Ouchterlony Tests Research Articles

Production of an Efficient Enzymatically Fab Fragment Antivenom against Cobra Snake (Naja naja oxiana) Venom.

Since around 100 years ago, the best treatment for millions of global snakebite victims has been polyclonal antivenoms. However, common antivenoms need continuous improvement to reduce rare, their side effects and get better performance. In the present study, Fab antivenom was produced through papain digestion of anti-cobra venom plasma, multi-step purification, and optimization, including ammonium sulfate precipitation and DEAE-cellulose column chromatography. Then, the existence of the corresponding Fab fragment antibody was seen and confirmed by SDS-PAGE method and double immunodifusion (Ouchterlony) test. In addition, the potency test in NIH laboratory mice revealed that each milliliter of the new Fab antivenom was able to neutralize 624 micrograms and (80LD50) of cobra venom, which is about 15% more efficient than the primary plasma of the same concentration, and 1.57 times more effective than the cobra antivenom found in commercial hexavalent antivenom of Razi Institute. According to the findings, it seems that this new Fab antivenom can be used as a new candidate for treatment of cobra snakebite victims.

APLICACIÓN DE TÉCNICAS INMUNOLÓGICAS DE DEPREDACIÓN EN TRIATOMA INFESTANS (HEMIPTERA: REDUVIIDAE)

An immunological technique used to study the interrelation between prey and predator is described.Immune serum was obtained by injecting extracts of eggs of Triatoma infestans of one week of age into rabbits and rats. The presence of antibodies was verified with Ouchterlony test.The specificity of the serum and antiserum was tested in extracts of all stages of Triatoma infestans, in eggs and lst. instar nymphs of Rhodnius proxilus and in adults of Periplaneta americana.Only triatomines showed a positive reaction in Ouchterlony test, indicated by clear precipitation bands. No positive reaction was obtained for Periplaneta americana.These results suggest that the antiserum used presents a marked specificity towards the subfamily Triatominae. This immunological method can ben used, therefore, in the study of triatominophagy by cockroaches and in the study of the role of commonpredator of triatomines and cockroaches.

Inhibition of local effects induced by Bothrops erythromelas snake venom: Assessment of the effectiveness of Brazilian polyvalent bothropic antivenom and aqueous leaf extract of Jatropha gossypiifolia

Bothrops erythromelas is a snake of medical importance responsible for most of the venomous incidents in Northeastern Brazil. However, this species is not included in the pool of venoms that are used in the Brazilian polyvalent bothropic antivenom (BAv) production. Furthermore, it is well known that antivenom therapy has limited efficacy against venom-induced local effects, making the search for complementary alternatives to treat snakebites an important task. Jatropha gossypiifolia is a medicinal plant widely indicated in folk medicine as an antidote for snakebites, whose effectiveness against Bothrops jararaca venom (BjV) has been previously demonstrated in mice. In this context, this study assessed the effectiveness of the aqueous extract (AE) of this plant and of the BAv against local effects induced by B. erythromelas venom (BeV). Inhibition of BeV-induced edematogenic and hemorrhagic local effects was assayed in mice in pre-treatment (treatment prior to BeV injection) and post-treatment (treatment post-envenomation) protocols. Inhibition of proteolytic, phospholipase A2 (PLA2) and hyaluronidase enzymatic activities of BeV were evaluated in vitro. BAv cross-reactivity and estimation of antibody titers against BeV and BjV were assessed by Ouchterlony double diffusion test. The results show that in pre-treatment protocol AE and BAv presented very similar effects (about 70% of inhibition for edematogenic and 40% for hemorrhagic activities). However, BAv poorly inhibited edema and hemorrhage in post-envenomation protocol, whilst, in contrast, AE was significantly active even when used after BeV injection. AE was able to inhibit all the tested enzymatic activities of BeV, while BAv was active only against hyaluronidase activity, which could justify the low effectiveness of BAv against BeV-induced local effects in vivo. Ouchterlony's test showed positive cross-reactivity against BeV, but the antibody titers were slightly higher against BjV. Together, these data indicate that despite the presence of immunological cross-reactivity, Brazilian polyvalent bothropic antivenom presented low inhibitory potential against biological and enzymatic effects of BeV, illustrating the need for new strategies in the production of antivenom with broad neutralizing potential in the treatment of Bothrops spp. envenomation throughout the country. Together, the results highlight the antiophidic potential of J. gossypiifolia, suggesting that it can be considered a potential adjuvant in the treatment of bothropic envenomation local effects.

