Double Immunodiffusion Technique Research Articles

Modernization of the Pseudomonas syringae Pathovars Serogrouping Scheme

In 1979, L.T. Pastushenko and I.D. Symonovych developed a scheme of serogrouping phytopathogenic bacteria of the Pseudomonas genus, which is still used now. However, today’s using this serogrouping scheme is complicated by the lack of all data accumulated over the years of its application. Moreover, the scheme does not correspond to the modern taxonomy of phytopathogenic bacteria of the Pseudomonas genus. Aim. On the basis of own experimental results and data of scientific literature, to carry out modernization of the serogrouping scheme of phytopathogenic bacteria of the Pseudomonas genus. Methods. The strains of Pseudomonas syringae pathovars such as atrofaciens, coronafaciens, tabaci, which were isolated from plants of wheat, rye, oats, tobacco, and various species of affected weeds in different regions of Ukraine have been studied in the work. Antigenic properties of bacterial strains were studied by agglutination and precipitation reactions (the Ouchterlony double immunodiffusion techniques) using antisera to P. syringae strains of nine serological groups (I, II, III, IV, V, VI, VII, VIII, IX). To carry out the precipitation reaction, O- and OH-antigens were obtained by a modified Grasse’s method. The presence of the same number of precipitation lines of the studied antigens as the number of lines with homologous antiserum of the corresponding serogroup testified to their belonging to this serogroup according to the known serogrouping scheme of phytopathogenic bacteria developed in 1979 by L.T. Pastushenko and I.D. Symonovych. Results. It has been proved that strains of P. syringae pathovars isolated from different cereals (rye, wheat, oats) and segetal vegetation differ in antigenic composition. The antigenic composition of P. syringae strains depends on the host plant from which the pathogen was isolated. Strains of the causative agent of basal glume rot P. syringae pv. atrofaciens isolated from wheat belong to four serological groups (II, IV, V, VI), from rye — to five serological groups (I, II, IV, V, VI), as well as strains of this pathogen isolated from segetal vegetation of wheat agrophytocenosis. Strains of the halo bligth P. syringae pv. coronafaciens from affected oat plants belong to two serological groups (I, V). The serogrouping scheme has been supplemented by new data on the antigenic properties of P. syringae pv. tomato, the causative agent of the bacterial speck disease of tomatoes (Solanum lycopersicum), which is classified as serogroup IV. It has been found that P. syringae pv. tabaci strains, which cause wildfire of tobacco, are part of three serogroups — VII, VIII, IX, and not of two ones, as was presented in the known scheme (1979). Conclusions. Therefore, on the basis of our own research and literature data, the serogrouping scheme of phytopathogenic bacteria of the Pseudomonas genus developed by L.T. Pastushenko and I.D. Symonovych has been modernized. In the renovated scheme, current species’ names of phytopathogens are present, this scheme introduces new information about the serogroups of P. syringae pv. atrofaciens isolated from rye seeds and plants, weeds, P. syringae pv. coronafaciens — from oat plants, P. syringae pv. tabaci — from tobacco, and P. syringae pv. tomato — from affected tomato plants.

PUBLIC HEALTH HAZARDS ASSOCIATED WITH NATURALLY OCCURRING TOXINS IN LEGUME SEEDS - RAPID DETECTION AND CHARACTERIZATION OF A LECTIN FROM A KOREAN CULTIVATED KIDNEY BEAN (PHASEOLUS VULGARIS L.)

Objective: The delicacy of kidney beans is highly appreciable but, at the same time, their toxicity has raised an alarming concern. In this regard, more public awareness is needed about bean poisoning.Methods: This work aimed at promptly investigating the chemical and biological properties of a lectin derived from a Korean cultivar of the common bean (Phaseolus vulgaris L.).Results: Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed the presence of two major polypeptide bands around 31.00 kDa. Hemagglutination assay and Ouchterlony double immunodiffusion technique confirmed the presence of lectin in dry seeds.Conclusion: It was evident that the Korean bean cultivar contains biologically active lectins, which may pose a risk to the consumer if the beans are eaten in raw or undercooked state.

