Double Diffusion Test Research Articles

Purification and immunoreactivity of monoclonal antibodies against myeloma-λ chain

In this study, two murine IgM-monoclonal antibodies (IgM-McAbs) against lambda chain of myeloma protein from hybridoma ascites were purified by Sephadex G200 chromatography. The eluate of the first peak on absorption at 280 nm formed one precipitation band with rabbit anti-mouse IgM or anti-mouse immunoglobulin in agar gel double diffusion test; it also formed a detectable precipitation line in the IgM reaction area in immunoelectrophoresis. These findings showed that the eluate of the first peak on absorption at 280 nm contained purified IgM-McAbs. It was found in indirect ELISA that the immunoreactivity of the eluate of the first peak was four to five fold higher than that of the original ascites with equimolar protein concentration. Double antibody sandwich ELISA analyses of the immunoreactivity of HRP-conjugated IgM-monoclonal antibodies with lambda chain denoted that the purified IgM-McAb-HRP-conjugate might be of practical value in quantitative as well as qualitative assay of lambda chains.

Purification and characterization of a hemolysin of Vibrio mimicus that relates to the thermostable direct hemolysin of Vibrio parahaemolyticus

A hemolysin (designated Vm-rTDH) from Vibrio mimicus (AQ0915-E13) was purified by ammonium sulfate fractionation and successive column chromatography with DEAE-Sephadex A-25, hydroxyapatite, Mono Q, Superose 12 and Phenyl-Superose. The Mr of the subunit was estimated to be about 22,000 by sodium dodecyl sulfate-slab gel electrophoresis. The isoelectric point of Vm-rTDH was approximately pH 4.9. The hemolytic activity of Vm-rTDH was stable upon heating at 100 degrees C for 10 min, similar to that of the thermostable direct hemolysin (Vp-TDH) of V. parahaemolyticus. Vm-rTDH also showed lytic activities similar to those of Vp-TDH. Immunological cross-reactivity between Vp-TDH and Vm-rTDH was demonstrated by the Ouchterlony double-diffusion test. Thus we conclude that V. mimicus produces a newly discovered type of hemolysin (Vm-rTDH) which is similar to Vp-TDH.

Purification and characterization of a hemolysin ofVibrio mimicusthat relates to the thermostable direct hemolysin ofVibrio parahaemolyticus

A hemolysin (designated Vm-rTDH) from Vibrio mimicus (AQ0915-E13) was purified by ammonium sulfate fractionation and successive column chromatography with DEAE-Sephadex A-25, hydroxyapatite, Mono Q, Superose 12 and Phenyl-Superose. The Mr of the subunit was estimated to be about 22,000 by sodium dodecyl sulfate-slab gel electrophoresis. The isoelectric point of Vm-rTDH was approximately pH 4.9. The hemolytic activity of Vm-rTDH was stable upon heating at 100 degrees C for 10 min, similar to that of the thermostable direct hemolysin (Vp-TDH) of V. parahaemolyticus. Vm-rTDH also showed lytic activities similar to those of Vp-TDH. Immunological cross-reactivity between Vp-TDH and Vm-rTDH was demonstrated by the Ouchterlony double-diffusion test. Thus we conclude that V. mimicus produces a newly discovered type of hemolysin (Vm-rTDH) which is similar to Vp-TDH.

Purification and Characterization of Rice Peroxidases

Four peroxidase components, named RP-2, 4, 6, and 7, were isolated from rice (Oryza sativa L.) green leaves. Isoelectric focusing indicated that each preparation was homogeneous. The molecular weights of RP-2, 4, 6, and 7 estimated by SDS-PAGE were 48,000, 48,000, 40,000, and 39,500, and their isoelectric points were 5.4, 8.1, 9.3, and 9.2, respectively. The activity of every preparation was maximum around pH 5.0. Antisera against these purified enzymes were raised in rabbits. Ouchterlony double diffusion tests with these antisera suggested that RP-6 and 7 were immunochemically identical and RP-2 and 4 were identical in parts and that RP-6 and 7 were quite different from RP-2 and 4. Analysis of the N-terminal amino acid sequences also showed that these peroxidase components were classified into two groups. The polymerase chain reaction showed that RP-2 and/or RP-4 contained an active central region, which is homologous to other plant peroxidases.