Evaluation of immunodominant proteins of Mycobacterium avium paratuberculosis cell wall by Western blot analysis.

Mycobacterium avium subspecies paratuberculosis (M. paratuberculosis) is a slow growing mycobactin, whose dependence on mycobacterial species is known to be the causative agent of Johne's disease (paratuberculosis) in all species of domestic ruminants worldwide. The organism is transmitted via close contact, ingestion, or transplacentally from mother to fetus and occurs commonly in grazing domestic animals. Johne's disease (JD) is characterized by gradual weight loss, decreased milk production, and diarrhea due to the chronic, progressive, granulomatous enteritis and lymphadenitis. The disease can cause serious economic damage to the dairy industry due to the loss of milk production and early culling of infected animals. In recent years, researchers have focused on the identification of a specific antigen of M. paratuberculosis to use in diagnosis test and preparation of effective vaccine. The goal of this study is evaluation of the immunodominant proteins of M. paratuberculosis cell wall. The amount of protein was determined with a Lowry assay (22.68 μg/100 μL). For production of polyclonal antibody against proteins of M. paratuberculosis cell wall, a New Zealand white rabbit was immunized with antigen and Freund's adjuvant. After immunization, the rabbit was bled to produce enriched serum. Antibodies were purified from serum with ion exchange chromatography. In the Ouchterlony test, the reactions between antigen and antibodies were seen in dilutions of one quarter for serum, one quarter for Ig, and one half for IgG by clear precipitation lines due to the well immunization of the rabbit. Electrophoresis and Western blot analysis were used and subsequently a sharp band appeared in nitrocellulose paper; these bands were about 25, 37, 50, 75, and 150 kDa molecular weight, which indicated immunodominant proteins.

Alginate Nanoparticles as a Promising Adjuvant and Vaccine Delivery System

During last decades, diphtheria has remained as a serious disease that still outbreaks and can occur worldwide. Recently, new vaccine delivery systems have been developed by using the biodegradable and biocompatible polymers such as alginate. Alginate nanoparticles as a carrier with adjuvant and prolong release properties that enhance the immunogenicity of vaccines. In this study diphtheria toxoid loaded nanoparticles were prepared by ionic gelation technique and characterized with respect to size, zeta potential, morphology, encapsulation efficiency, release profile, and immunogenicity. Appropriate parameters (calcium chloride and sodium alginate concentration, homogenization rate and homogenization time) redounded to the formation of suitable nanoparticles with a mean diameter of 70±0.5 nm. The loading studies of the nanoparticles resulted in high loading capacities (>90%) and subsequent release studies showed prolong profile. The stability and antigenicity of toxoid were evaluated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and ouchterlony test and proved that the encapsulation process did not affect the antigenic integrity and activity. Guinea pigs immunized with the diphtheria toxoid-loaded alginate nanoparticles showed highest humoral immune response than conventional vaccine. It is concluded that, with regard to the desirable properties of nanoparticles and high immunogenicity, alginate nanoparticles could be considered as a new promising vaccine delivery and adjuvant system.

Microscopic and polyclonal antibody-based detection of yellow leaf disease of arecanut (Areca catechu L.)