ISOLATION, CHARACTERIZATION AND IMMUNOCHEMICAL STUDIES ON FIBROUS PROTEINS FROM COWRY SHELL (CYPRAEA MONETA, LINNAEUS)

Background:Biomaterials are non-drug substances used to treat, enhance or replace functions of body tissues or organs. Natural sources of biomaterials have recently become the focus of several research activities. Cowry shell constitutes one of the most promising natural sources of biomaterials because of its chemical stability, biodegradability and biocompatibility in the body. However, its applications may be limited due to immunogenic and toxic responses that may occur following implantation, hence this study.Materials and Methods:Crude fibrous protein extracted with citrate buffer from pulverised cowry shells (Cypraea moneta (L)), was resolved into two components (CSP1 and CSP2) by gel filtration. Immunological studies were performed with antisera obtained from rabbits by double immunodiffusion and immunoelectrophoresis techniques. Mice treated with the proteins were observed for signs of toxicity and their liver, kidney, lungs and spleen were processed histologically.Results:The native molecular weight of CSP1 and CSP2 determined by gel filtration were 91kDa and 33kDa respectively. CSP1 and CSP2 displayed single bands on SDS-PAGE with subunit molecular weight values of 19kDa and 19.5kDa respectively. Antisera obtained from rabbits immunised with the crude citrate buffer extracts precipitated the antigen in double immunodiffusion tests. Histopathological examinations revealed a dose-dependent damaging effect of the shell proteins on liver, kidney, lung and spleen tissues of the treated mice.Conclusion:This study showed that cowry shells contain fibrous proteins which are immunogenic and toxic in mice at relatively high concentrations, causing visible organ damage without concurrent physical manifestations.

Frequency of antinuclear antibodies in mestizo Mexican children with morphea

Morphea is a disease that affects connective tissue and microvessels. Its pathogenesis is unknown, but several autoimmune factors participate. Our objective was to determine the frequency of antinuclear antibodies (ANAs) in pediatric patients with morphea and to establish their relation with the clinical variants and disease activity. A cross-sectional study was carried out from January 1999 to January 2008 in patients with morphea seen at the Instituto Dermatologico de Jalisco. ANAs were determined through an indirect immunoflourescent method, and the immunospecificity was done with a double immunodiffusion technique in agarose gel. A total of 34 children were included in the study, 74% of the female gender. Plaque morphea was the most common variant, present in 44% of the cases, followed by linear morphea in 38%, and generalized morphea in 18%. ANAs were positive in 29%, with homogenous immunoflourescense as the most frequent pattern (70%). Of the ANA-positive patients, 83% had generalized morphea, and in 70% of the cases the disease were considered as active. The frequency of ANA-positive children with morphea was 29%, and seems to be related to more extensive disease. No previous studies exist on this topic in the mestizo Mexican population.

Quantitative determination of high molecular weight serum proteinase inhibitors in human semen.

Immunochemical determinations of serum proteinase inhibitors in human semen showed the presence of alpha1-antitrypsin and alpha1,x-antichymotrypsin, whereas inter-alpha-trypsin inhibitor, antithrombin III, alpha2-neuramino-glycoprotein and alpha2-macroglobulin could not be detected. Both serum proteinase inhibitors were determined in the seminal vesicle secretions of two patients with prostatic cancer. Employing the Ouchterlony double immunodiffusion technique pattern of identity was found between alpha1-antitrypsin resp. alpha1,x-antichymotrypsin in seminal plasma, seminal vesicle secretions and serum. Mean alpha1-antitrypsin concentration in seminal plasma of 129 andrological patients was 97.7 mug/ml and that of alpha1,x-antichymotrypsin 32.8 mug/ml. There were no differences in the mean alpha1-antitrypsin concentrations of normozoospermic and oligozoospermic ejaculates and those with seminal plasma fructose deficiency. Azoospermic ejaculates, however, showed a significant decrease of the mean alpha1-antitrypsin concentration (p less than 0.05). Alpha1,x-antichymotrypsin concentrations of normozoospermic ejaculates were significantly higher compared to those of oligozoospermia and azoospermia (p less than 0.05). Alpha1,x-antichymotrypsin levels in semen samples were fructose deficiency were not different from those of the total ejaculate population. The cause and significance of the observed differences in the inhibitor concentrations within the different ejaculate types is not known. However, there are no indications for the involvement of both proteinase inhibitors in male reproductive processes.

