glucuronidase had three distinct bands which consisted of one major and two minor bands corresponding to molecular weight of 79000, 74000, and 70000, respectively. A broad pH activity curve with a single optimum at pH 4.4 was observed in both the microsomal and the lysosomal beta-glucuronidases. Immunological gel diffusion technique with rabbit antiserum against rat liver lysosomal beta-glucuronidase revealed that both enzymes had the same or quite similar antigenic determinants.