Phytoplasma, the pathogen of yellow leaf disease (YLD) of arecanut (Areca catechu L.) was detected by transmission and scanning electron microscopy. Tissues of YLD affected palms contained phytoplasmas in the phloem sieve elements, but not in symptomless healthy palm tissues. Phytoplasma was purified from tissues of diseased palms employing percoll density gradient centrifugation and confirmed by transmission electron microscopy. Using the purified phytoplasma preparation, a polyclonal antiserum was raised in rabbits and used for standardisation of agar gel double diffusion (Ouchterlony) test and DAC-ELISA. Clear precipitin line was observed in Ouchterlony test between the antigen from diseased palms and the pathogen-specific antibodies after 48 h incubation and only undiluted antiserum showed best result in the test. However, in ELISA, 1:10 antigen dilution and 1:400 pathogen-specific antibodies dilution produced sensitive detection of the pathogen with a difference of >3.5 times absorption values between healthy and diseased samples. The results thus confirmed the ability of antiserum to distinguish healthy and infected plants and utility of ELISA for effective diagnosis of YLD.

The Immunological Properties of Stroma-free Polyhemolysate Containing Catalase and Superoxide Dismutase Activities Prepared by Polymerized Bovine Stroma-free Hemolysate

Crosslinking of ultrapure hemoglobin, crystalline catalase, and superoxide dismutase resulted in a soluble nanodimensional complex of polyhemoglobin-catalase-superoxide dismutase. A less expensive and more convenient way is to crosslink bovine stroma-free hemolysate (stroma-free hemolysate) that already contains hemoglobin, catalase, and superoxide dismutase into polyhemoglobin with catalase and superoxide dismutase activities (stroma-free polyhemolysate) [21]. The objective of the present study is to evaluate the immunological properties of this stroma-free polyhemolysate. Each of three groups of rats received weekly subcutaneous injections of one of the stroma-free polyhemolysate, stroma-free hemolysate, and saline for four weeks. One week after the four cycles of weekly immunization, serum and plasma were collected for C3a complement activation tests and Ouchterlony antibody-antigen precipitation tests, respectively. Results show that stroma-free polyhemolysate retained significant antioxidant enzyme activity. The C3a complement activation test and Ouchterlony test show that four weekly subcutaneous injections of bovine stroma-free polyhemolysate did not result in any immunological reaction in rats when tested this way.

Occurrence of bacterial black shoot disease of European pear in Yamagata Prefecture

Black lesions on shoots of European pear trees observed in an orchard in Yamagata Prefecture in May 2007 were suspected to be caused by a bacterial pathogen. The surface of the colonies isolated on a high sucrose medium did not have the crater morphology that is characteristic of E. amylovora bvs. 1–3, and a specific DNA fragment was amplified from the isolates in the PCR using the EprpoD primer set. The partial sequences of the 16S rRNA gene placed the isolates in the genus Erwinia. The isolates differed serologically from E. amylovora biovars and E. pyrifoliae in an Ouchterlony double-diffusion test although their bacterial properties suggested that they are closely related to E. amylovora biovars and E. pyrifoliae. In a DNA–DNA hybridization test, the relatedness between the isolates and E. amylovora biovars or E. pyrifoliae did not exceed 70% level, indicating that they are independent species. Thus, the isolates belongs to the genus Erwnia but are not E. amylovra or E. pyrifoliae. After succulent pear shoots were injected with bacterial suspensions (109, 108, 107 and 106 cfu/ml) of the isolates, lesions formed with 109 and 108 cfu/ml, but the disease incidence with 108 cfu/ml was much lower than with E. amylovora and E. pyrifoliae. Virulence of the present isolates is thus thought to be very weak. On the basis of these results, we consider that this is a new shoot disease of European pear. In the 2007 season, all affected trees were pulled out after harvest. No symptoms have been observed in field surveys since the fruitlet season in 2007.