Practical Evaluation of Methods for Detection and Specificity of Autoantibodies to Extractable Nuclear Antigens

Detection and specificity of autoantibodies against extractable nuclear antigens (ENA) play a critical role in the diagnosis and management of autoimmune disease. Historically, the detection of these antibodies has employed double immunodiffusion (DID). Autoantibody specificity was correlated with diagnoses by this technique. Enzyme immunoassays have been developed by multiple manufacturers to detect and identify the specificity ENA autoantibodies. To address the relationship of ENA detection by DID and enzyme immunoassay, the performances of five immunoassays were compared. These included two DID and three enzyme-linked immunoassays (ELISA) (both screening and individual antigen profile kits). The sample set included 83 ENA-positive, antinuclear-antibody (ANA)-positive specimens, 77 ENA-negative, ANA-positive specimens, and 20 ENA- and ANA-negative specimens. Sensitivity and specificity were calculated by two methods: first, by using the in-house DID result as the reference standard, and second, by using latent class analysis, which evaluates each kit result independently. Overall, the results showed that the ELISA methods were more sensitive for detection of ENA autoantibodies than DID techniques, but presence and/or specific type of ENA autoantibody did not always correlate with the patient's clinical presentation. Regardless of the testing strategy an individual laboratory uses, clear communication with the clinical staff regarding the significance of a positive result is imperative. The laboratory and the clinician must both be aware of the sensitivity and specificity of each testing method in use in the clinical laboratory.

Identificação sorológica de espécies de vírus que infetam cucurbitáceas em áreas produtoras do Maranhão

Os vírus representam sérios obstáculos para o sucesso da olericultura no mundo inteiro, constituindo a identificação daqueles de maior incidência numa região, papel fundamental para o estabelecimento de estratégias de controle. Visitas de campo foram realizadas a plantios de espécies de cucurbitáceas em áreas produtoras do Maranhão e amostras foliares foram coletadas de 118 plantas com sintomas ou suspeita de sintomas de vírus, sendo 46 de abóbora (Cucurbita moschata), 30 de melancia (Citrullus lanatus), 23 de maxixe (Cucumis anguria), 13 de pepino (C. sativus) e seis de melão (C. melo). Todas as amostras foram testadas contra anti-soros específicos para os principais vírus das famílias Bromoviridae, Comoviridae e Potyviridae que infetam cucurbitáceas no Nordeste, mediante "enzyme-linked immunosorbent assay" (ELISA) indireto e dupla difusão em agar. Os resultados revelaram a identificação sorológica de Papaya ringspot vírus (PRSV) em 64,4% das amostras analisadas, seguido de Watermelon mosaic virus-2 (WMV-2) em 15,2%, Cucumber mosaic virus (CMV) em 6,8%, Squash mosaic virus (SqMV) em 3,4% e Zucchini yellow mosaic virus (ZYMV) em 3,4%. Este levantamento confirma a predominância do PRSV em espécies de cucurbitáceas cultivadas no estado do Maranhão.

