Double Immunodiffusion Technique Research Articles

Immunochemical studies of high and low molecular forms of urokinase.

The immunochemical properties of high and low molecular forms of urokinase (HMW-UK, MW 53,000, 124,000 IU/mg protein; LMW-UK, MW 32,000, 230,000 IU/mg protein) were studied with specific antisera against the functionally active heavy chain (H chain, MW 31,000, 201,000 IU/mg protein) and the light chain (L chain, MW 18,000) of HMW-UK. Using a double immunodiffusion technique, LMW-UK did not demonstrate L chain antigenicity in the molecule. Anti-L-chain serum exerted no effect on LMW-UK and the H chain, but anti-H-chain serum strongly inhibited the fibrinolytic activity of all the active enzymes (HMW-UK, LMW-UK, and H chain). Anti-L chain serum was found to exert an antifibrinolytic effect on HMW-UK.

Caseous lymphadenitis in goats.

Two dairy goat stud herds were surveyed for Corynebacterium ovis infection by clinical examination and serum tests using both the haemolysis inhibition and double immunodiffusion techniques for detection of C. ovis antitoxin. In one herd, of 53 goats 11 had abscesses from which C. ovis was recorded; 33 and 29 goats were positive to the haemolysis inhibition and double immunodiffusion tests respectively. Serums from 10 and 9 of 23 kids gave reactions positive to the respective tests with 19 of 23 kids having been born to does with demonstrable antibody. In a second herd of 57 goats 16 had superficial abscesses containing C. ovis; serums from 34 and 45 goats gave positive reactions to the haemolysis inhibition and double immunodiffusion tests respectively. The prevalence of caseous lymphadenitis in these stud herds was a cause of economic loss to the owners through euthanasia of severely affected goats, reduced milk production and emaciation, the cost of labour and drugs for treatment, jeopardisation of income from boarding or mating with outside stock and the appearance of goats at stud shows, and sale of valuable breeding stock to local and export markets.

Qualitative and quantitative abnormalities of argininosuccinate synthetase in citrullinemia

Enzymological and immunochemical analyses of the liver were performed in seven Japanese patients with citrullinemia. Among the urea cycle enzymes in the liver, only the activity of argininosuccinate synthetase was specifically decreased to 2 to 50% of normal controls. Liver argininosuccinate synthetase of patients was indistinguishable from that of controls when tested immunochemically by Ouchterlony's double immunodiffusion technique with anti-rat argininosuccinate synthetase antiserum. Immunochemical analysis by means of the single radial immunodiffusion revealed that the decrease in the activity of liver argininosuccinate synthetase was explainable by a decrease in the amount of the enzyme protein in five patients, while the decrease in the activity in the other two patients was not accompanied by a decrease of enzyme protein. The K m values for the substrates of liver argininosuccinate synthetase of the former five were similar to those of the control, while the kinetic properties of the latter two were quite different in terms of higher K m values and negative cooperativity. From these results, we consider that citrullinemia may consist of more than one type including qualitative or quantitative abnormalities of argininosuccinate synthetase caused by some defects in certain genes or in the epigenetic processes in the liver.

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.

Dependence of diffusion coefficients and immunoprecipitating titers on pH: Albumin and IgG in human serum and their rabbit antibodies

A new two-dimensional double-immunodiffusion technique in gel plates, the two-cross technique, has been applied to the investigation of immunosystems in the pH range from pH 5.0 to pH 8.5. The results can be summarized as follows: (a) With increasing pH values of the medium, precipitating titers of all the proteins involved in the antigen-antibody systems examined increased. Consequently, the solubility of aggregates of the primary antigen-antibody complex decreased. (b) In the human albumin-rabbit antibody system the stoichiometry of the primary complex varies in the pH range from 5 to 7, the antigen-to-antibody ratio at pH 5 being double the ratio at pH 7. (c) The antigen-to-antibody ratio of the system human IgG in serum-rabbit antibodies is constant from pH 5 up to pH 7, and then slightly changes in favor of the ratio. (d) Human albumin in serum steadily increased its molecular association with the rising pH, reaching a mean molecular association number N = 2.2 at pH 8.5, while the human IgG in serum was in a monomeric form from pH 5 up to pH 7.5, and then associated up to N = 1.6 at pH 8.5. (e) The mean association number of rabbit antibody preparations used was approximately N = 1.85 between pH values 5 and 7 and then increased up to approximately N = 3.2 at pH 8.5, depending on the batch and on the preparation procedure used.