Molecular cloning and characterization of a single-chain variable fragment antibody specific for benzoylecgonine expressed in Escherichia coli

Benzoylecgonine is a major metabolite of cocaine. We generated hybridoma cells (C1303) producing anti-benzoylecgonine monoclonal antibody (mAb) with a single-chain variable fragment (scFv) and an antigen-binding domain from the C1303 cells. Genes encoding an scFv antibody and constant region (Fc) were amplified from a cDNA library of C1303 cells using PCR. The two frameworks built for scFv and scFv-Fc consisted of HL [(heavy chain variable region, V(H)) - linker - (light chain variable region, V(L))] and HL-Fc, respectively. A 45 base-pair-long sequence encoding (Gly(4)-Ser)(3) was used as the linker, and the mouse IgG1 constant region sequence (225 amino acids) was used as the Fc domain. These two types of recombinant Abs were determined to be 750 bp in length (which corresponds to a 30 kDa protein) in the HL and 1,432 bp in length (which corresponds to a 65 kDa protein) in the HL-Fc, respectively. The parental Ab and HL-Fc affinities against benzoylecgonine were measured by ELISA and found to be nearly equal to the Ab concentration. We were also able to measure HL affinity using an agarose diffusion assay (Ouchterlony test). The affinity of the recombinant single-chain antibody against benzoylecgonine was sufficiently comparable to that of the parent antibodies to be used for the immunodetection of specific drug compounds or the detoxification of drug abusers by immunotherapy.

Serotype IX, a Proposed New Streptococcus agalactiae Serotype.

We identified three isolates of Streptococcus agalactiae (group B streptococcus [GBS]), of human origin, which failed to react with antisera against any of the nine known GBS serotypes. Polyclonal rabbit antisera raised against these isolates and standard GBS typing sera were used in capillary precipitation and Ouchterlony tests to compare the strains with known GBS serotype reference strains. All three previously nontypeable isolates reacted with all three new antisera, producing lines of identity in the Ouchterlony test. Weak cross-reactions with antisera against several GBS serotypes were observed but were removed by absorption with corresponding antigens. The new antisera were used to test 227 GBS isolates that had been nontypeable or difficult to type using standard antisera. Of these, five reacted with the new antisera. These results suggested that all eight isolates belong to the previously unrecognized GBS serotype. They were tested by Western blotting for the Calpha and Cbeta proteins and by PCR to identify molecular serotypes and surface protein antigen genes. Two segments of the cps gene cluster (3' end of cpsE-cpsF and 5' end of cpsG, approximately 700 bp; 3' end of cpsH and 5' end of cpsM, approximately 560 bp) were sequenced. All eight isolates expressed Calpha, and seven expressing the Cbeta protein and the corresponding genes, bca and bac, respectively, were identified. They all share the same, unique partial cps sequence. These results indicate that these eight isolates represent a new S. agalactiae serotype, which we propose should be designated serotype IX.

Ein neues serologisches Screeningverfahren zur Diagnostik der Zimmerspringbrunnenalveolitis

A new ELISA is presented here, which detects the IgG antibodies against the antigens in the moldy water of misting fountains. In the sera of 30 of 32 patients suffering from humidifier lung from these new developed humidifiers, IgG antibodies were detected by this screening test. Positive results of this ELISA using coated pooled moldy humidifier water from 4 patients should be confirmed by the Ouchterlony test using patients humidifier water or by IgG tests against the single antigens of humidifier lung as Aureobasidium pullulans, Cephalosporium acremonium, Penicillia and other known molds and bacteria.

Ubiquity of parasporin-1 producers in Bacillus thuringiensis natural populations of Japan

Parasporin, a Bacillus thuringiensis parasporal protein, is unique in having a strong cytocidal activity preferential for human cancer cells. In this study, we characterized parasporin activities associated with three novel geographical isolates of B. thuringiensis. Parasporal inclusion proteins of the three isolates were highly toxic to human uterus cervix cancer cells (HeLa), but not to non-cancer uterine smooth muscle cells (UtSMC). Inclusions of the isolates lacked insect toxicity and hemolytic activity against sheep erythrocytes. Ouchterlony immunodiffusion tests revealed that the proteins of the three isolates are immunologically closely related to parasporin-1 (Cry31A), but dissimilar to the three other existing parasporin groups. Our results provide evidence that the parasporin-1-producing organism is a common member in B. thuringiensis populations occurring in natural environments of Japan.

