A Rapid Extraction Method Combined with a Monoclonal Antibody-Based Immunoassay for the Detection of Amatoxins.

Amatoxins (AMAs) are lethal toxins found in a variety of mushroom species. Detection methods are needed to determine the occurrence of AMAs in mushroom species suspected in mushroom poisonings. In this manuscript, we report the generation of novel monoclonal antibodies (mAbs, AMA9G3 and AMA9C12) and the development of a competitive, enzyme-linked immunosorbent assay (cELISA) that is sensitive at 1 ng mL-1 and shows selectivity for α-amanitin (α-AMA) and γ-amanitin (γ-AMA), and less for ß-amanitin (ß-AMA). In order to decrease the overall time needed for analysis, the extraction procedure for mushrooms was also simplified. A rapid (1 min) extraction procedure of AMAs using solvents as simple as water alone was successfully demonstrated using Amanita mushrooms. Together, the extraction method and the mAb-based ELISA represent a simple and rapid method that readily detects AMAs extracted from mushroom samples.

A New Conjugation Method Used for the Development of an Immunoassay for the Detection of Amanitin, a Deadly Mushroom Toxin.

One of the deadliest mushrooms is the death cap mushroom, Amanita phalloides. The most toxic constituent is α-amanitin, a bicyclic octapeptide, which damages the liver and kidneys. To develop a new tool for detecting this toxin, polyclonal antibodies were generated and characterized. Both α- and β-amanitin were coupled to carrier proteins through four different linking chemistries, one of which has never before been described. These conjugates were evaluated for their effectiveness in generating antibodies specific for the free toxin, as well as their utility in formatting heterogeneous assays with high sensitivity. Ultimately, these efforts yielded a newly described conjugation procedure utilizing periodate oxidation followed by reductive amination that successfully resulted in generating sensitive immunoassays (limit of detection (LOD), ~1.0 µg/L). The assays were characterized for their selectivity and were found to equally detect α-, β-, and γ-amanitin, and not cross-react with other toxins tested. Toxin detection in mushrooms was possible using a simple sample preparation method. This enzyme-linked immunosorbent assay (ELISA) is a simple and fast test, and readily detects amatoxins extracted from A. phalloides.

Feasibility of immunodiagnostic devices for the detection of ricin, amanitin, and T-2 toxin in food.

Qualitative and quantitative comparisons were conducted of commercially available immunodiagnostic devices for the detection of three select agents with oral LD50 values > or = 0.1 mg/kg of body weight. Ricin (oral LD50 > 1 mg/kg), amanitin (oral LD50 approximately 0.1 mg/kg), and T-2 toxin (oral LD50 > 1 mg/kg) were spiked into beverages, produce, dairy, and baked goods and assayed using commercially available enzyme-linked immunosorbent assays (ELISAs) and lateral flow devices. In all cases, the commercial diagnostic kits successfully detected all three select agents at concentrations below what might be a health concern. The considerable difference between the limit of detection of the immunodiagnostic devices employed (typically < or = 0.020 microg/g) and the amount of the select agent necessary to pose a health threat in a single serving of food facilitated the design of protocols for the high throughput screening of food samples. These protocols entailed simple extraction methods followed by sample dilution. Lateral flow devices and sandwich ELISAs for the detection of ricin had no significant background problems due to the food matrices. Competitive ELISAs, which typically have unacceptably high background reactions with food samples, successfully detected amanitin and T-2 toxin.

Diagnostic accuracy of urinary amanitin in suspected mushroom poisoning: a pilot study.

