Development and validation of streptavidin-biotin-based double antibody sandwich ELISA for ricin diagnosis.

BACKGROUND: Ricin is a potential biowarfare agent. It is a phytotoxin isolated from castor seeds. At present there is no antidote available for ricin poisoning, patients only get supportive treatment based on their symptoms. This highlights the importance of early detection to avoid severity of accidents and reduce the risk factor. Considering this, our study aimed to develop a highly sensitive and specific sandwich ELISA for the detection of ricin. METHODS: Ricin was purified from castor seeds. Anti-ricin polyclonal and monoclonal antibodies were generated from rabbit antisera and hybridoma cell (1H6F1) supernatant using a protein A/G column. Antibody titer estimation was done using Indirect ELISA. A streptavidin-biotin-based sandwich ELISA was developed and the limit of detection (LOD), linear range, intra and inter-assay coefficient of variation (CV), and cross-reactivity with other similar toxins were determined. Interference of human plasma samples spiked with ricin was also checked. RESULTS: The LOD of the ELISA was found to be 0.45 ng/ml, with a linear range of 0.90-62 ng/ml, intra and inter-assay CV ranged from 3.34 % to 5 % and 5.17 % to 10.80 % respectively. The assay was not cross-reactive with other similar ribosome-inactivating protein (RIP) toxins. Ricin was detected in spiked plasma samples. CONCLUSION: The developed assay is highly sensitive and specific for detecting ricin and is not cross-reactive with other similar types of toxins. The assay can detect ricin in spiked plasma samples, so it has the potential to be used for the analysis of clinical samples after ricin poisoning.

[Green coffee bean allergy and ultrafine particles exposure in Trieste dock workers]. / Allergia a caffè verde ed esposizione a particelle ultrafini nei lavoratori del porto di Trieste.

SUMMARY: The paper reviews allergy to green coffee bean and castor bean in dock workers and in coffee processing workers from '80 to nowadays in Trieste (NE of Italy). The avoidance of use of jute sacks contaminated with castor bean caused a decrease in sensitization to castor bean and the better work practices to handle jute sacks permitted to reduce airborne exposure to green coffee been powders, that resulted below occupational exposure limits. However, the measurement of ultrafine particles emitted during the handling of sacks showed exposure to high level of particles below 40 nm and permitted to identify some work tasks that can cause a more elevated exposure. Moreover, some sacks, coming from Tanzania, are still contaminated with castor bean, causing mild allergic symptoms. The work condition in dock workers in Trieste improved in years with a reduction of exposure to these allergens. However, the adoption of protective measures as well as periodical medical surveillance are needed to prevent sensitization or to detect the early onset of new cases.

Identification of critical amino acids in the IgE epitopes of Ric c 1 and Ric c 3 and the application of glutamic acid as an IgE blocker.

BACKGROUND: The allergenicity of Ricinus communis L. (castor bean, Euphorbiaceae) is associated with components of its seeds and pollen. Castor bean allergy has been described not only in laboratory workers, but also in personnel working in oil processing mills, fertilizer retail, the upholstery industry and other industrial fields. In the present study, we describe the critical amino acids in the IgE-binding epitopes in Ric c 1 and Ric c 3, two major allergens of R. communis. In addition, we also investigate the cross-reactivity between castor bean and some air and food allergen extracts commonly used in allergy diagnosis. METHODOLOGY/PRINCIPAL FINDINGS: The IgE reactivity of human sera from atopic patients was screened by immune-dot blot against castor bean allergens. Allergenic activity was evaluated in vitro using a rat mast cell activation assay and by ELISA. Cross-reactivity was observed between castor bean allergens and extracts from shrimp, fish, gluten, wheat, soybean, peanut, corn, house dust, tobacco and airborne fungal allergens. We observed that treatment of rat and human sera (from atopic patients) with glutamic acid reduced the IgE-epitope interaction. CONCLUSIONS/SIGNIFICANCE: The identification of glutamic acid residues with critical roles in IgE-binding to Ric c 3 and Ric c 1 support the potential use of free amino acids in allergy treatment.

