Double Immunodiffusion Technique Research Articles

Vitellogenin and vitellin of a millipede Spirostreptus asthenes: Occurrence, isolation and partial characterization

Vitellogenin (Vg), the yolk precursor protein and its product vitellin (Vn) have been identified in hemolymph and fat body of females and mature oocytes in the millipede Spirostreptus asthenes employing double immunodiffusion technique. These two proteins are absent in males indicating that they are female specific. Immunoelectrophoresis has shown that there is only one vitellogenic protein present in S. asthenes. Vg and Vn were isolated by gel filtration. Polyacrylamide gel electrophoresis (PAGE) analysis revealed that Vg and Vn are glycolipoproteins. Vg contains 48.8% protein, 2.2% carbohydrate and 48.9% lipid. Vn is comprised of 52% protein, 2.3% carbohydrate and 45.4% lipid. The lipid components of Vg and Vn include mainly phospholipids such as phosphatidic acid, sphingomyelin, phosphatidyl choline, phosphatidyl ethanolamine and cholesterol. On SDS-PAGE analysis both Vg and Vn yielded five sub units each. The molecular weight of the sub units of Vg was found to be 135, 115, 105, 73 and 56 kDa and those of Vn were 125, 110, 100, 68, and 53 kDa. The vitellogenic system of S. asthenes resembles that of insects. The phylogenetic relationship of the vitellogenic system of this millipede with other arthropod groups is discussed.

Observations on the natural diet of Cymatia bonsdorfi (C. Sahlb.) (Heteroptera: Corixidae): an immunological analysis

Immunological analyses of the gut contents of 1769 adult and 486 nymphal instars of the lesser waterboatman, Cymatia bonsdorfi, from Lough Corrib, western Ireland, were performed using a double immunodiffusion technique. Antisera against six potential prey categories (Oligochaeta, Cladocera, Copepoda, Gammarus, Diptera and Ephemeroptera), identified on the basis of field and laboratory observations, were utilized. Diptera larvae formed over 90% of the diet of adult bugs and were also the principal prey group of all nymphal instars.

Isolation and purification of a low molecular weight protein from bovine urine.

A low molecular weight protein was separated from urine samples obtained from a heifer with spontaneous renal disease and from cows with CaNa2EDTA-induced renal dysfunction. The molecular weight and electrophoretic mobility of the separated protein were examined. The low molecular weight protein collected by gel filtration chromatography was further separated into two fractions by ion exchange chromatography using DEAE-cellulose. One of the two fractions, the lowest molecular weight protein showed a single band in SDS-PAGE, and its molecular weight was approximately 12,000. An antiserum against this protein formed a single precipitin line with the urine from cows with experimentally induced renal dysfunction and a heifer with spontaneous renal disease by the double immunodiffusion technique. However, the antiserum did not form any precipitin line with the concentrated urine of healthy cow and human beta 2-microglobulin. In cellulose acetate membrane electrophoresis, this protein migrated in the same position as that of serum gamma-globulin from healthy cow.

Differences in distribution of myotoxic proteins in venoms from different Bothrops species

A. M. Moura da Silva, D. F. Cardoso and M. M. Tanizaki. Differences in distribution of myotoxic proteins in venoms from different Bothrops species. Toxicon 28, 1293–1301, 1990.—Antigens with high myotoxic activity were isolated from Bothrops jararacussu venom by Sephadex G-75 and SP-Sephadex C-25. These antigens were recognized using western blotting by B. jararacussu, B. moojeni, B. neuwiedi and B. pradoi antivenoms, and weakly by B. jararaca antivenom. B. alternatus, B. atrox, B. cotiara and B. erythromelas antivenoms failed to recognize these antigens. Antisera raised against these antigens recognized bands with mol. wt around 18,000 in the venoms of B. jararacussu, B. moojeni, B. neuwiedi and B. pradoi and reacted in ELISA with non-denaturated B. jararaca venom. However it failed to react in ELISA with non-denatured B. alternatus, B. atrox, B. cotiara and B. erythromelas venoms. The myotoxicity induced by these crude venoms confirmed that these antigens are possibly the only major myotoxin as the levels of creatine phosphokinase activity in mice serum released by intramuscular injection of B. jararacussu, B. moojeni, B. neuwiedi and B. pradoi venoms (myotoxin +) were five to eight-fold higher than those obtained with B. alternatus, B. atrox, B. cotiara, B. erythromelas and B. jararaca venoms. Using the double immunodiffusion technique the myotoxins of B. jararacussu, B. neuwiedi and B. pradoi showed total identity while B. moojeni myotoxin behaved as a partially identical antigen.

Monoclonal antibodies recognizing the main surface antigens of newly excysted metacercaria or adult flukes of Paragonimus ohirai.