BACKGROUND: Amatoxin-containing species are responsible for the most severe cases of mushroom poisoning, with high mortality rate. Therefore, this poisoning should be ruled out in all patients presenting gastrointestinal symptoms after wild mushroom ingestion. OBJECTIVE: To determine sensitivity, specificity, positive predictive value, negative predictive value, and diagnostic efficacy (DE) of urinary amanitin analysis in cases of suspected mushroom poisoning. METHODS: All cases of mushroom ingestion referred to a Poison Center during a one-month period were analyzed. Amanitin measurements were performed by ELISA method (functional least detectable dose 1.5 ng/ml; cut-off value not clearly established). Gastrointestinal symptoms latency and initial clinical assessment were considered alternative diagnostic tools. Definitive diagnosis was used as the reference standard. RESULTS: Among 61 patients included in the study, amatoxin poisoning was diagnosed in 10 cases. Urine samples were collected 5.5 to 92 hours after mushroom ingestion. Urinary amanitin DE was 91.8%, 93.4%, and 80.3%, based on the cut-off value considered (1.5, 5.0, and 10.0 ng/ml, respectively). Symptoms latency longer than 6 hours and initial clinical assessment DE were 70.5% and 67.2%, respectively. To identify amatoxin poisoning, initial clinical assessment resulted more sensitive and urinary amanitin analysis more specific. CONCLUSIONS: Urinary amanitin analysis is a valuable diagnostic tool and may significantly contribute to the management of suspected mushroom poisoning. At present, the best diagnostic accuracy can be obtained taking advantage of both the high sensitivity and negative predictive value of the clinical assessment performed by an experienced toxicologist, and the high specificity and positive predictive value that characterize urinary amanitin analysis.

A beta-turn in alpha-amanitin is the most important structural feature for binding to RNA polymerase II and three monoclonal antibodies.

Four amatoxin-binding proteins with KD values in the nanomolar range, 3 monoclonal antibodies and RNA polymerase II, were studied with respect to their affinities to 24 alpha-amanitin derivatives with modified side chains. From KD values we estimated the amounts of binding energy that single side chains of the amatoxins contribute to complex formation. Ile6, previously identified by X-ray analysis to be part of a beta-turn (Kostansek EC, Lipscomb WN, Yocum RR, Thiessen WE, 1978, Biochemistry 17:3790-3795) proved to be of outstanding importance in all complexes. Replacement of the isoleucine with alanine reduced the affinity to all binding proteins to < 1%, suggesting a strong hydrophobic interaction. A strong effect was also seen when Gly5 was replaced with alanine, suggesting that the absence of a side chain in proximity to the beta-turn is likewise important. In addition to the beta-turn, each of the proteins showed at least 2 other points of strong contact formed by hydrogen bonds. Donors are the indole NH of 6'-hydroxy-Trp4 and OH of hydroxy-Pro2 and dihydroxy-Ile3. All the antibodies, but not RNA polymerase II, recognized the indole nucleus of 6'-hydroxy-Trp4. The geometric arrangement of the 4 strongest contact points suggests that the amatoxin binding site is different in each of the 4 proteins, except for the 2 antibodies raised in the same animal. Here, most of the contact points were identical but differed in strength of interaction. The method of structural analysis presented in this study is useful for identifying contact sites in complexes of proteins with peptides of rigid conformation. Furthermore, the method complements X-ray data by providing information on the amount of binding energy contributed by single structural elements.

Polyclonal amanitin-specific antibodies: production and cytoprotective properties in vitro.

The amanitins found in several mushroom species are responsible for many deaths every year. Based on its successful application to cardiac glycoside overdose, immunotherapy could be applicable to amanitin toxicity. Therefore, we produced polyclonal amanitin antibodies by immunizing rabbits with a novel conjugate of alpha-amanitin. Purified antibodies had an average association constant for alpha-amanitin of 1.3 x 10(9) M-1. A partially protective effect of the antibodies against amanitin toxicity in vitro in Chang cells was evident at a molar ratio of antibody binding sites to alpha-amanitin of 4:1. Together with reported studies in vivo, these investigations indicate the potential of immunotherapy for amanitin poisoning.

Identification of structural features involved in binding of alpha-amanitin to a monoclonal antibody.