A new 2S albumin from Jatropha curcas L. seeds and assessment of its allergenic properties.

Significant effort has been made world-wide to boost biofuels with the expectation of a positive contribution to renewable fuel and greenhouse gas reduction. Jatropha curcas L. has proved to be an opportunistic crop in tropical areas, particularly in unfavorable environments. For this reason, analyses of toxicity and allergy caused by its seeds and pollen are necessary. A 12kDa, allergenic 2S albumin, denoted Jat c 1, was isolated from Physic nut (J. curcas) seeds. Jat c 1 binds IgE attached to rat mast cells, inducing histamine release. It also showed strong cross-reactivity with the major allergens from castor bean, Ric c 1 and Ric c 3.

Allergic memory of patients sensitized to castor bean after a long stimulation-free period.

We have taken advantage of the temporary exposure of Marseilles population to castor bean seed proteins to follow 26 allergic patients more than 20 years after sensitization. Skin tests, specific immunoglobulin E (IgE) antibody assays, and specific immunoblots were performed. Skin test reactivity to Ricinus Communis and specific IgE concentrations decreased progressively and almost completely disappeared after 20 years. Specific IgE concentration displayed a fairly exponential decrease, with a half-life of 4.7 years. Thus, in the absence of any antigenic stimulation, directly by castor bean, or indirectly by cross-reactivity to other Euphorbiaceae, especially latex, IgE sensitization is bound to disappear.

Ric c 1 and Ric c 3, the allergenic 2S albumin storage proteins of Ricinus communis: complete primary structures and phylogenetic relationships.

The 2S albumin storage protein of Ricinus communis consists of the two heterodimeric proteins Ric c 1 and Ric c 3 each of which is composed of a small and a large subunit linked together by disulphide bridges. The complete primary structures of both heterodimeric proteins were determined by enzymatic degradation and automated Edman degradation. The sequences of all four chains correspond to the known cDNA sequence of the gene of a presumed precursor molecule and to the previously determined partial sequences for Ric c 1 and Ric c 3. In addition, few differences in amino acid positions were found which seem to be related to different varieties of R. communis. Sequence comparisons with 2S albumin from other plant genera revealed high degrees of homology and support the view of a common genetic origin of this protein family. Ric c 1 and Ric c 3 which have 11,212 and 12,032 daltons, respectively, share a similar molecular size, biological function and allergenicity with the 2S albumins from Brassica juncea (Braj 1E) and Sinapis alba L (Sin a 1). Ric c 1 and Ric c 3 may be classified as isoallergens if, additionally, the high degree of similarity in the position of polar residues is taken into account.

Immunobiochemical characterization of Putranjiva roxburghii pollen extract and cross-reactivity with Ricinus communis.

Putranjiva roxburghii (PR) pollen has been found to be an important aeroallergen for type I hypersensitivity. In the present study, the IgE binding proteins of PR pollen have been characterized and compared with pollen allergens of Ricinus communis (RC) belonging to family Euphorbiaceae. On isoelectric focusing, PR pollen extract resolved into 35 bands (pI 3-9), whereas SDS-PAGE separated it into 18 protein components (MW 14-100 kD). Pooled patient's sera (ID +ve to PR) recognized 12 allergenic proteins in Putranjiva and five of them (MWs 92, 80, 55, 43 and 30 kD) showed immunologic reactivity to most of the sera samples tested individually by immunoblot. A number of shared allergenic proteins (MWs 92, 80, 66, 50, 43 and 14 kD) were observed between PR and RC pollen extracts on immunoblot using Putranjiva allergic serum pool. Inhibition in the binding for most of PR pollen allergenic proteins was obtained with higher concentration of RC extract than PR itself, depicting the presence of cross-reacting allergens in both. Putranjiva pollen extract was fractionated by a combination of DEAE Sephadex-A 50 and Sephadex-G 200 column chromatography. Periodate deglycosylation of western blotted PR extract and Put I fraction indicated the involvement of carbohydrate moieties in the allergenic activity. Of the two fractions from Put I (Ia and Ib), Put Ib was found to be the most allergenic protein by ELISA inhibition. Dot blot analysis with individual patients sera identified it as a major allergen of PR.

