Gel Double Immunodiffusion Research Articles

Stability of serum precipitins to Aspergillus fumigatus for the diagnosis of allergic bronchopulmonary aspergillosis

BackgroundAllergic bronchopulmonary aspergillosis (ABPA) reflects hypersensitivity and an exaggerated immune response to Aspergillus fumigatus. ABPA typically occurs in individuals with airway diseases such as asthma or cystic fibrosis and is associated with worse outcomes for individuals with these conditions. Each year, physicians across the province of British Columbia submit over 2600 diagnostic testing requests to a centralized location in Vancouver, requiring specimen collection, storage, and shipment from different clinics across the province. Timely and reliable testing of Aspergillus precipitins is critical to optimizing diagnosis and management of ABPA. At our centre, we analyzed sample stability in varying storage conditions to provide guidance to those using this routine diagnostic test.MethodsTo determine temperature and time stability, 31 serum specimens positive for Aspergillus fumigatus precipitins from routine clinical testing were each aliquoted and incubated at 4 and 37 °C. Samples were repeatedly assayed for precipitins to Aspergillus fumigatus via agarose gel double immunodiffusion (AGID) at 7, 14, and 28 days post-incubation.To determine freeze–thaw stability, 39 serum specimens submitted for routine clinical testing for Aspergillus precipitins were randomly selected. Each specimen was aliquoted and stored at 4 or −20 °C. 4 °C samples were maintained at 4 °C while −20 °C samples were split into three groups corresponding to one, two, or three freeze–thaw cycles. −20 °C samples were thawed at room temperature in the morning and then immediately frozen overnight for up to a total of three freeze–thaw cycles.ResultsRegarding temperature and time stability, median stability time was 47 and 34 days at 4 and 37 °C, respectively. The log-rank model indicates no statistically significant difference between the two temperature storage conditions (p = 0.14) with a hazard ratio of 0.61 (95% CI, 0.31–1.2).In terms of freeze–thaw stability, no indication of serum degradation with regards to Aspergillus fumigatus precipitins was found with repeated freeze–thaw cycles as compared to refrigerated storage.ConclusionsThe stability of serum precipitins to Aspergillus fumigatus was found to be dependent on time, but not temperature and freeze–thaw cycles. Specimens for Aspergillus fumigatus precipitins testing should be shipped at ambient temperature and tested within 2 weeks from collection.

Detection of Aspergillus-specific antibodies by agar gel double immunodiffusion and IgG ELISA in feline upper respiratory tract aspergillosis

Feline upper respiratory tract aspergillosis (URTA) is an emerging infectious disease. The aims of this study were: (1) to assess the diagnostic value of detection of Aspergillus-specific antibodies using an agar gel double immunodiffusion (AGID) assay and an indirect immunoglobulin G (IgG) ELISA; and (2) to determine if an aspergillin derived from mycelia of Aspergillus fumigatus, Aspergillus niger and Aspergillus flavus can be used to detect serum antibodies against cryptic Aspergillus spp. in Aspergillus section Fumigati. Sera from cats with URTA (group 1: n = 21) and two control groups (group 2: cats with other upper respiratory tract diseases, n = 25; group 3: healthy cats and cats with non-respiratory, non-fungal illness, n = 84) were tested. Isolates from cats with URTA comprised A. fumigatus (n = 5), A. flavus (n = 1) and four cryptic species: Aspergillus felis (n = 12), Aspergillus thermomutatus (Neosartorya pseudofischeri, n = 1), Aspergillus lentulus (n = 1) and Aspergillus udagawae (n = 1).Brachycephalic purebred cats were significantly more likely to develop URTA than other breeds (P = 0.013). The sensitivity (Se) of the AGID was 43% and the specificity (Sp) was 100%. At a cut-off value of 6 ELISA units/mL, the Se of the IgG ELISA was 95.2% and the Sp was 92% and 92.9% for groups 2 and 3 cats, respectively. Aspergillus-specific antibodies against all four cryptic species were detected in one or both assays. Assay Se was not associated with species identity. Detection of Aspergillus-specific antibodies by IgG ELISA has high Se and Sp for diagnosis of feline URTA.