Évaluation d'une technique d'immunoblot pour la détection des anticorps anti-SSA et des anticorps anti-SSB

Immunoblotting and ELISA methods are now employed for the detection of anti Ro/SSA and anti La/SSB antibodies sometimes more often than double immunodiffusion or counter-immunoelectrophoresis. We have developed an immunoblotting technique for the detection of anti Ro/SSA and anti-La/SSB antibodies. This assay named IB-Hela, which uses a Hela cell extract, was compared with the double immunodiffusion technique like two other commercial immunoassays (Elisa test from Sanofi-diagnostic Pasteur and Line immunoassay from Innogenetics). This evaluation shows a good agreement especially in the case of Ro/SSA antibodies detection. IB-Hela seems to be useful in the La/SSB detection with a better sensitivity than the double immunodiffusion technique. We conclude that the IB Hela is helpful for the search for anti-Ro/SSA and anti-La/SSB autoantibodies and that it can be used as an alternative method to the classical techniques of immunodiffusion and counter immunoelectrophoresis.

Identificación del origen de especie animal en carne fresca utilizando inmunodifusión doble

Garcia MJA, Erosa VGE, Prieto C, Nunez GFA. Tec Pecu Mex 2000,38(3)231-237. The immunodiffusion technique was used to determine the frequency of horsemeat sold without authorization in the meat retails of Chihuahua city. This technique was achieved by the obtention of fresh antigen and the production of specific antisera for the horsemeat. This antisera was sufficiently specific and powerful enough to detect a minimum of 5% contamination of fresh horsemeat mixed with beef. Samples were taken from 15 meat retails in the city, divided into three strata: I) Four meat retails that sell selected meat (n=40), II) Four commercial meat retails (n=40) and III) Seven popular meat retails (n=70). Samples were processed and analyzed with the double immunodiffusion technique. From a total of the 150 samples, frequency of positive samples was 33, representing 22%, these positive samples were from stratus III and therefore the positive samples represented 47% of the sample in this stratus. Of the 15 meat chops, four were positive (26.6%). According to the results, the double immunodiffusion technique is a good technique for species identification of fresh meat and it is also easy to implement.

Reference sera for antinuclear antibodies. II. Further definition of antibody specificities in international antinuclear antibody reference sera by immunofluorescence and western blotting

To define the fine specificity of the 10 reference sera used for determination of antinuclear antibodies (ANA) and ANA subsets which are available from the Arthritis Foundation (AF) and from the Centers for Disease Control and Prevention (CDC). AF/CDC sera were assessed by experienced laboratory staff, using indirect immunofluorescence and Western blotting. The original assignment of fluorescence patterns to 4 reference sera was confirmed, and the fluorescence intensities were determined using reference fluorescent beads. On Western blots, sera AF/CDC2 (anti-SS-B/La) and AF/CDC7 (anti-SS-A/Ro) did not detect Ro antigens, sera AF/CDC9 and AF/CDC10 appeared to be monospecific anti-Scl-70 and anti-Jo-1 sera, respectively, serum AF/CDC4 (anti-U1 small nuclear RNP) recognized the 70-kd band, and serum AF/CDC5 recognized the Sm antigen with its multiple bands. Semiquantitative analyses revealed that AF/CDC5, AF/CDC2, and AF/CDC10 were strongly reactive sera, whereas AF/CDC4 and AF/CDC9 were much weaker and should be used at lower dilutions on Western blots. The AF/CDC ANA reference sera, originally described as reference reagents for indirect immunofluorescence and double immunodiffusion techniques, are also useful for Western blotting. The data presented herein further support the use of these sera for reference purposes.

Electrophoretic detection of myeloperoxidase, protease, lactoferrin and lysozyme in buffalo polymorphonuclear granular acid extracts.