Specific IgE to castor bean (Ricinus communis) pollen in the sera of clinically sensitive patients to seeds.

Human sensitization to castor bean seeds in occupational workers and people living close to oil processing factories has been acknowledged for a long time. In view of the cross-reactivity among different plant parts of the same species, we studies crossreactivity between seeds of castor bean and its pollen at the molecular level. Sera from 26 seed-positive atopics, when analyzed for ELISA against seed and pollen extracts of castor bean, showed binding with both seed and pollen extracts, but binding was stronger with seed extracts as compared to pollen. ELISA inhibition revealed partial similarity as pollen extract could not achieve 90% inhibition even at 100 micrograms/ml, and remained the same after protein concentrations of 40 micrograms/ml. Antigenic extracts of seeds and pollen separated into 12 and 20 fractions on SDS-PAGE, respectively. The 26 sera studied for specific IgE binding to different fractions of seed and pollen extracts showed IgE binding in 17 and 16 cases respectively, but with weak binding to pollen fractions. The crossreactivity was confirmed with pooled sera by blot inhibition. Seed antigen completely inhibited the sera for specific IgE at 10 mg/ml protein while pollen antigen showed only partial inhibition. Crossreactivity and presence of common epitopes between seed and pollen extracts are confirmed.

Microglioma, a histiocytic neoplasm of the central nervous system.

Neuropathologists have long suspected the existence of a tumor derived from the microglia, which are the resident immunocompetent cells of the central nervous system. Previously, definitive characterization of this rare putative tumor was hampered by the lack of precise immunohistochemical reagents. We herein report on a patient with microglioma, and we define the immunohistochemical characteristics of the tumor. The patient was a 50-year-old white woman who presented with a 1-year history of progressive paresthesia, visual difficulties, and cranial nerve abnormalities. The patient died in June 1972. At autopsy, the brain weighed 1540 grams and was remarkable for a diffusely infiltrating periventricular tumor, which extended from the rostral tip of the lateral ventricles through the spinal cord. Microscopically, the tumor cells had extremely long, slender, twisted nuclei, and the cells diffusely infiltrated the brain parenchyma so that the extent of the tumor was difficult to determine. Formalin-fixed, paraffin-embedded tissue blocks from the neuropathology archives were studied. The neoplastic cells stained intensely with CD68 (KP1) and Ricinus communis agglutinin-120 markers for microglia and also with HAM-56, a marker for macrophages. The tumor cells stained negative for glial fibrillary acidic protein. The recent availability of precise immunohistochemical reagents has clearly defined this rare neoplasm and has facilitated reliable distinction from lymphoma and gliomatosis cerebri.

Factors related to the development of sensitization to green coffee and castor bean allergens among coffee workers.

BACKGROUND: Occupational allergic respiratory symptoms in coffee workers have been frequently reported, but the ultimate cause of sensitization is still debated, castor bean being considered besides green coffee beans. Atopy and cigarette smoking have been suggested as promoting factors of sensitization for several occupational allergens. OBJECTIVE: This study was carried out to assess the prevalence of allergic respiratory symptoms and of sensitization to both green coffee beans and castor bean in the whole workforce of a coffee manufacturing plant. Furthermore we wanted to ascertain both the presence of castor bean antigens in the settled dust of the green coffee beans warehouse and the possible crossreactivity between the two beans. Meanwhile, the effect of smoking and atopy was considered. METHOD: Two-hundred and eleven workers were examined. A questionnaire on oculorhinitis and asthma was administered and skin-prick tests for green coffee beans, castor bean and 15 common inhalant allergens were carried out. Isoelectric focusing, isoelectric focusing immunoblot and radioallergosorbent assay (RAST) inhibition were performed on samples of settled environmental dust from the green coffee area, as well as on castor bean and green coffee beans. RESULTS: Ten per cent of the workers complained of oculorhinitis alone and 16% of asthma (nearly always associated with oculorhinitis). The overall prevalence of skin-sensitization was: 15% for green coffee beans, 22% for castor bean, 22% for common allergens. Evidence of sensitization to occupational allergens was more common in smokers, with a more than twofold increase in relative risk. The strong association between skin positivity to common and occupational allergens suggests that atopy acts as an enhancing host factor towards occupational sensitization. The analysis of the dust confirmed the presence of castor bean antigens. CONCLUSION: Our findings indicate that castor bean is the major cause of occupational sensitization among coffee workers, whereas smoking and atopy act as enhancing factors.