Characterization of a New Fimbrial Antigen Present in Escherichia coli strains Isolated from Calves*

Thirteen Escherichia coli strains isolated from calves with diarrhoea, supposed to carry a common antigen were examined for their hemagglutinating activity and compared by bacterial agglutination, double diffusion in two dimensions and by crossed immunoelectrophoresis (CIE). Two of the strains were examined also in the electron microscope. Most of the strains agglutinated red blood cells of horse, ox, guinea pig and chicken, of which the agglutination of ox erythrocytes was mannose-resistant (MRHA). None of the strains agglutinated human erythrocytes. All strains with MRHA of ox red blood cells, regardless to their O:K:H antigens could be agglutinated in unabsorbed or absorbed antisera produced against cultures C1209 (020:K-:H9) and C1213 (09:K36:H-) when live cells as antigens were used. None of these sera agglutinated reference strains carrying K88, K99, 987P, F41 or FY (Att25) antigen respectively. By the double gel diffusion test and by CIE in extracts (60 degrees C) of the strains a common heat labile antigen, responsible for the MRHA of ox red blood cells was identified. Electron microscopy revealed that this common antigen was represented by thin, long, hair-like fimbriae on cells of E. coli C1213, and that specific homologous antibodies attached to these fimbriae.

Two New Strains of Melon Necrotic Spot Virus.

Two different isolates of melon necrotic spot virus (MNSV), designated as MNSV-NK and MNSV-NH were isolated from infected melons in Nagasaki Prefecture. The host ranges of these isolates were restricted to the Cucurbitaceae and were similar to that of an isolate of MNSV from Shizuoka Prefecture (MNSV-S), but they produced a different type of local lesion on cotyledons of Cucumis melo L. The rates of systemic infection induced by MNSV-NK, MNSV-NH and MNSV-S in C. melo L. were 9.1%, 90.1% and 3.6%, respectively. MNSV-NK and MNSV-NH were designated as a new serotype N, because these viruses were serologically related but not identical to MNSV-S (serotype S) with double diffusion test in agar gel. All three isolates had a single coat protein with molecular weight of 41k dalton. MNSV-NK and MNSV-NH had two species of single-stranded RNAs (RNA1 and RNA2), while MNSV-S had three (RNA1, RNA2 and RNA3). Only RNA1 (MW 1.51×106) of each isolate retained pathogenicity. The origin of other RNAs was unknown yet. From these pathogenccal, serological and physical properties, three isolates were designated as NK, NH and S strains of melon nectoric spot virus.

Seroprevalence of Echinococcus granulosus infection in a Uruguayan rural human population

Serological tests using hydatid cyst fluid (HCF) are useful as a primary screen for detecting asymptomatic infections due to Echinococcus granulosus in human populations. An enzyme-linked immunosorbent assay ( elisa) performed with whole sheep HCF was used to determine the prevalence of hydatid antibodies in a rural human population in Uruguay. In order to eliminate cross-reactions with sheep proteins in HCF, 1% normal sheep serum was added to each human serum before testing. Elisa seropositives were further tested using the double diffusion test (DD5) and confirmed where possible by ultrasound and X-ray examination. Serum samples were obtained from 420 individuals inhabiting rural areas in the Department of Paysandu (160), Rivera (21) and San José (239). An overall seroprevalence of 1·24% was obtained. Two of 17 seroreactors were subsequently proven to have hydatidosis. These results confirm and extend previous studies indicating an unusually high prevalence of E. granulosus infection in the Uruguayan population.

Autoantibodies and red blood cell membrane proteins in a case of canine autoimmune hemolytic anemia.

Autoantibodies and erythrocyte membrane proteins were analyzed in a case of a dog with autoimmune hemolytic anemia (AIHA). In crisis phase, antiglobulin test was positive. The eluate from the erythrocytes of the dog with AIHA gave agglutination against autologus erythrocytes. Immunoglobulin subclasses in the eluate were revealed to be IgG and IgA by the double diffusion test. Comparing the SDS polyacrylamide gel electrophoretic patterns of erythrocyte membrane proteins between the crisis and remission phases, there was a change in the protein on protein 4.1 region. However, there were no changes in blood group typings in two phases.