Manifestações cutâneo-mucosas da coccidioidomicose: estudo de trinta casos procedentes dos estados do Piauí e Maranhão

Coccidioidomycosis is a systemic mycosis that usually presents as a benign infection. Patients generally recover spontaneously; however, a small proportion of infected individuals develop progressive complications that may affect the skin in the form of disseminated pleomorphic lesions and may become fatal. To identify and describe skin and mucous membrane manifestations of coccidioidomycosis, to identify occupational hazards associated with the disease and to determine its associated clinical presentation. A study conducted between 2003 and 2006 involving 30 patients from the Brazilian states of Piauí and Maranhão with coccidioidomycosis diagnosed by direct microscopy, sputum culture or screening serology using agar gel double immunodiffusion, in association with anamnesis and physical examination. Extrapulmonary lesions were found in 13 cases (43.3%), consisting predominantly of dermatological manifestations of hypersensitivity: erythema nodosum (26.6%), exanthema with erythematosquamous lesions (26.6%) and erythema multiforme (23.3%), as well as ulcerations of the tongue (13.3%), lip ulcers (6.6%) and subcutaneous abscess (3.3%). These manifestations were seen during the acute phase of the disease. Skin manifestations associated with an acute respiratory infection reinforce the hypothesis of a diagnosis of coccidioidomycosis, particularly in individuals who hunt armadillos or in those exposed to soil excavation.

Differentiation of Bulgarian Isolates from Cucumber Mosaic Virus by Serological Methods

Cucumber mosaic virus (CMV) is of great importance to the Bulgarian economy and hence a detailed knowledge of its diversity under local geographic and climatic conditions is required. An extended study was carried out on CMV strains the currently occur in Bulgaria. Fifty‐one isolates and strains found in different regions and various crops were biologically characterized and serologically differentiated into subgroups I and II using different variants of enzyme‐linked immunosorbent assay (ELISA) [double antibody sandwich (DAS)‐, antigen‐coated plate (ACP)‐, triple antibody sandwich (TAS)‐ with poly and monoclonal antibodies] and immunodiffusion tests. The ELISA modifications with monoclonal antibodies individually (ACP) or in combination with polyclonal antibodies (TAS‐ELISA) are suitable for mass screening of CMV isolates. The hyperimmune sera against strains from CMV subgroups I and II were very efficient for use in isolate differentiation via gel double immunodiffusion. The results obtained correlated with the polymerase chain reaction and restriction fragment length polymorphism data reported by other authors. The majority of the isolates belonged to subgroup I, whereas 10, mainly from tomato and pepper, belonged to subgroup II. Most of the subgroup II isolates came from the north of Bulgaria. The results of the present study will help to clarify the virus epidemiology and to develop specific control measures.

Immunochemical characterisation of species-specific antigens in bovine crude heparin

In order to develop immunoassays for the control of the species origin of crude heparins, polyclonal antisera were produced in rabbits against samples obtained from the last purification steps of bovine intestinal crude heparin. The reactivity of the antisera was analysed by agar gel double immunodiffusion, immunoelectrophoresis, crossed and line immunoelectrophoresis. Up to 13 antigenic components were detected in the effluents of the ion-exchange chromatographic step, and three in the final crude heparin. The major and most anodic antigen (Ag1) was recovered in bovine crude heparin purified by the two different industrial processes. This bovine specific antigen was found in high concentrations in lung, liver, small intestine, spleen and kidney. It displayed an apparent molecular weight of 45 kDa by size-exclusion chromatography. Though the identification of Ag1 is not yet fully elucidated, a single radial immunodiffusion assay has been developed for the quantification of this antigen, allowing the detection of 6 p 1000 bovine crude heparin in porcine heparin (50 mg/ml) after 2 h diffusion.

Synthesis of novel sialic acid-containing polymers as inhibitors of hemagglutination

Influenza viruses are serious pathogens for humans. They are known to infect host tissues by first binding to sialoconjugates on the cell surface through their hemagglutinin. α-Sialoside-containing polymers have been demonstrated to be potent inhibitors of the hemagglutination on human erythrocytes by influenza viruses. Moreover, they have showed superior antigenic properties than their corresponding natural sialoconjugates from the basis of multivalent and cluster effects. For the viewpoint of drug targeting, we have tried to develop efficient synthesis of novel sialopolymers in which the inter-sialoside distances could be controlled. We report the novel synthesis of sialocopolymers using condensation polymerization. The condensation of branched α-sialoside, prepared from p-nitrophenyl α-sialoside and 3,3′-iminobis(propylamine) with 1,4-diisocyanatobutane and 1,6-diisocyanatohexane, produced copolymers without any promoters. These copolymers retained their specific lectin-binding properties, as determined with wheat germ agglutinin (WGA), a plant lectin known to bind sialosides and glucosaminides on the agar gel double immunodiffusion test. Each copolymer showed clear binding. On micro-turbidimetric assay, they exhibited strong cross-linking properties with WGA, and copolymer including the hexyl group showed stronger binding properties than the butyl one.