Defining carbohydrate specificity of Ricinus communis agglutinin as Gal beta 1-->4GlcNAc (II) > Gal beta 1-->3GlcNAc (I) > Gal alpha 1-->3Gal (B) > Gal beta 1-->3GalNAc (T).

To define carbohydrate specificity of Ricinus communis agglutinin (RCA1), the combining site of RCA1 was further characterized by quantitative precipitin (QPA) and precipitin-inhibition assays (QPIA). Among the oligosaccharides tested for QPIA, Gal beta 1-->4GlcNAc (II, human blood group type II precursor sequence) was found to be 7.1 times more active than Gal beta 1-->3GalNAc (T, Thomsen-Friedenreich sequence) and about 1.7 times more active than the other three disaccharides tested--Gal beta 1-->4Man, Gal beta 1-->3DAra and Gal beta 1-->6GalNAc. Gal alpha 1-->4Gal, the receptor of the uropathogenic E. coli ligand was 3.6 times less active than the II sequence. These results indicate that the beta 1-->4 linkage of the terminal Gal to subterminal GlcNAc is important as this beta 1-->4GlcNAc sequence is at least 1.6 times more active than other types of disaccharides. Among the glycoproteins examined for QPA, native and desialized bovine submandibular glycoproteins, native and desialized human plasma alpha 1-acid glycoproteins, as well as crude hog stomach mucin and its three mild acid hydrolyzed products reacted well with the lectin. These glycoproteins precipitated over 75% of the lectin nitrogen added indicating that RCA1 has the ability to recognize Gal beta 1-->4/3GlcNAc and/or the related residues at the non-reducing ends and at positions in the interior of the chains. However, Tn (GalNAc alpha 1-->Ser/Thr sequence) rich glycoproteins such as desialized ovine submandibular glycoprotein and desialized armadillo salivary glycoprotein, in which over 90% of the carbohydrate side chains are Tn determinants with none or only a trace of I/II or T determinants, precipitated poorly with RCA1. From the present and previous results obtained, the carbohydrate specificity of RCA1 can be constructed and summarized in decreasing order by lectin determinants as follows: II (Gal beta 1-->4GlcNAc) > I (Gal beta 1-->3GlcNAc) > E (Gal alpha 1-->4Gal) and B (Gal alpha 1-->3Gal) > T (Gal beta 1-->3GalNAc), while Tn (GalNAc alpha 1-->Ser/Thr) is a poor inhibitor.

Factors relating to the development of respiratory symptoms in coffee process workers.

After several cases of occupational asthma had been reported in a coffee processing factory in England, 197 coffee workers representing 80% of the production workforce were studied to determine the factors affecting the development of work related respiratory symptoms of wheeze, cough, and dyspnoea. Two computer administered questionnaires concerning the presence of respiratory symptoms and the occurrence of work related respiratory symptoms were used. Workers underwent skin prick testing to green coffee bean extract (GCB) and 11 common inhalant allergen extracts and bronchial provocation testing with methacholine. The presence of specific immunoglobulin E (IgE) antibodies to GCB and castor bean extract (CAB) were determined by a radioallergosorbent test (RAST). The prevalence of work related respiratory symptoms was 12.7%, bronchial hyperresponsiveness 30%, atopy 54%, positive GCB skin prick test 14.7%, positive GCB RAST 14%, and positive CAB RAST 14.7%. None of the workers was sensitised to fungi present in the factory and the numbers of certain species of fungi, despite being greater than may be found out of doors or in an uncontaminated indoor environment, were fewer than are generally associated with the presence of work related respiratory symptoms among agricultural workers. Storage mites were not isolated. Green coffee bean extract and CAB RAST were significantly correlated using the McNemar test but there was limited allergenic cross reactivity in RAST inhibition studies of the two extracts. The only factors that were significantly and independently associated with work related symptoms were CAB RAST and duration of employment. Bronchial hyperresponsiveness was not independently associated with work related respiratory symptoms. The significant independent associations of bronchial hyperresponsiveness included GCB RAST, duration of employment, and resting forced expiratory volume in one second. Exposure to CAB, a highly potent antigen, may be overriding the effects of other factors such a GCB, atopy, bronchial hyperresponsiveness, and smoking. This study suggests that CAB contamination remains a potential problem in the coffee processing industry and all efforts to eliminate it from the working environment should continue.