Occupational airborne contact dermatitis caused by thyme dust.

The aim of the study was to assess occupational hazards to the farmer's skin associated with processing thyme (Thymus vulgaris L.). 46 farmers were studied during the threshing of dried thyme. They were questioned about work-related skin problems and examined before and after work. In all persons, serum thyme-specific IgE was measured. Skin prick tests, the Ouchterlony test and the migration inhibition test were carried out with allergens of airborne bacteria and fungi present in the working environment. Of the 46 farmers studied, 4 showed skin symptoms after 5-30 min of exposure to thyme dust. Thyme-specific IgE was found in 1 person with work-related symptoms, but also in 2 asymptomatic farmers. Therefore, the importance of IgE seems to be questionable in eczema related to thyme dust. Skin and blood tests with microbial allergens also showed no significant differences between the symptomatic and asymptomatic farmers. To our knowledge, this is the 1st description of occupational airborne contact dermatitis caused by thyme dust. The etiology of thyme-related skin symptoms remains obscure, although an irritant mechanism seems probable.

Antigen-Antibody Interaction: Application of the Ouchterlony Method for Determining Evolutionary Relationships

The purpose of this experiment is to illustrate how antigen-antibody interactions can be applied to the study of evolution. Antigens are substances that stimulate an immune response from the body. There are two general categories of antigens: pathogens and allergens (Karp 1996). Pathogens include viruses, bacteria, fungi, protozoan parasites and toxins. Allergens include pollen, spores, and food additives. Antibodies are globular proteins produced by B lymphocytes that bind to an antigen and target the antigen for destruction. Antibodies are located in interstitial fluid and plasma and are mostly effective against pathogens that have not invaded host cells. The Ouchterlony test is a double diffusion method on an agar plate that clearly shows any reaction between antigens and antibody (Ouchterlony & Nilsson 1973). Antigens and antibody are inoculated onto an agar plate into separate wells where they diffuse through the medium. As long as the pore sizes within the agar gel substance are large enough, an assumption is made that diffusion of randomly moving particles can freely occur. The agar must be an inert material that does not react with the testing materials. If the antibody recognizes and binds to an antigen on the plate, a white precipitate will form. The precipitate itself is too large to continue to diffuse-once a precipitate is formed, it is stationary. The antigens for this experiment are albumins from various ungulates: horses, pigs, cows and sheep. Albumins are small globular proteins isolated from blood. Albumin is produced by the liver and participates in homeostasis by influencing the osmotic balance of plasma. Antiserum is a liquid containing antibodies that have been exposed to a specific antigen. For example, antihorse serum includes antibodies that will react against antigens isolated from a horse. Antisera were purchased from Sigma, a chemical supply company (PO Box 14508, St. Louis, MO 63178, USA; 800-325-3010). Students learn in lecture and lab that an outstanding characteristic of antibodies is their property of specificity. The general hypothesis being tested is: a specific antigen will react with a specific antibody that has been formed against it, and form a precipitate. Therefore, horse albumin should react only with anti-horse serum, pig albumin with anti-pig, and so on. The results can be used to support the current classification of the ungulates at the order and suborder level. To accomplish this, students must become informed about the following subjects:

Evidence of immunological Responses by a Host Fish (Ambloplites rupestris) and Two Non-Host Fishes (Cyprinus carpio and Carassius auratus) to Glochidia of a Freshwater Mussel (Villosa iris)