Heterogeneity observed in the components of hemolysin BL, an enterotoxin produced by Bacillus cereus

Hemolysin BL (HBL), a diarrheal enterotoxin originally isolated from Bacillus cereus strain F837/76, is composed of three antigenically distinct proteins designated B, L 1, and L 2. All three components are required for biological activity. Here, we report antigenic and physical variations in HBL components produced by other B. cereus isolates. Reactions of partial identity were observed in double gel immunodiffusion assays using antibodies to highly purified B, L 1, and L 2 components of F837/76 and culture supernatants of strains F837/76 and S1C. Western blot analysis showed that F837/76 produced one 38-kDa B protein, one 38-kDa L 1, and one 43-kDa L 2 protein. In strain S1C, two B (38 and 42 kDa), two L 1 (38 and 41 kDa), and one L 2 (43 kDa) proteins were detected. Further Western blot analysis of 127 B. cereus isolates showed that 90 produced one or more of the three HBL components. Approximately half of these 90 isolates (43/90; 48%) produced protein profiles which differed from that of F837/76. A total of four B, two L 1, and three L 2 component profiles with proteins of different sizes were observed. Individual strains produced various combinations of single or multiple bands of each component. In addition, some strains produced only one or two of the three HBL components. The public health significance of these strains is unknown, as all three components are required for biological activity. The data presented here demonstrates a high degree of heterogeneity in HBL and provide the basis for further studies to characterize the variations in HBL and to determine the role of the variant components in pathogenicity.

Isolation and characterization of monospecific antibodies to β2-microglobulin

Monospecific polyclonal antibodies were isolated from sheep antiserum to β2-microglobulin by affinity chromatography and ammonium sulfate fractionation followed by ion-exchange chromatography. Antibodies were characterized by polyacrylamide gel electrophoresis, immunoblotting, and double radial immunodiffusion according to Ouchterlony. The isolated antibodies are highly specific and can be used for measuring β2-microglobulin in human serum by Mancini's radial immunodiffusion method.

Serotyping of Canadian isolates of Treponema hyodysenteriae and description of two new serotypes

A total of 30 isolates of Treponema hyodysenteriae collected in the Saint-Hyacinthe (Quebec, Canada) area were serotyped by agar gel double immunodiffusion by using extracted lipopolysaccharide and hyperimmune rabbit antisera. Only 17% (5 of 30) of the isolates were typed with antisera specific for each of the seven known serotypes of T. hyodysenteriae. Antisera raised against 11 untypeable local isolates were then produced and tested against each lipopolysaccharide extract. Results showed two serologically distinct groups among 21 of the 25 untypeable isolates. The isolates in each group shared identical antigens. No detectable reactions could be observed between antisera raised against these 11 isolates and the antigens extracted from 7 reference serotype strains. On the basis of these results, two new serotypes of T. hyodysenteriae, serotypes 8 and 9, are proposed. We also propose isolate FM 88-90 as the reference strain for serotype 8 and isolate FMV 89-3323 as the reference strain for serotype 9. These two new serotypes, which represented 70% of the isolates tested, seem to be the major serotypes found in the province of Quebec.

Serological grouping ofTreponema hyodysenteriae

Two Australian isolates of Treponema hyodysenteriae which did not fit within the current serological grouping system for these bacteria were examined by agarose gel double immunodiffusion tests (AGDP). Isolate Vic1 was serologically unique, and we propose that it becomes the type organism for a new sixth serological group of T. hyodysenteriae (Group F). Isolate Q1 was unusual in that lipopolysaccharide (LPS) extracted from it reacted strongly in AGDP with serum raised against the type organism for serogroup D (A1), and also weakly with serum raised against the type organism for serogroup B (WA1). The nature of this cross-reactivity was examined by using cross-absorbed antisera in AGDP, and by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot analysis. The pattern of serological cross-reactivity between Q1, A1 and WA1 was complex and was not fully defined, but the isolate Q1 apparently shared low molecular weight 'serogroup' LPS antigens with A1, and shared higher molecular weight LPS antigens with WA1. On this basis Q1 was designated as belonging to serogroup D, although it was recommended that this be qualified as D (B) to indicate the presence of weak cross-reactivity with serogroup B. Such serological cross-reactivity may have significance in relation to the development of immunity to T.hyodysenteriae. Isolate Q1 may be a potentially useful organism for vaccine development because of its ability to induce a good serological response to LPS of treponemes from both serogroups D and B.