Cyanoprotein: Immunological properties and content changes during the development of non-diapause female bean bugs, Riptortus clavatus

Immunological properties and content changes of cyanoprotein (CP) were investigated in the bean bug, Riptortus clavatus. Anti-CPegg serum was prepared by immunizing a rabbit with CP purified from eggs (CPegg). In the Ouchterlony double diffusion test, the precipitin line between CPegg and anti-CPegg serum fused with that of non-diapause and diapause female hemolymph and anti-CPegg serum. Rocket immunoelectrophoresis (RIE) using anti-CP serum showed two types of rockets (A and B) depending on the samples. Namely, CPegg and non-diapause female adult hemolymph formed A rockets (heavy-stained with Coomassie Brilliant Blue) and early diapause female adult hemolymph formed B rockets (light-stained), but hemolymph from fifth instar nymphs formed both A and B rockets. Both rockets A and B were demonstrated by SDS-PAGE analysis of the precipitin lines to be formed from the same CP subunit (MW, 76,000). CP-1, 2 and 3 bands from native PAGE of nymphal hemolymph formed A rockets and CP-4 formed B rockets. The contents of CP-A (CP-1 to 3) and CP-B (CP-4) were separately determined by measuring the sizes of rocket A and B. CP-A and CP-B content were demonstrated to increase during the development of the last instar nymph and decrease at adult emergence by RIE analysis of non-diapause female whole body extracts. CP-B is predominant in the nymphal stage. In the early adult stage (day 2 and 3 after emergence), neither CP-A nor CP-B were detected. Only CP-A appeared again at day 4 after emergence and increased during development and vitellogenesis of non-diapause females.

Detection of antibodies against Echinococcus granulosus major antigens and their subunits by immunoblotting

An immunoblot assay was tested to evaluate its ability to diagnose human hydatidosis and to analyse the reactivity of hydatid patients' sera with the subunits of the 2 major Echinococcus granulosus antigens (5 and B). In all, 308 sera were examined: 166 sera from patients with clinically diagnosed hydatidosis, 100 sera from healthy control subjects, and 42 sera from patients with diseases other than hydatidosis. The sensitivity of the method was 90%, as compared to 78% with the immunoelectrophoresis/ double diffusion test for antigen 5. No reactivity was found with 15 sera from patients with schistosomiasis, 7 sera from patients with trichinellosis, or 20 sera from patients with non-parasitic diseases. Analysis of serum reactivities showed the presence in all positive sera of antibodies directed against the 39 kDa molecule of the antigen 5 complex. A lower reactivity (55% of all hydatid sera) was observed with the subunits of the antigen B complex.

Purification and Characterization of Rice Peroxidases.

Four peroxidase components, named RP-2, 4, 6, and 7, were isolated from rice (Oryza sativa L.) green leaves. Isoelectric focusing indicated that each preparation was homogeneous. The molecular weights of RP-2, 4, 6, and 7 estimated by SDS-PAGE were 48,000, 48,000, 40,000, and 39,500, and their isoelectric points were 5.4, 8.1, 9.3, and 9.2, respectively. The activity of every preparation was maximum around pH 5.0. Antisera against these purified enzymes were raised in rabbits. Ouchterlony double diffusion tests with these antisera suggested that RP-6 and 7 were immunochemically identical and RP-2 and 4 were identical in parts and that RP-6 and 7 were quite different from RP-2 and 4. Analysis of the N-terminal amino acid sequences also showed that these peroxidase components were classified into two groups. The polymerase chain reaction showed that RP-2 and/or RP-4 contained an active central region, which is homologous to other plant peroxidases.

The use of DNA probes for confirming enterotoxin production by Staphylococcus aureus and micrococci

DNA-DNA colony hybridization was employed to evaluate the results obtained by different immunological methods for detection of staphylococcal enterotoxin. Staphylococcus aureus strains tested for staphylococcal enterotoxin production by immuno-assays and micrococci not previously tested for staphylococcal enterotoxin production were examined for presence of the genes encoding for staphylococcal enterotoxin A, B, C and E by using three corresponding DNA probes. The staphylococcal enterotoxin A probe also detected staphylococcal enterotoxin E gene because of 100% homology. The optimal sensitivity plate method showed the best accordance between the immuno-assay and the hybridization reactions. The enzyme-linked immunosorbent assay detected 12.5 to 17% staphylococcal enterotoxin producers without hybridization reactions. The microslide gel double diffusion test and the reversed passive latex agglutination test showed rather poor accordance with the hybridization reactions. All 17 strains of different micrococci investigated were negative in hybridization with all three DNA probes.