A simplified double immunodiffusion technique for detection of Corynebacterium ovis antitoxin

A method is described for obtaining yields of Corynebacterium ovis exotoxin sufficiently high to allow use of unconcentrated supernatant and serum as the reactants in a double immunodiffusion test. C ovis supernatant with a haemolytic titre or rabbit dermal necrosis titre of 1:16,394 or greater produced readily detectable precipitation lines with specific antitoxin in the range of concentrations commonly found in sera from cases of natural C ovis infection. When used as a diagnostic screening test, sera from 32 confirmed cases of caseous lymphadenitis in sheep and goats were positive whereas sera from 16 sheep free of caseous lymphadenitis were negative. Sera from 10 out of 16 kids which had been suckled by mothers from an affected flock of goats were positive.

Precipitation of animal plasma proteins by puff-adder venom

1. 1. Plasma and serum samples obtained from various animals never previously exposed to snakes or snake venom were diffused against different concentrations of puff-adder, Bitis arietans, venom using the double immunodiffusion technique. 2. 2. Depending upon venom concentration, two precipitin arcs could be produced in the case of all plasma samples used. No serum samples showed any arcs except pigeon serum, where one precipitin line was observed. 3. 3. By altering the concentration of snake venom between 1% and 10% when immunodiffusing against plasma a change in position of the precipitin lines was observed and also the disappearance of one or both of the two bands at higher concentrations. This indicates that the arcs observed are in all probability due to precipitation of plasma protein fractions. 4. 4. Previous results indicated that one of the two bands observed when diffusing venom against plasma was due to the precipitation of fibrinogen. By diffusing snake venom against heparin we have now shown that the second band involves this molecule and is not due to another coagulation factor as was suggested previously.

Antigenic relationship betweenHaemophilus influenzae andHaemophilus isolated from porcine pleuropneumonia

Comparative double immunodiffusion techniques were used to study capsular and O antigenic relationships betweenHaemophilus influenzae types a-fandH. pleuropneumoniae types 1–5 and a strain (202) closely related toH. pleuropneumoniae. Culture fluids or culture supernatants were used as antigens and rabbit antisera were produced against cell suspensions of the strains tested. A reaction of identity was obtained between the capsular precipitate ofH. influenzae c and a precipitate formed by strain 202, when developed with anti-H. influenzae c serum or the serum produced against strain 202. Mutual cross-absorption of capsular antibodies was also demonstrable. No other capsular or O antigenic cross-reactivity was demonstrable between the strains tested.

Cross-reactivity of the O antigens among Haemophilus influenzae type b strains.

The double-immunodiffusion technique was used to compare the O antigens of 18 meningeal Haemophilus influenzae type b strains with each other and with H. influenzae strains representing the capsular types a-f. The O antigens of the meningeal strains and the reference type b strain (strain Rab) cross-reacted to a large extent, while only a few cross-reactions were demonstrable with the other serotypes.

A serological comparison of some species of microsporida

Double immunodiffusion techniques were used to investigate the taxonomic relationships between six different microsporidian isolates. Microsporidia used included Nosema bombycis, N. algerae, N. plodiae, and three organisms morphologically similar to N. necatrix. Antigens were extracted from spores after disruption in an MSK Braun cell homogenizer. Cross-reactions were seen between N. plodiae and two of the N. necatrix isolates, while the third N. necatrix, N. bombycis, and N. algerae were antigenically unrelated. One of the N. necatrix isolates revealed temperature-related antigenic differences, but no antigenic differences resulted from aging spores for 5 months before disruption.

Immunogenic capacity of ribosomes of Salmonella typhi interfered with a flagellin-like material contaminant.

The double-immunodiffusion technique and sodium dodecyl sulfate-polyacrylamide electrophoresis were used to demonstrate the presence of flagellin-like material strongly attached to ribosomes of Salmonella typhi Ty 2. This flagellin-like material contaminating the ribosome preparation interferes with the induction of antiribosome serum promoting the formation of antisera reacting either only with flagellin or in some cases with flagellin and ribosomes, but giving a very weak reaction with the latter. The interference is also observed when purified ribosomes from a nonflagellated mutant of S. typhi (S. typhi O-901) mixed with purified S. typhi Ty 2 flagellin are utilized as antigens. The antiribosome sera obtained with ribosomes from S. typhi O-901 have a considerably higher titer than those that are interfered with. These sera were able to react with ribosomes obtained from several related species and did not react with flagella-derived flagellin of S. typhi Ty 2.