Proposed revisions to the serological typing system forTreponema hyodysenteriae

Antisera were prepared in rabbits against seven well-characterized strains of Treponema hyodysenteriae of known serotype, and reacted in agarose gel double immunodiffusion tests (AGDP) with lipopolysaccharide (LPS) extracted from 18 Western Australian isolates of the organism. Eight isolates were provisionally typed by this method, but sera raised against one 'typed' and two 'untypable' local isolates reacted in an unexpected fashion with LPS from other local and type strains. Serum raised against the 'typed' local isolate reached with LPS from other previously untyped local isolates: this indicated the presence of more than one major LPS antigen amongst certain local isolates, and was confirmed by cross-absorption of sera. Sera raised against apparently untypable local isolates reacted with LPS from certain type organisms, thus suggesting the presence of complex antigenic relationships between LPS antigens. The serotyping system for T. hyodysenteriae which was proposed by Baum & Joens (1979) uses unabsorbed antisera and is made unworkable by these observations. Instead we propose placing organisms which share common LPS antigens into serogroups A to E, members of which are defined by their reactivity with unabsorbed sera raised against a type organism for the group. We suggest strains B78, WA1, B169, A1 and WA6 respectively as being the most suitable type organisms for the five serogroups identified so far. Isolates possessing additional unique LPS antigens can be regarded as serotypes within the serogroup. However the serotype of an isolate can only be established if antiserum is prepared against it, and this serum continues to react homologously after cross-absorption with bacteria from other serotypes within the serogroup.

Enterotoxin B production and phage typing patterns of Staphylococcus aureus.

A total of 253 clinical strains of Staphylococcus aureus collected from six hospitals of Kaohsiung-Pingtung district were under investigation of their enterotoxin B producing capability and phage typing patterns. As high as 47.0% of enterotoxinogenic strains were detected by means of geldouble immunodiffusion method. Among 119 positive strains, 60.6% were found to be in pus specimen and most of enterotoxinogenic strains(66.4%)were prevalent in the period of May-July. Typing by the Basic Set of phages at 100 times the strength of the Routing Test Dilution (RTD), the strains could be grouped into 23 types and the types of 55,77,79/55/77,77/85,79/80/77,80/77/85/7and 55/77/85 were predominant. Enterotoxin B producing strains were distributed in the types of 55/85,55/77/85,55/54,79/80/77,79/55/77and 77. Of all strains tested, 14.2% was selected out as methicillin resistant strains (MRSA). The phage typing patterns of MRSA were predominantly fall into types of 80/81,77 and 79/55/77. In addition, the types of 80/81 and 77 were especially correlated with enterotoxin B production.

Nature and reactivity of staphylococcal enterotoxin A monoclonal antibodies.

Monoclonal antibodies from four clones (C5, C3, B2II, and B2I) directed against staphylococcal enterotoxin A were tested by the indirect enzyme-linked immunosorbent assay and double-gel immunodiffusion (micro-Ouchterlony) assay for the nature of heavy and light chain types. The reactivities of monoclonal antibodies were also tested by indirect enzyme-linked immunosorbent assay with various levels of purified staphylococcal enterotoxin A and various levels (dilutions) of monoclonal antibodies and saturation analysis-competitive indirect enzyme-linked immunosorbent assay. The heavy-chain isotype of monoclonal antibodies was found to be an unspecified subclass of immunoglobulin G1, and the light chain was the kappa type. Monoclonal antibodies from all of the clones exhibited high reactivity and nearly the same affinity to staphylococcal enterotoxin A in saturation analysis-competitive enzyme-linked immunosorbent assay. Purified immunoglobulin G from B2I yielded very high absorbance (1.2) at 405 nm with 1 ng of staphylococcal enterotoxin A as the coating antigen in the enzyme-linked immunosorbent assay. Monoclonal antibodies from B2I also neutralized the biological activity of staphylococcal enterotoxin A when tested by the kitten bioassay.

Immunological responses and the role of the paralyzing toxin in rabbits infested with Rhipicephalus evertsi evertsi.