Immunization of cattle ( Bos indicus × Bos taurus) against Hyalomma anatolicum anatolicum using antigens derived from tick salivary gland extracts

Studies were conducted on 41 cross-bred ( Bos taurus x Bos indicus) calves which were less than 1 year old. Three types of salivary gland antigens (SG Ag), viz. SG Ag-I (whole), SG Ag-II (supernatant) and SG Ag-III (sediment), prepared from the ixodid tick, Hyalomma anatolicum anatolicum, were used for immunizing these calves. The calves were divided into five groups. The first three groups were immunized with SG Ag-I, -II and -III with Freund's complete adjuvant (FCA), respectively. The fourth group was immunized with SG Ag-I (without adjuvant), and the fifth group remained as an unimmunized control inoculated with FCA only. In all the groups, immunization was carried out on days 1 and 14 and the animals were challenged on the 21st day. Significant resistance developed in the calves of the SG Ag-I and SG Ag-II (both with FCA) groups. Of all, SG Ag-I with FCA proved the most potent immunogen. Both humoral and cellular immune responses were demonstrated by in vivo and in vitro tests. The capillary tube agglutination test and double diffusion test gave positive reactions 21 days after first immunization. The immunized calves showed a significant increase in the levels of serum gamma globulin and per cent ‘E’ rosettes. Tick salivary gland antigens therefore seem to have promising potentials in inducing resistance in calves.

Influence of adjuvants on immunity in rabbits vaccinated with infective larval somatic proteins of Trichostrongylus colubriformis

Adult New Zealand rabbits were vaccinated at 2-week intervals with three doses of 100 μg of infective larval somatic proteins (L 3SP) administered subcutaneously with Freund's complete adjuvant (FCA) or beryllium hydroxide and then challenged orally with 10 000 L 3. Groups of rabbits immunized orally with three doses of 5000 or 2000 L 3 served as vaccination controls. Intestinal worm burdens on Day 21 after challenge revealed that beryllium hydroxide effectively potentiates the protective immunogenicity of L 3SP. The level of protection obtained using the beryllium adjuvant (94.8%) was nearly as high as that in rabbits immunized with three doses of 5000 L 3 (99.8%). Rabbits vaccinated using FCA showed very poor immunity (29.5%). Local and systemic antibody levels detected by radioimmunoprecipitation tests using 125I-L 3-SP showed very little correlation with the degree of protection. The beryllium hydroxide-treated group demonstrated significantly higher bile IgA antibody levels than other experimental rabbits. FCA-treated rabbits developed a much higher serum precipitating antibody response, detectable using gel double diffusion tests, than the beryllium group. Also, mucosal IgA antibody levels detected on Day 21 after challenge were significantly higher in the FCA group than in other groups.

Studies on monoclonal anti-isotypic and anti-idiotypic antibodies against leukemia and myeloma: I. preparation and characteristics of monoclonal anti-isotypic antibodies to igm from B chronic lymphocytic leukemia

Five hybridomas producing McAbs against human serum IgM-isotype were obtained by fusing the myeloma NS-1 cell and the BALB/C murine spleen cell which had been immunized by serum IgM of patient with B chronic lymphocytic leukemia (B-CLL). These McAbs only reacted with human IgM, not with other immunoglobulins in ELISA and immune double diffusion test. An approximate positive rate of peripheral blood lymphocyte (PBL) was got when these McAbs and the McAbs against B cell were tested by indirect immunofluorescent assay. The positive rate was similar to that obtained by direct immunofluorescent test. Immunoblotting showed that the molecular weight of the antigen to these McAbs was 70-90 Kd, indicating that the antigen was IgM. The practical value of the McAbs against human IgM was also discussed.

Serological Comparison of Isolates of Cherry Leaf Roll Virus from Diseased Beech and Birch Trees in a Forest Decline Area in Germany with Other Isolates of the Virus

Abstract Two isolates of cherry leaf roll virus (CLRV), one from diseased beech and one from diseased birch trees in an area with forest decline near Bonn in West Germany, were found to be serologically closely related, but not indentical as assessed by spurformation of precipitin lines in agarose gel double diffusion tests. Such tests also distinguished these German CLRV isolates from ten other distinct CLRV isolates obtained from different natural hosts and from various countries. The German beech isolate was most similar serologically to isolates from walnut and from birch in England and the German birch isolate to an English cherry isolate and an isolate from Sambucus racemosa in Finland. These results provide further evidence of the antigenicdiversity of CLRV.