Polymorphonuclear (PMN) cells of more than 90% viability and 92% purity were isolated from the peripheral blood of buffaloes. The cationic proteins were extracted with 0.2 mol/L sodium acetate, pH 4.0 from the granules in the PMN and subjected to both non-denaturing and denaturing acid urea polyacrylamide gel electrophoresis (AUPAGE) for identification of myeloperoxidase (MPO), lysozyme, protease activity and lactoferrin. Protease was identified using alpha-naphthyl acetate as substrate, while lactoferrin was identified using a reference lactoferrin from bovine milk in AUPAGE, and by double immunodiffusion and Western blot techniques. Based on AUPAGE, lysozyme was found to be most cationic of all the proteins and peptides from the PMN granules as was evident from reference lysozyme run. The results indicated that the granules in buffalo PMN cells have lysozyme, protease, MPO and lactoferrin.

Immunosuppressive Effect of Honey on the Induction of Allergen-Specific Humoral Antibody Response in Mice

Our study with honey for its possible immunomodulatory activity reveals the immunosuppressive activity on induction of murine humoral antibody responses against different allergens as determined by passive cutaneous anaphylaxis and Ouchterlony double immunodiffusion techniques. Ovalbumin (OVA)-specific IgE antibody responses elicited with various doses were completely suppressed by different sources of commercial honeys. Honey is also found to have suppressed the induction of OVA-specific humoral antibody responses in different strains of mice. The results obtained in this work confirm the immunosuppressive activity of honey and suggest its possible applicability in conditions requiring immunosuppression.

Molecular Cloning and Expression of Rat Liver Aminopeptidase B

We isolated, by immunological screening of a Uni-ZAP XR cDNA library constructed from rat liver mRNAs, a cDNA clone with 2212 base pairs encoding aminopeptidase B (EC 3.4.11.6). The open reading frame encodes a 649-amino acid protein with a theoretical molecular mass of 72,545 Da and bears the consensus sequence of the zinc metalloexopeptidases, indicating that the enzyme belongs to this family, which includes aminopeptidase A, aminopeptidase N, and leukotriene-A4 hydrolase. Escherichia coli SOLR cells infected with the pBluescript phagemid excised from the Uni-ZAP XR vector containing the aminopeptidase B cDNA had a high L-arginyl-beta-naphthylamidase activity. The recombinant protein was purified to homogeneity from the recombinant E. coli extracts. The enzyme had Cl--dependent aminopeptidase activity specifically restricted to the Arg and Lys derivatives and contained 1 mol of zinc per mol of the enzyme.

Tissue distribution and characterization of a 30-kDa cysteine proteinase inhibitor from bovine skeletal muscle

Gel chromatography on a Sephadex G100 column of a crude extract obtained from bovine diaphragma muscle separated four fractions (F-I, F-II, F-III and F-IV) in the range of 12–70 kDa that were active against either papain, trypsin or both. From the F-III fraction, a cysteine proteinase inhibitor was purified by two successive anionic exchange chromatographies on Q-Sepharose and Mono Q columns. The pooled active fraction had a Mw of ∼ 30 kDa, and isoelectrofocusing revealed one band with a pI of 6.7. The papain-inhibiting activity was unaffected by dithiothreital or 2-mercaptoethanol treatment, and only one band was obtained after SDS-poly acrylamide gel electrophoresis under both reducing and non-reducing conditions. These results suggest that the 30-kDa muscle cysteine proteinase inhibitor did not contain disulphide bonds essential for activity and the protein was a monomer. This proteinase inhibitor is stable between 40 and 80°C and pH 5–12. Furthermore, the 30-kDa inhibitor is stable to papain proteolysis. The tissue distribution of this inhibitor was investigated using double immunodiffusion and Western blot techniques that provided evidence for its presence in bovine heart, spleen, liver and lung and its absence in bovine plasma.

Paracoccidioides brasiliensis. A mycologic and immunochemical study of a sample isolated from an armadillo (Dasipus novencinctus).