Twenty-nine rabbits were exposed to 4 infestations with 20, 50, and 100 adult red-legged ticks (Rhipicephalus evertsi evertsi). Upon challenge, 16 rabbits (55%) contracted paralysis, and 14 of these (88%) died. Most deaths occurred in rabbits challenged with 100 ticks. Immunity to paralysis and subsequent resistance to tick feeding developed in the hosts that survived the initial challenge. Antibody titers to a salivary gland antigen increased progressively as determined by the following tests: passive hemagglutination, agar gel double immunodiffusion, and enzyme-linked immunosorbent assay. Rabbits challenged with 20 ticks displayed declining titers with advanced frequency of challenge and few of the rabbits in this group developed paralysis. There was no decline in antibody titers in groups of rabbits exposed to 50- and 100-tick loads. It is suggested that a certain quantum of antigen injected by the ticks is necessary for induction of immunity to paralysis.

Presence of toxic shock toxin in toxic shock and other clinical strains of Staphylococcus aureus.

Toxic shock toxin (TST), also known as pyrogenic exotoxin C (Schlievert et al., J. Infect. Dis. 143:509-516, 1981) and staphylococcal enterotoxin F (Bergdoll et al., Lancet i:1017-1021, 1981), was purified from toxic shock strains of Staphylococcus aureus by preparative isoelectric focusing and by chromatofocusing. Neither method produced an absolutely pure protein as determined by silver staining of sodium dodecyl sulfate-acrylamide gels, although chromatofocusing was the better method of the two. Three molecular weight variants of the protein were found in the two toxic shock syndrome strains that were studied, regardless of the purification method that was used. An isoelectric point of 7.15 and molecular weights of 21,400, 22,100, and 23,200 were determined for the different forms of the protein from electrophoresis data. A sedimentation coefficient of 2.3S was determined by sucrose gradient centrifugation, and a Stokes radius of 2 X 10(-7) cm was determined by gel filtration. An average molecular weight of 18,900 for all of the TST forms was calculated from these data by the Stokes-Einstein equation. A survey for TST in 32 control and 46 toxic shock strains of S. aureus by isoelectric focusing and by agarose gel double immunodiffusion with specific rabbit antiserum revealed that the isoelectric focusing method tends to overestimate the number of TST-positive strains because of the detection of non-TST, neutral staphylococcal proteins. Based on immunodiffusion data, the association of TST with toxic shock strains was found to be 100% in vaginal isolates and 62% in non-vaginal isolates. In the control strains, TST was found in 16% of the vaginal strains and 23% of the non-vaginal strains. The value of this toxin as a marker for toxic shock and its relationship to the pathogenesis of this disease are discussed.

Hydrophilic form of dopamine beta-hydroxylase from purified noradrenergic vesicles. Activities and comparisons.

Bovine splenic nerve and adrenal medulla were used as homologous sources of dopamine beta-hydroxylase permitting the isolation of enzyme specific to a purified fraction of large dense cored noradrenergic vesicles and chromaffin granules, respectively. The hydrophilic (water soluble) form of the enzyme was purified to homogeneity on the bases of gel electrophoresis, isoelectric focusing, and double immunodiffusion tests from the physical lysates of the vesicles and granules. Amino acid analyses suggest that the hydrophilic dopamine beta-hydroxylase is the predominant form in the nerve vesicles. It has higher neutral and lower hydrophobic amino acid group residues when compared to the adrenomedullary enzyme prepared in this and most other laboratories. Among the neutral amino acids, this difference appears to reflect approximately 40% higher serine and glycine contents, and among the hydrophobic amino acids it may reflect in part approximately 25% lower leucine content. Although the terms hydrophilic and amphiphilic can be properly applied to certain chemical properties of the D beta H forms, it is not at all certain that these terms can be used quantitatively to describe the matrix and membrane associated forms of the enzyme, respectively.

Comparison of ultracentrifugation and gel filtration for the isolation of bovine lipoproteins.