This study was undertaken to prepare monoclonal antibodies which recognized the tegumental antigens of newly excysted metacercaria (NEM) and adult flukes of Paragonimus ohirai. From splenic cells of mice immunized with either the Triton X-100 extract of adult fluke surface or NEM sonicate, monoclonal antibodies were produced by ELISA screening followed by trial immunostaining of fluke sections. We prepared a monoclonal antibody (AS-Mab) recognizing the tegumental antigen of the adult fluke and a monoclonal antibody (MS-Mab) for the NEM tegumental antigen. AS-Mab also bound to the tegument of 1-week-old juveniles but not to that of NEM. MS-Mab bound the tegument from the metacercaria to adult stages, but the degree of immunostaining appeared to decrease with maturation of the flukes. By the double immunodiffusion technique, the two monoclonal antibodies formed crossing precipitin lines against a mixture of the adult surface antigen extract and the NEM antigen extract. MS-Mab and AS-Mab gave precipitin line against the incubation fluid from NEM and adult flukes, respectively.

Biological studies of the flesh flySarcophaga (Parasarcophaga) misera and its effects as a predator of the snailIndoplanorbis exustus

Attempts were made to identify a natural predator of the freshwater snailIndoplanorbis exustus, vector of schistosomiasis. Ochterlony's double immunodiffusion technique was used. The flySarcophaga (Parasarcophaga) misera was identified as a predator ofI. exustus. Various aspects of the biology of this fly and its effects as a predator of snailI. exustus were studied in the laboratory. The possible use of the larvae of this fly to controlI. exustus is discussed as an additional control measure to chemical control.

Contaminating Antibodies May Produce Unexpected Reactions in Counter-Current Immunoelectrophoresis

ABSTRACT: The possibility of contaminating antibodies in an antiserum causing reactions in countercurrent immunoelectrophoresis at low antigen concentrations, though not at higher concentrations, was raised, and also argued against, in a recent, widely-publicized legal case. This study has demonstrated that, in this technique, precipitin bands can be produced in tests involving low concentrations of antibodies only where there are also low concentrations of antigen, not where there are higher concentrations of antigen. These results indicate that, under appropriate circumstances, in counter-current Immunoelectrophoresis, a low concentration of contaminating antibodies in an antiserum could produce a precipitin band with a low concentration of an unsuspected antigen. This possibility points up the necessity of establishing the identities of the reacting antigens and antibodies in such tests, using a different procedure, such as the double radial immunodiffusion (Ouchterlony) technique. It is suggested that the value of counter-current immunoelectrophoresis in forensic testing should be given consideration.

Fibronectin in Neoplastic Peritoneal Effusions in Dogs

Fibronectin is a glycoprotein found in soluble form in plasma and in an insoluble form in connective tissue matrices. Fibronectin isolated from canine peritoneal effusions by affinity chromatography on gelatin-Sepharose was found to be immunologically indistinguishable from the plasma form, as shown by double immunodiffusion techniques. Utilizing an enzyme immunoassay technique, 7 patients with disseminated splenic hemangiosarcoma were found to have peritoneal fibronectin concentrations of 309 ± 133 (mean ± SD) μg/ml. In 8 patients with widespread lymphoma the peritoneal fibronectin concentrations were 323 ± 147 μg/ml. In 5 patients with hepatoma the peritoneal fibronectin concentrations were 361 ± 121 μg/ml. In 6 patients, 2 with mesothelioma, 2 with pancreatic adenocarcinomas, and 2 with prostatic adenocarcinoma, the peritoneal fibronectin concentrations were 315 ± 115 μg/ml. The lowest concentrations 117 ± 46 μg/ml, were found in patients with malignant disease and concurrent peritoneal thrombus formation. These data suggest that decreased fibronectin concentrations in peritoneal effusions are associated with, at least, a locally hypercoagulable state.

Some applications of the surface-spreading technique to virological studies.

By application of the surface-spreading technique, virus particles in infected cells and viremia serum, and precipitates in agar plates of the double immunodiffusion technique of Ouchterlony could easily and clearly be visualized without any purification process.

Glutamine synthetase of the human brain: purification and characterization.

Glutamine synthetase (GS) isolated from human brain formed a single band on sodium dodecyl sulfate-polyacrylamide gel with a molecular weight of 44,000. The enzyme had a specific activity of 179.2 U/mg protein when assayed by measuring the rate of the formation of gamma-glutamylhydroxamate using hydroxylamine as a substrate. In the presence of manganese ions, the relative activity of human brain GS was much lower than that of the sheep brain enzyme. The suppression of activity by increasing the ADP concentration, however, was less marked in the human enzyme than that in the sheep enzyme. Antibodies were raised in rabbits against the purified enzyme. The double-immunodiffusion technique disclosed cross-reactivities among GSs isolated from human, sheep, and rat brains, but the enzymes were not immunologically identical. Immunohistochemically, GS was localized in the cytoplasm of astrocytes in the human and rat brains and in pericentral hepatocytes of the liver.