A sample of P. brasiliensis isolated from the spleen and the liver of an armadillo (Dasipus novencinctus) has been analysed under a mycological and immunochemical viewpoint. The armadillo was captured in an area of Tucuruí (State of Pará, Brazil), the animal being already established as an enzootic reservoir of P. brasiliensis at that region of the country. This sample maintained in the fungal collection of the Tropical Medicine Institute of São Paulo (Brazil) numbered 135, has got all the characteristics of P. brasiliensis, with a strong antigenic power and low virulence for guinea-pigs and Wistar rats. The specific exoantigen of P. brasiliensis--the glycoprotein with a molecular weight of 43 kDa--was easily demonstrated with double immunodiffusion, immunoelectrophoresis, SDS-PAGE and immunobloting techniques.

Angiotensinase A (aminopeptidase A): properties of chromatographically purified isoforms from human kidney

Angiotensin-II-cleaving angiotensinase A (aminopeptidase A, E.C. 3.4.11.7, ATA) plays an important role in glomerular haemodynamics, the pathophysiology of essential arterial hypertension and the induction of vascular disorders. In order to study biochemical and immunological properties of ATA, two isoforms (I and II) of the glycoprotein were isolated for the first time from human kidney cortex. Kidney cortex homogenate, digested with bromelain, was fractionated by ammonium sulphate precipitation and subsequent hydrophobic interaction chromatography, using a fast protein liquid chromatographic (FPLC) system. By anion-exchange FPLC (Mono Q column), the isoforms of ATA were eluted in two distinct peaks and were further purified by size-exclusion FPLC and preparative polyacrylamide gel electrophoresis. Biochemical, immunological and immunohistological characterization disclosed differences in the intrarenal localization, glycosylation, Michaelis constant and apparent molecular mass (native and sodium dodecyl sulphate gel electrophoresis) but similar properties in the double-immunodiffusion technique. Polyclonal rabbit antibodies, raised against ATA isoforms I and II, precipitated an analogous antigen in urine from patients with renal tubular damage.

General principles of retrovirus immunodetection tests

Summary Because infecting retroviruses contain protein and glycoprotein antigenic determinants that can be readily distinguished from host cell determinants, the development of immunologic detection systems, immunodetection tests, or immunoassays capable of identifying antigens of some retroviruses (oncoretroviruses) in blood, body fluids, or cells is possible. Conversely, detection of antibodies produced by animals against some infecting retroviruses can also be used to identify current infections of lentiretroviruses and some oncoretroviruses. Studies of various microorganisms by various immunodetection systems indicate that the most specific and sensitive assays are immunofluorescence, radioimmunoassay, and immunoblot (western blot) analysis, followed by sensitive but less specific elisa and agglutination assays, and finally by even less sensitive but very specific isolation in culture and double immunodiffusion techniques. The first test used routinely for clinical detection of any retrovirus was the immunofluorescent antibody test, introduced in 1972, for detection of FeLV infection in pet cats. Since then, tests for human retroviruses, the human immunodeficiency virus types 1 and 2 (hiv-1 and hiv-2 and the human T-cell lymphotropic virus types I and II (htlv-I and htlv-II) have been introduced for routine use in human medicine. Recently, retroviral tests for a second feline retrovirus, the feline immunodeficiency virus (fiv) have been introduced in veterinary medicine. General principles of sensitivity, specificity, true-positive and -negative rates, false-positive and -negative rates, and positive and negative predictive values apply to all methods used for detection of retroviral infections.

Development of the immunofluorescent antibody test for detection of feline leukemia virus infection in cats

Summary Studies of the immunodetection of various microorganisms by various assay systems indicated that the most specific and sensitive assays are immunofluorescence, radioimmunoassay, and immunoblot analysis (western blot), followed by sensitive but less specific elisa and agglutination assays and, finally, by even less sensitive but very specific virus isolation and double immunodiffusion techniques. The first test for the clinical detection of FeLV infection in pet cats was the immunofluorescent antibody (ifa) test, which was introduced in 1972. The FeLV test is used for detection for FeLV infection and not as a test for leukemia or any other feline disease. The ifa test was compared with an immunodiffusion (id) test and with tissue culture isolation (tci) of the virus in 26 cats to establish a standard for FeLV tests. Excellent correlation was observed between the ifa and the id tests (100%) and between the ifa and id tests, compared with tci (96.2%). From these studies, it is clear that the ifa test is more accurate and more practical, and that results can be obtained faster than can those for the id or tci tests for FeLV.