Serological comparison of three strains of Aedes aegypti

Young adults of three strains of the mosquito, Aedes aegypti, were compared serologically by means of the double-immunodiffusion technique. 1. 1. Strains and sexes were serologically distinguishable. 2. 2. Differences in antigenic composition were evident among the strains and sexes. 3. 3. Degree of intraspecific serological relationship varied with sex.

Human fetal intestinal alkaline phosphatase

The properties of meconium and neonatal fecal alkaline phosphatases have been compared with those of purified human intestinal ALP in order to study the properties of human fetal intestinal ALP. The results obtained were as follows: 1. (1) The electrophoretic mobility of meconium ALP was different from that of adult intestinal ALP. 2. (2) The active band of neonatal fecal ALP migrated toward the cathode depending on the postnatal period. The later the specimens were obtained, the further the active band moved towards the cathode until the 5th postnatal day, when the mobility became similar to that of adult intestinal ALP. 3. (3) Meconium and neonatal fecal ALPs are identical with purified human intestinal ALP in the following properties: (a) sensitivity to L-phenylalanine and imidazole, (b) heat stability at 56° C and 65°C, (c) K M value using phenylphosphate as substrate, (d) the double immunodiffusion technique, (e) reaction after treatment with anti-human intestinal ALP antibody, and (f) radioimmunoassay in the intestinal ALP assay system. 4. (4) The electrophoretic mobility of meconium and neonatal fecal ALPs were altered by neuraminidase treatment, and the active band moved towards the cathode to the same electrophoretic position with the same breadth as that of purified human intestinal ALP, which showed no migration by this treatment. These results show that the human fetal intestinal ALP is not a new ALP isoenzyme but modified enzyme of adult intestinal ALP. The difference in electrophoretic mobilities between fetal and adult intestinal ALPs is probably due to the difference in their content of sialic acid.

Nitration of tyrosyl residues in human alpha-lactalbumin. Effect on lactose synthase specifier activity.

Alpha-Lactalbumin isolated from human milk was reacted with tetranitromethane in molar excess of 8-32 mol/mol of tyrosine. After gel filtration on Sephadex G-75, followed by chromatographic fractionation using DEAE-Sephadex A-25, three main components were separated, which differed from one another in the extent of nitration. These protein fractions were found to contain, respectively, one and two nitrotyrosine residues, or two nitrotyrosine residues together with one nitrotryptophan. The lactose synthase specifier activity of each of these components was measured and compared with that of unsubstituted alpha-lactalbumin. Comparison of kinetic parameters showed the chemically modified proteins to be only slightly less active when tyrosines were the sole residues modified. In sharp contrast the additional nitration of a single tryptophan residue totally abolished the specifying activity of alpha-lactalbumin. Circular dichroism spectra of the tryptophan derivative revealed some structural alteration when compared with the other two and with the native protein. The conclusion could also be confirmed by using a double-immunodiffusion technique. After hydrolysis of the derivatives with thermolysin, it was possible to localize the substituted residues in the known sequence of human alpha-lactalbumin. Tyrosine-103 was found to be more easily nitrated than tyrosine-18. These two residues seem, therefore, to be on the outer surface of the molecule and more exposed than tyrosine-36 and tyrosine-50. Some precautions are indicated in the use of tetranitromethane as a nitrating agent on the basis of complex products observed in the nitration of the free amino acids tyrosine and tryptophan and their derivatives.

Improved Estimation of Urinary Myoglobulin by Counterimmunoelectrophoresis, as Compared with the Double Immunodiffusion Technique

We describe a simple, sensitive, and relatively inexpensive counterimmunoelectrophoretic technique for detection and estimation of myoglobin. The test can be completed within 90 min, compared with 12-48 h required by double immunodiffusion. With the counterimmunoelectrophoretic technique we could detect myoglobin in concentrations of 2-3 mg/liter, while the detection limit by double immunodiffusion was about 9 mg/liter.