Immunochemical Comparison of Glutamine Synthetases from Some Solanaceae Plants

The presence of different glutamine synthetase isoenzymes in different Solanaceae plants and their relative antigenicities against antiglutamine synthetase from tomato leaf serum were studied. All the plants tested showed one glutamine synthetase isoenzyme except for Mandragora autumnalis, which showed two, after discontinuous polyacrylamide gel electrophoresis and specific in situ assay. Antigenicities were compared by the double immunodiffusion technique. The Nicotiana glauca enzyme showed equal reactivity to that of Lycopersicon esculentum, but its antigenicity was higher than Withania frutescens, Datura stramonium, and Hyoscyamus niger. The study of relative antigenicities permitted differentiation of the glutamine synthetase enzymes from uncultivated species of Solanaceae.

Phospholipid transfer protein from maize seedlings is partly membrane-bound

Specific antibodies raised against phospholipid transfer protein from maize seeds, react with mitochondria or microsomes solubilized by sodium deoxycholate. A single precipitin line was observed with both types of solubilized membranes when the double immunodiffusion technique was used. When the solubilized membranes were separated by fast-protein liquid chromatography (FPLC) on a reverse phase column, prior to the immunodiffusion, only fractions co-migrating with the pure phospholipid transfer protein, reacted with the antibody. Alternatively, solubilized membranes were submitted to sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), followed by immunoblotting; the detection of antigen-antibody complexes by a peroxydase reagent revealed the presence of a band co-migrating with the pure protein. All these observations strongly suggest that phospholipid transfer proteins are membrane-bound. The physiological significance of this finding will be discussed.

Monoclonal antibodies can precipitate low-density lipoprotein. I. Characterization and use in determining apolipoprotein B.

We produced 20 mouse monoclonal antibodies against human plasma low-density lipoprotein (LDL). Individually they failed to precipitate LDL in agarose gel by the double-immunodiffusion technique; collectively they did, or as few as two combined monoclonal antibodies could do so. To mimic polyclonal antibodies in determination of apolipoprotein B (apo B) by radial immunodiffusion, a combination of four particular monoclonal antibodies (clones A, B, C, and D) was necessary. We characterized these four clones with respect to temperature dependency, affinity, total binding to 125I-labeled LDL, and specificity to the different species of apolipoprotein B. Two monoclonal antibodies (B and C) bound 100% of 125I-labeled LDL; clones A and D bound 80% and 87%, respectively. All four clones bound maximally to LDL at 4 degrees C. The affinity constants for clones A, B, C, and D were 0.6, 2.1, 3.8, and 2.3 X 10(9) L/mol, respectively. By the Western blotting technique, the four monoclonal antibodies all reacted with the species B-100 and B-74 of apolipoprotein B, and to various degrees with B-48 and B-26. Radial immunodiffusion (chi) and direct enzyme-linked immunosorbent assay (y) with a mixture of the four monoclonal antibodies gave almost identical results for 70 patients: y = 0.921 chi-2.58; r = 0.933.

The identification of diphtheria, tetanus and pertussis vaccines by single radial and double immunodiffusion techniques

The identification tests for adsorbed diphtheria, tetanus and pertussis vaccines, which are required by the European Pharmacopoeia to be undertaken in animals, may be replaced by precipitation tests, for instance in agaros gels. Such in vitro tests eliminate the use of animals and are less expensive and time-consuming. The single radial immunodiffusion technique is a suitable semiquantitative test, while the double diffusion test is necessary for the investigation of complete or partial identity. The precipitates obtained in the single radial diffusion tests and in double diffusion tests with diphtheria toxoid were visible without staining; those obtained in the double diffusion tests with tetanus toxoid were weaker and staining was sometimes needed.

Subunit structure and immunological properties of wheat carboxypeptidase

Wheat carboxypeptidases I, II, III and IV from wheat seeds with isoelectric points of 4.8, 5.6, 6.0 and 6.5, respectively, were found to be homogeneous by the Ouchterlony double immunodiffusion technique using an antiserum of the enzyme III. In a previous paper [1], the native enzyme III (MW = 118 k) was separated into two 58 k subunits (MW = 58 k) and further divided into the 35 k and 25 k fragments (MW = 35 k and 25 k, respectively). The native enzyme III and the 58 k subunit produced a single precipitin line against the antiserum. The 35 k and 25 k fragments did not cross-react with the antiserum. The amino acid compositions of the 35 k and 25 k fragments were similar to each other. Amino-terminal amino acids of the 35 k and 25 k fragments were both glutamic acid. Carboxy-terminal groups of the 35k and 25k fragments were determined to be -(Gly, Ser)-Glu-OH and -Thr-Pro-Glu-OH, respectively. The Ouchterlony double immunodiffusion technique revealed the presence of a common antigen in a carboxypeptidase from wheat seeds and one from germinated wheat. Comparison of both enzymes is discussed.