Bovine Albumin‐Like Protein in Commercial Human Albumin for Clinical Use

We have demonstrated that several lots of commercial human serum albumin (HSA), prepared for clinical use by two manufacturers, reacted with anti-bovine whole serum in the double-diffusion test. The antigenic proteins were purified by affinity chromatography from these HSA lots and their chemical and immunological properties characterized. The purified proteins showed the same molecular weight as HSA and bovine serum albumin (BSA). By CNBr treatment and trypsin digestion, the purified protein produced the same fragments as BSA. Moreover, the purified protein had an amino acid composition very similar to BSA and also 30 residues identical to those of the N-terminal amino acid sequence of BSA. In addition, it was shown that heterophilic Hanganutziu-Deicher antigenicity was present in the purified protein. The protein reactive against-bovine whole serum, contained in commercial HSA, was called BSA-like protein.

A comparison of two enzyme linked immunosorbent assays for enzootic bovine leucosis serology

Abstract Extract The double gel diffusion test (GDT) is the internationally accepted standard method for detection of antibodies against bovine leukemia virus (BLV)(1). The method has been standardised (within certain limits) and a reference serum has been introduced by the European Commission to control the level of sensitivity of the test.

Farmer's Lung: Thermophilic Actinomycetes as a Source of "Farmer's Lung Hay" Antigen

Mouldy hay was produced in the laboratory by sterilising good hay, inoculating with aqueous suspensions of microorganisms, and incubating at 40 degrees or 60 degrees C. Extracts were tested for presence of farmer's lung hay (F.L.H.) antigen by agar-gel double-diffusion and immunoelectrophoresis tests against sixteen to twenty sera from patients with farmer's lung. F.L.H. antigen developed in hay after: (1) inoculating with mixed microbial suspensions from antigenically active hay; (2) inoculation with mixed suspensions of pure cultures of thermophilic actinomycetes, after raising the pH of the hay to 70 either by prior inoculation with fungi or by infiltration with ammonia vapour; and (3) inoculation at pH 70 with pure cultures of Thermopolyspora polyspora or with Micromonospora vulgaris. F.L.H. antigen did not develop in hay after inoculation with fungi only, or with six other actinomycetes tested, or after prior heating (though some sera reacted to fungal antigens in all these extracts). T. polyspora is the richest source yet found of F.L.H. antigen, and inhalation of an extract by affected subjects produces some of the features of farmer's lung. Pure cultures can produce F.L.H. antigen on artificial media without hay. Spores and mycelium are rich in F.L.H. antigen, and inhalation of the spores may play a part in farmer's lung disease. Other antigens relevant to farmer's lung may be found in other actinomycetes, not yet cultured.

Microsomal redox systems in brown adipose tissue: high lipid peroxidation, low cholesterol biosynthesis and no detectable cytochrome P-450

The presence of redox systems in microsomes of brown adipose tissue (BAT) in cold exposed rats was investigated and compared with liver. BAT microsomes showed high activity of lipid peroxidation measured both by the formation of malondialdehyde (MDA) and by oxygen uptake. NADH and NADPH dependent cytochrome c reductase activity were present in both BAT and liver microsomes. Aminopyrine demethylase and aniline hydroxylase activities, the characteristic detoxification enzymes in liver microsomes could not be detected in BAT microsomes. BAT minces showed very poor incorporation of [1-14C]acetate and [2-14C]mevalonate in unsaponifiable lipid fraction compared to liver. Biosynthesis of cholesterol and ubiquinone, but not fatty acids, and the activity of 3-hydroxy-3-methyl glutaryl CoA reductase appear to be very low in BAT. Examination of difference spectra showed the presence of only cytochrome b5 in BAT microsomes. In addition to the inability to detect the enzyme activities dependent on cytochrome P-450, a protein with the characteristic spectrum, molecular size in SDS-PAGE and interaction with antibodies in double diffusion test, also could not be detected in BAT microsomes. The high activity of lipid peroxidation in microsomes, being associated with large oxygen uptake and oxidation of NADPH, will also contribute to the energy dissipation as heat in BAT, considered important in thermogenesis.