Lipoprotein of the Density 1. 006–1. 020 in the Plasma of Patients with Type III Hyperlipoproteinaemia in the Postabsorptive State

Using a rate zonal ultracentrifugal technique in 10 patients with type III hyperlipoproteinaemia, very low density lipoproteins (VLDL) (d less than 1.006, S(f) greater than 20), an intermediate lipoprotein density class with a S(f) range of 15 - 20 (Lp III), and low density lipoproteins (LDL) (S(f) 6 - 8) could be isolated from the plasma. The Lp III was found as a symmetrical peak between VLDL and LDL; its plasma concentration was 248 mg/100 ml plasma (mean), 92 - 469 mg/100 ml plasma (range); the Lp III/LDL ratio was 0.65 (mean), 0.25 - 2.3 (range); the chemical composition w/w was 35.8 percent total cholesterol, 19.3 percent triglycerides, 24.3 percent phospholipids, and 20.4 percent protein (mean). In agarose gel electrophoresis, the Lp III migrated faster than LDL, but slower than VLDL. By means of a double immunodiffusion technique and immunoelectrophoresis, apo-Lp B and apo-Lp C were detectable. In an angle head rotor at 1.4 x 10-8 g. min. Lp III can be separated from VLDL at a solvent density of 1.006, and from LDL at a density of 1.025. In the postabsorptive state in one patient with type I hyperlipoproteinaemia, five with type II and 20 with type IV, and in 50 healthy persons of both sexes, this Lp III peak was not found. The finding of a high concentration of Lp III and a rather low concentration of LDL in the plasma of patients with type III hyperlipoproteinaemia can be regarded as further evidence for an impaired catabolism of VLDL to LDL at Lp III ("intermediate" lipoprotein).

Free and bound carcinoembryonic antigen in neoplasms and in normal adult and fetal tissue

Using a radioimmunoassay, CEA or an immunologically indistinguishable substance is found in normal adult as well as fetal tissue. The highest level in normal adult tissue was found in the lung (average 300 ng/g) and in fetal tissue, the colon (average 1000 ng/g). Of many carcinomas, the highest levels were found in primary and metastatic carcinomas of the colon (range 850–92,800 ng/g). Blood levels of CEA may reflect the concentration of CEA in the cancer as well as the volume of the cancer. Purification of CEA through polyacrylamide gel electrophoresis and isoeletric focusing indicated the presence of at least six antigens which differ in isoeletric points or mol. wt. A perchloric acid extract of one gastric hepatic metastatic adenocarcinoma contained no demonstrable CEA using double immunodiffusion techniques. Following exposure of this perchloric acid precipitate to 15% NaCl, CEA could be demonstrated by double immunodiffusion and the radioimmunoassay showed an increase from 53 to 5750 ng/g. This phenomenon existed in a number of carcinomas to a varying extent as well as in normal adult tissue. Thus it may be importance to include dissociation techniques when studying the level of CEA in blood or tissue.

Excretion of alpha-foetoprotein in the urine of pregnant rats and hepatoma-bearing animals.

Urine of normal rats, pregnant animals and animals bearing chemically induced hepatoma was tested with antisera to foetoproteins by the double immunodiffusion technique. Antigens were not detected in the urine of normal rats. Alpha-foetoprotein was demonstrated in the urine of pregnant rats and hepatomabearing animals.

IMMUNOCHEMICAL STUDY ON THE SPECIES SPECIFICITY OF SPERM-BINDING PROTEIN FROM THE SURFACE OF THE SEA URCHIN EGG.

Immunological species specificity of sperm-binding protein from eggs of the 4 sea urchin species, Hemicentrotus pulcherrimus, Pseudocentrotus depressus, Anthocidaris crassispina and Temnopleurus toreumaticus, was examined by means of double immuno-diffusion technique in agar. Ca-soluble fraction of sperm-binding protein which is considered to be responsible for initial sperm-egg bonding at fertilization, has species-specific antigenic component. Correlations in antigenic constituents among the 4 species are described.

Effect of formaldehyde on the immunochemical and biological activity of staphylococcal enterotoxin B.

Staphylococcal enterotoxin B was treated with 0.15 or 0.3% formaldehyde at 37 C and pH 7.3. As early as 8 hr after addition of HCHO, the toxin was unable to precipitate as much antibody from antiserum as untreated toxin; greater amounts of treated toxin were required to reach equivalence and to inhibit precipitation. With increasing time, its reactivity decreased so that at 32 weeks it could precipitate only 20% as much antibody as untreated toxin. When other factors were kept constant, the degree of inactivation increased as the HCHO concentration increased from 0.6 to 1.4%. Treated toxin gave a "reaction of partial identity" by the Ouchterlony double-immunodiffusion technique. The emetic effect of the toxin for the monkey remained essentially unchanged for about 48 hr of treatment, but the amount required to cause death by the intravenous route increased from 25 to over 1,000 mug/kg of body weight. HCHO destroyed the pyrogenic effect for rabbits. The treated toxin remained immunogenic for rabbits even after 4 to 5 weeks of exposure to 0.3% HCHO at 37 C.