The sensitivity, specificity and efficiency values of some serological tests used in the diagnosis of paracoccidioidomycosis

This work reports on the results of double immunodiffusion (ID), counterimmunoelectrophoresis (CIE), complement fixation (CF) and indirect immunofluorescence (IIF) techniques in the serodiagnosis of paracoccidioidomycosis. The study was undertaken on four groups of individuals: 46 patients with untreated paracoccidioidomycosis, 22 patients with other deep mycoses, 30 with other infectious diseases (tuberculosis and cutaneous leishmaniasis) and 47 blood donors as negative controls. Data were obtained using Paracoccidioides brasiliensis antigens, i.e., a yeast culture filtrate for ID, CIE and CF, and a yeast cell suspension for IIF. The sensitivity, specificity and efficiency values were measured according to GALEN & GAMBINO. The gel precipitation tests (ID and CIE) showed the greatest sensitivity (91.3 and 95.6%, respectively), maximum specificity (100%) and the highest efficiency values when compared to the CF and IIF tests.

Propionyl-CoA condensing enzyme from Ascaris muscle mitochondria : I. Isolation and characterization of multiple forms

The condensation of two propionyl-CoA units or a propionyl-CoA with acetyl-CoA is required for the synthesis of 2-methylvalerate or 2-methylbutyrate, respectively, two of the major fermentation products of Ascaris anaerobic muscle metabolism. An enzyme that preferentially catalyzes the condensation of propionyl-CoA rather than acetyl-CoA has been purified from the mitochondria of the parasitic intestinal nematode Ascaris lumbricoides var. suum. The purified enzyme is over 10 times more active with propionyl-CoA than with acetyl-CoA as substrate. It also catalyzes the coenzyme A-dependent hydrolysis of acetoacetyl-CoA at a rate four times higher than the propionyl-CoA condensation reaction. The purified Ascaris condensing enzyme preferentially forms the 2-methyl-branched-chain keto acids rather than the corresponding straight chain compounds. The native molecular weight of the purified enzyme was estimated to be 160,000 by gel filtration chromatography and 158,000 by high pressure liquid chromatography. The enzyme migrated as a single protein band with M r 40,000 during sodium dodecyl sulfate-polyacrylamide electrophoresis, indicating that the enzyme is composed of four subunits of the same molecular weight. Chromatography on CM-sephadex resulted in the isolation of two separate peaks of activity, designated as A and B. Both A and B had the same molecular weight and subunit composition. However, they differed in their specific activities and isoelectric points. The p Is of condensing enzymes A and B were 7.6 and 8.4, respectively. Propionyl-CoA was the best substrate for the condensation reaction with both enzymes. However, the specific activity of enzyme B for both propionyl-CoA condensation (3.4 μmol/min/mg protein) and acetoacetyl-CoA thiolysis (13.8 μmol/min/ mg protein) was 2.4 times higher than that obtained with enzyme A. Similarly, chromatography on phosphocellulose resolved the Ascaris condensing enzyme activity into one minor and two major peaks. All of these components had the same molecular weight and subunit composition, but differed in their specific activities. The two major phosphocellulose peaks cross-reacted immunologically when examined by the Ouchterlony double immunodiffusion technique. In addition, antiserum against the phosphocellulose most active form cross-reacted with forms A and B isolated by chromatography of the enzyme on CM-Sephadex, indicating that all forms were immunochemically related.