ABSTRACT Immunological responses of fishes to glochidia were evaluated using glochidia of the rainbow mussel (Villosa iris) to infest a host species, rock bass (Ambloplites rupestris), and two nonhost species, common carp (Cyprinus carpio) and goldfish (Carassius auratus). Ouchterlony double-diffusion tests showed that host and nonhost species expressed a humoral defense factor specific to glochidial antigens after induced infestation with glochidia. Precipitin bands were observed in tests on infested fishes but not in tests on uninfested fishes. Microagglutination tests showed that host and nonhost species that were uninfested, infested, or reinfested with glochidia all expressed some agglutination response to glochidial antigens. Experimental fishes had specific humoral defense factors that reacted immunologically to glochidia tissue.

A CASE OF HUMAN FASCIOLIASIS

We experienced a case of human fascioliasis diagnosed by a resected specimen and postoperative immunoserological tests. A 71-year-old man was admitted to the hospital because of transient upper abdominal pain and a hepatic abnormal shadow on abdominal ultrasonography and computed tomography. The laboratory data revealed eosinophilia and elevations in biliary emzymes and carcinoembryonic antigen. Imaging procedures showed multiple nodular masses localized in left lobe of the liver. On suspicion of malignant tumor, a left lobectomy of the liver was performed. The lesion was found by microscopic examination to be multiple abscesses with remarkable infiltration of eosinophils. Though parasitic disease was suspected, no parasitic body nor egg was detected in the resected specimen and feces. The diagnosis of fascioliasis was established by immunoselorogical methods (Ouchterlony test and immunoelectrophoresis). After the operation and subsequent medication with prasziquantel, the laboratory data were normalized.

Measurement of beta 2-microglobulin in bovine serum and urine by radioimmunoassay.

A radioimmunoassay (RIA) system for quantification of bovine beta 2-microglobulin (beta 2-M) in serum and urine was developed. The protein isolated from bovine colostrum showed a single band in SDS-PAGE, and its molecular weight was approximately 11,600. Amino acid sequences for the first 24 residues and the amino acid composition of the protein were in agreement with those in the bovine beta 2-M of previous research works. In an Ouchterlony test, a single precipitation line was formed between the protein and the antiserum made by the protein. From these results, it was confirmed that the protein isolated from the colostrum was pure bovine beta 2-M. For creation of an RIA calibration curve for urine, a urine void of beta 2-M, as much as possible (beta 2-M-free urine), and a PBS were used as diluents. Intraassay (n = 10) and interassay (n = 3) variances were 1.7-4.6% and 7.1-11.5% in the PBS dilute method, and were 1.4-5.1% and 12.3-13.5% in the beta 2-M-free urine dilute method, respectively. Mean recoveries were 160 +/- 19% (mean +/- SD) and 98.4 +/- 7.9% in PBS and beta-M-free urine, respectively. It was found that the method using the beta 2-M-free urine as a diluent was more accurate than using PBS. The beta 2-M concentrations in serum and urine of healthy Holstein cows measured by this RIA system showed a logarithmic normal distribution for urine and a normal distribution for serum. The mean beta 2-M concentrations were 0.0305(+0.04443)(-0.0210) mg/l (Geometric mean +/- S.D., n = 43) in urine and 2.87 +/- 0.45 mg/l (Arithmetic mean +/- S.D., n = 26) in sera. Further, we could not observe the particular tendency of daily variation in urinary beta 2-M concentrations of healthy cows (Holstein, n = 3 x 2 days).

Ein Vergleich von vier Immunoblot-Systemen zur Differenzierung von Antikörpern gegen nukleäre und zytoplasmatische Antigene

Since recently, immunoblots for detection and differentiation of antibodies against nuclear antigens (ENA), other nuclear antigens and cytoplasmic antigens, blotted on nitrocellulose strips (Western blot technique) became commercially available. The aim of our analysis was to compare the specifity and sensitivity of four different immunblot systems (blots A, B, L, V). We examined 20 sera of patients with various clinically confirmed connective tissue diseases. The sera contained a total of 32 different antigens. The antibodies in the sera were defined by double immunodiffusion (Ouchterlony) test, immunofluorescence (HEp2 cells) and by comparison with WHO reference sera.