Comparison of Dermatophilus Congolensis Precipitating Antigens Prepared by Three Methods

SUMMARY Dermatophilus congolensis antigens were prepared from nutrient broth and trypticase soy broth cultures by three methods: ammonium sulphate precipitation, trichloroacetic acid extraction of the bacterial cells and culture supernatant ultrafiltration. The ultrafiltration antigens were concentrated using either Amicon XM50 or YM10 membranes. The three antigen types were tested by the reverse single radial immunodiffusion (RSRID) and double immunodiffusion (DID) methods. Sharply defined multiple precipitation lines were demonstrated with serum from cattle with streptothricosis using the nutrient broth supernatant antigen concentrated on YM10 membranes. Only weak or diffuse lines were obtained with the XM50-concentrated and ammonium sulphate-precipitated antigens. No precipitins were demonstrated in sera from the vaccinated cattle. The RSRID method offered no advantage over the DID technique. Nutrient broth concentration was less complicated and more efficient than the other methods tested. However, ultrafiltration membranes with pore size of 50 000 daltons should be used to avoid antigen loss. Failure to demonstrate precipitins in serum from cattle vaccinated intradermally with D. congolensis may indicate that this route provides a different type of antigenic stimulus; other workers have elicited precipitin reactions using different vaccination procedures.

Acid stable protease inhibitor in ascites of ovarian carcinoma

In patients with ovarian tumors, a novel protease inhibitor which is very stable in acid (AS-PI, acid stable protease inhibitor) was identified in the ascites and tumor fluid as well as in the urine and plasma. The highest AS-PI activity was observed in the tumor fluid of ovarian carcinomas (10.7 ± 2.3 U/ml), followed by the ascites of ovarian carcinomas (8.2 ± 4.2 U/ml). There was a significant difference in activity of the tumor fluid and ascites between malignant and benign tumors ( P < 0.005, P < 0.05, respectively). The same antigenicity of AS-PI fractionated from ascites of ovarian carcinomas to urinary trypsin inhibitor was identified by double immunodiffusion and neutralization techniques. It migrated in the serum albumin fraction on immunoelectrophoresis. By gel filtration, the AS-PI in the ascites of ovarian carcinomas showed a molecular weight of 700008̄0000. Two active components with molecular weights of 61300 ± 2100 and 73300 ± 2500 were detected by SDS polyacrylamide gel electrophoresis.

Immunological relationship between carbonic anhydrase isoenzyme C and immature enamel matrix proteins of the rat incisor.

The antigenic relationship between amelogenins from developing rat enamel and carbonic anhydrase (CA) from rat erythrocytes was investigated by using a double immunodiffusion technique. Amelogenins ranging in size from 23, 000 to 30, 000 daltons formed a single precipitin line with an antibody against CA isoenzyme C. The results suggest that most of the amelogenins are derived from CA isoenzyme C.

Immunochemical evidence for the enzymatic difference of Δ 6-desaturase from Δ 9- and Δ 5-desaturase in rat liver microsomes

The enzymatic properties of the three types of microsomal acyl-CoA desaturases, Δ 6-, Δ 9- and Δ 5-desaturases, were immunologically compared using a monospecific antibody raised against the purified linoleoyl-CoA desaturase (Δ 6-desaturase). By the double immunodiffusion technique, the anti-Δ 6-desaturase antibody showed a single precipitin line to the purified Δ 6-desaturase and microsomes treated with Triton X-100, but no line was observed with the partially purified Δ 9-desaturase. The antibody even inhibited definitely Δ 6-desaturase activity in microsomes, but neither stearoyl-CoA (Δ 9-) nor eicosatrienoic acid (Δ 5-) desaturations were inhibited. By these immunological investigations it was confirmed that terminal Δ 6-desaturase is different enzyme from desaturases Δ 9- and Δ 5.

Immunological identification of milk-clotting enzymes

SUMMARYThe 6 most widely used milk-clotting enzymes, i.e. chymosin, bovine and porcine pepsins and proteinases from Mucor miehei, M. pusillus and Endothia parasitica, have been purified and used to prepare rabbit antisera against each of them. The antisera were adsorbed with appropriate cross-linked antigens to remove non-specific antibodies. Monospecific antisera thus obtained were used to identify enzymes eventually added to calf or bovine rennets, using the double radial immunodiffusion technique. The threshold of sensitivity was c. 1%, expressed as clotting activity. This qualitative method complements the quantitative procedure recently proposed for determining chymosin and bovine pepsin A in commercial extracts of bovine veils.