Isolation and partial characterization of antigen 5.1 from the seeds of Ricinus communis, L. (Var. Amarelo de Irece).

Antigen 5.1 was isolated from the most acidic fraction of castor bean allergens (CB-1A) by gel filtration on Sephadex G-75 followed by polyacrylamide gel electrophoresis (PAGE) (yield: 6.2 mg antigen 5.1/g CB-1A). This antigen was homogeneous by the criteria of PAGE, isoelectric focusing, SDS-PAGE, immunoelectrophoresis and gel filtration. The antigen has an apparent molecular mass of 12 +/- 2 kDa and an isoelectric point of pH 5.1. Antigen 5.1 lacks proline, phenylalanine, threonine and tryptophan. It was immunochemically identical to one of the three immunoprecipitation lines presented by CB-1A by the Ouchterlony technique, and was positive when tested (250 micrograms) by IgE-mediated passive cutaneous anaphylaxis in LOU.M rats.

Role of CD8 T cells in rat IgE responses.

The role of the cytokines interleukin-4 and interferon-gamma in the regulation of IgE responses in the mouse and man have focused on the role of CD4 T cells. In the rat, antigen-specific CD8 T cells, generated following inhalation of antigen, have been shown to be capable of suppressing IgE responses. Repeated intraperitoneal injections of 1 ng ricin and 1 microgram antigen established a long-lived IgE response in both low- and high-IgE responder rat strains (Wistar and Brown Norway). The duration of the IgE antibody response was 204 and 248 days, respectively. Total IgE levels rose from 30 +/- 20 to 39,000 +/- 7,500 ng/ml in the Wistar rat and from 120 +/- 100 to 47,000 +/- 8,000 ng/ml in the Brown Norway rat. An even greater (10(4)-fold) increase was seen in antigen-specific IgE antibody levels. Ricin alone had no effect and concomitant or prior stimulation with antigen was required. The proportion of CD4+ and CD8+ cells present in the spleen at the peak of the IgE response was markedly increased compared with animals given ricin or antigen alone. Furthermore, CD8 T cells were approximately 100 times more sensitive to ricin than CD4 T cells. These data suggest that enhancement of IgE responses in ricin-treated animals results from the selective deletion of T cells which suppress IgE and are of the CD8 phenotype.

Isolation and partial characterization of antigen 5.1 from the seeds of Ricinus communis, L. (var. amarelo de irece)

Antigen 5.1 was isolated from the most acidic fraction of castor bean allergens (CB-1A) by gel filtration on Sephadex G-75 followed by polyacrylamide gel electrophoresis (PAGE) (yield: 6.2 mg antigen 5.1/g CB-1A). This antigen was homogeneous by the criteria of PAGE, isoelectric focusing, SDS-PAGE, immunoelectrophoresis and gel filtration. The antigen has an apparent molecular mass of 12 ñ 2 kDa and an isoelectric point of pH 5.1. Antigen 5.1 lacks proline, phenylalanine, threonine and tryptophan. It was immunochemically identical to one of the three immunoprecipitation lines presented by CB-1A by the Ouchterlony technique, and was positive when tested (250 *g) by IgE-mediated passive cutaneous anaphylaxis in LOU.M rats