Interconversions of lipophorin particles by adipokinetic hormone in hemolymph of Panstrongylus megistus, Dipetalogaster maximus and Triatoma infestans (Hemiptera: Reduviidae)

Hemolymph of Panstrongylus megistus , Dipetalogaster maximus and Triatoma infestans (Hemiptera: Reduviidae), in the presence of adipokinetic hormone and fat bodies, has the molecular components for generating low density lipophorin (1.060–1.070 m ml −1 ) from high density lipophorin (1.130–1.140 g ml −1 ), similar to that observed in long-distance flight insects. Low density lipophorin showed a significant increase of acylglycerols and a higher amount of apolipophorin-III (mol.wt ∼ 18 kDa) with respect to the original fraction of the incubation media. This process was dependent upon the nutritional status of the insects, whose fat bodies with > 20 days of starvation had the highest levels of acylglycerols. Antiserum against high density lipophorin produced a single precipitation line when it reacted by using Ouchterlony's test with high and low density lipophorins from the three species, and no reaction was observed with other lipophorins.

Construction and characterization of recombinant Vibrio cholerae strains producing inactive cholera toxin analogs.

The catalytic A subunit of cholera toxin (CT-A) is capable of ADP-ribosylating the guanine nucleotide-binding protein, which regulates cell adenylyl cyclase, leading to the life-threatening diarrhea of cholera. Amino acids involved in the enzymatic activity of CT-A have previously been identified. By means of site-directed mutagenesis, an analog of the CT-A subunit gene was created with codon substitutions for both Arg-7 and Glu-112, each of which has been shown to produce subunits lacking ADP-ribosyltransferase activity. The mutated gene fragment was exchanged for the wild-type copy in the previously cloned ctxAB operon from El Tor biotype, Ogawa serotype Vibrio cholerae strain 3083, which produces CT-2. Further, the zonula occludens toxin gene, zot, was inactivated by an insertional mutation to create the new plasmid construct pCT-2*. Additionally, a DNA fragment encoding the B subunit of CT-1 (CT produced by classical biotype, Inaba serotype V. cholerae strain 569B) was exchanged for the homologous part in pCT-2*, resulting in the creation of pCT-1*. These plasmid constructs were introduced into the CT-negative V. cholerae mutant strain JBK70 (E1 Tor biotype, Inaba serotype); CT-A-B+ derivatives CVD101 and CVD103 of classical biotype Ogawa and Inaba serotype strains 395 and 569B, respectively; El Tor biotype Inaba and Ogawa serotype strains C6706 and C7258, respectively, recently isolated in Peru; and O139 (synonym Bengal) strain SG25-1 from the current epidemic in India. Recombinant toxins (CT-1* and CT-2*), partially purified from culture supernatants of transformed JBK70, were shown to be inactive on mouse Y1 adrenal tumor cells and in an in vitro ADP-ribosyltransferase assay. CT-1* and CT-2* reacted with polyclonal and monoclonal antibodies against both A and B subunits of CT. The toxin analogs reacted with antibodies against CT-A and CT-B on cellulose acetate strips and in a GM1 enzyme-linked immunosorbent assay; they reacted appropriately with B-subunit epitype-specific monoclonal antibodies in checkerboard immunoblots, and they formed precipitin bands with GM1-ganglioside in Ouchterlony tests. However, the reactions of the modified proteins with anti-A-subunit monoclonal antibodies were weaker than the reactions with wild-type holotoxins. V, cholerae strains carrying ctxA*, with either ctxB-1 or ctxB-2, and inactivated zot genes were created by homologous recombination. The recombinant strains and the purified toxin analogs were inactive in the infant rabbit animal model.(ABSTRACT TRUNCATED AT 400 WORDS)