Lipoproteins from the plasma of three nonlactating Holstein cows were isolated using either preparative ultracentrifugation or gel filtration chromatography. Lipoprotein classes obtained by ultracentrifugation were very low density plus chylomicra, less than 1.006 g/ml; low density, 1.007-1.039 g/ml; high density1, 1.040-1.063 g/ml; and high density, 1.064-1.22 g/ml. These lipoprotein classes were individually applied to an agarose gel column to determine at what volume they eluted in comparison to lipoproteins that were separated after applying total bovine lipoproteins to the column. Three major peaks corresponding to very low density lipoproteins plus chylomicra, low density, and high density lipoproteins resulted after gel filtration of total lipoproteins. Very low density lipoproteins plus chylomicra, obtained by ultracentrifugation, eluted as a single peak, as did low density and high density lipoproteins. However, high density1 lipoproteins eluted as two peaks. The first peak eluted at the same volume as low density lipoproteins, and the second peak eluted at a volume similar to that of the ascending slope of the high density lipoprotein peak. Results from disc polyacrylamide gel electrophoresis, immunoelectrophoresis and double immunodiffusion of lipoprotein fractions, and SDS polyacrylamide gel electrophoresis of their apoproteins, similarly indicated that the lipoproteins present in the 1.040-1.063 g/ml density interval are a mixture of low and high density lipoproteins rather than a unique class of lipoproteins.

On the possible presence of a β 2-microglobulin-like protein in extracts of livers from normal chickens and chickens with erythroblastosis—III. Immunological relationship to serum albumin of a small molecular weight human liver protein

1. 1. Human liver contains a small molecular weight protein (SMWP) previously shown to be biochemically homologous with a chicken liver protein in terms of its amino acid residues. 2. 2. This human protein, which reacts immunologically as a human serum protein, has been tested for its reactions against a battery of antisera that react specifically with many of the human serum proteins. 3. 3. Material prepared from four human livers gave strong reactions in double gel immunodiffusion with an antiserum against human albumin. One of the liver preparations reacted weakly with antiserum against human ferritin; this ferritin is assumed to be a contaminant. 4. 4. Because of the biochemical homology of human liver SMWP with chicken liver SMWP the latter would be expected to react immunologically as serum albumin.

Purification of two Clostridium perfringens enterotoxin-like proteins and their effects on membrane permeability in primary cultures of adult rat hepatocytes.

We isolated two proteins, ET-1 and ET-2, from the sporangial extracts of Clostridium perfringens type A. Both proteins had some properties in common with the well-known C. perfringens enterotoxin. ET-1 and ET-2 behaved as single and distinct entities in anion exchange chromatography and disk gel electrophoresis. ET-2 was the more anionic protein since it eluted more slowly from the anion exchange column and migrated faster toward the anode in polyacrylamide disk gel electrophoresis (pH 8.5, native gels). Additionally, in this electrophoretic system ET-2 was not distinguishable from the enterotoxin. The amino acid compositions of ET-1 and ET-2 were similar but differed in a few amino acid residues. The values for both proteins were also similar to the published reports of others for the enterotoxin. Both ET-1 and ET-2 showed lines of identity in agar gel double immunodiffusion against anti-enterotoxin antiserum. Both ET-1 and ET-2 were toxic for rat hepatocytes in primary monolayer culture as determined by accelerated exodus of L-[14C]glucose from preloaded cells and by the rapid uptake of 45Ca2+ after exposure to the proteins. In this regard, ET-1 and ET-2 appeared to be identical in mechanism of action to what has been regarded in the literature as "the" C. perfringens enterotoxin. Interestingly, ET-2 was 3 to 10 times more toxic on a weight basis than ET-1 was.

Specificities of antisera directed against soybean lipoxygenases-1 and -2 and purification of lipoxygenase-2 by affinity chromatography

Antiserum directed against lipoxygenase-1 from soybean ( Glycine Max (L.) Merr. var. Williams) was developed in rabbits with electrophoretically pure lipoxygenase-1 as an antigen. To remove traces of lipoxygenase-1 from a lipoxygenase-2 preparation the latter was chromatographed over a column of Sepharose 4B to which antibodies directed against lipoxygenase-1 were coupled. During affinity chromatography of lipoxygenase-2 no protein and only a small amount of activity were lost. Affinity-purified enzyme was used for immunization of rabbits to produce lipoxygenase-2 antibodies. Results with double gel immunodiffusion tests with the two soybean lipoxygenases and antisera directed against them demonstrated that there is no immunological relationship between the isoenzymes. Lipoxygenases from different species of leguminosae crossreacted only with antiserum directed against soybean lipoxygenase-2, and not with anti soybean lipoxygenase-1 serum. No crossreaction could be detected between soybean lipoxygenase antisera and lipoxygenases from species of Gramineae, Linaceae and Solanaceae.