Twenty-four derivatives of the cyclic octapeptide alpha-amanitin were assayed for their affinities to the monoclonal antibody beta A1/1. The derivatives were of natural, semisynthetic, and synthetic origin and had KD values ranging from 2 nM to > 70 microM. In the majority of the derivatives the chemical modifications had no detectable influence on the overall shape of the double-ring peptide. Given this condition, binding factors could be calculated from KD values of the amatoxin derivatives, which were valid for all amatoxins for estimating the contribution made by single structures to complex formation. The complex between alpha-amanitin and the immunoglobulin involves at least eight sites of contact. Four of them are responsible for strong interactions: (1) the OH group of hydroxyproline2 (binding factor 413), (2) the lipophilic side chain of isoleucine6 (binding factor 131), (3) the -CH2- moiety of the adjacent glycine5 or the absence of a side chain in this position (binding factor 361), and (4) the proton at the indole nitrogen of hydroxytryptophan4 (binding factor 140). The residual four interactions are hydrogen bonds of lower strength corresponding to binding factors of 1.5-8. The key role of the unique conformation of the amatoxins in determining their binding properties was shown by two amatoxin derivatives in which changes in the conformation were associated with virtually complete loss of affinity. For all amatoxin derivatives with conformations similar or identical to that of alpha-amanitin, we found empirical evidence that those structures of the peptide involved in binding make their contributions virtually independent of each other. It is a consequence of this rule that structural features that cooperate in binding could be characterized by the numerical product of their binding factors.

[The characteristics of phallotoxin-specific antibodies studied by immunization with a phallacidin-concanavalin A conjugate]. / Prouchvane i kharakteristika na falotoksinspetsifichni antitela posredstvom imunizatsiia s falatsidin-konkanavalin A koniugat.

High titre antiserum was obtained by immunization of rabbits with phalacidine-concanavaline A. Specific antiphalacidine antibodies were isolated from this serum on immobilized phalacidine. It was established by testing with antirabbit globulin sera that these antibodies were of IgG class only. IgG fraction was isolated from the immune serum by ion-exchange chromatography, but specific antibodies, which represented 8% of IgG of the serum, were isolated from it by affinity chromatography. The antibodies isolated by affinity, were tested against other phalatoxines and amotoxines. The results showed that they reacted with phalatoxines, but not with amatoxines and could be used in tests for quantitative determination of phalatoxines in mushroom extracts and body fluids.

Strongly enhanced toxicity of the mushroom toxin alpha-amanitin by an amatoxin-specific Fab or monoclonal antibody.

A monoclonal antibody, with high affinity against the mushroom toxin alpha-amanitin, was prepared. Administration of the Fab fragment of the monoclonal antibody to mice caused a 50-fold increase in alpha-amanitin toxicity. Electron micrographs showed normal appearance of hepatocytes but typical, amanitin-induced lesions in cells of the proximal convoluted tubules of the kidney. The pronounced nephrotoxicity is mainly explained by glomerular filtration and tubular reabsorption of the Fab-amatoxin complex and, to a lesser extent, of the immunoglobulin-amatoxin complex, which is still c. Twice as toxic as free alpha-amanitin. To our knowledge this is the first reported case where immunoglobulins or their fragments enhance rather than decrease the activity of a toxin. Accordingly, immunotherapy of Amanita mushroom poisoning in humans does not appear promising.

Purification of amatoxin-specific antibodies from rabbit sera by affinity chromatography, their characterization and use in toxicological studies.

Rabbits were immunized with fetuin-beta-amanitin. They produced amatoxin-specific immunoglobulins of various classes, predominantly IgG. The crude IgG fraction was isolated by gel filtration on Sephacryl S-300. By polyethylene glycol precipitation in the presence of tritiated amatoxin, as well as by a solid phase radioimmunoassay technique, the portion of amatoxin-specific antibodies in the IgG fraction was determined to be 5-13%. The affinity of the amatoxin-specific IgG for a tritiated amatoxin derivative was measured by equilibrium dialysis and calculated according to the method of Scatchard. The dissociation constant was 2.6 X 10(-9) M. The amatoxin-specific immunoglobulins were extracted by their affinity to alpha-amanitin-Sepharose 4B and eluted with excess alpha-amanitin. The complex was isolated in 95% yield with a ratio immunoglobulin:toxin of c. 2:1. Alternatively, the uncomplexed IgG could be eluted from the alpha-amanitin-Sepharose 4B with 5 M guanidine hydrochloride; this treatment, however, decreased the binding capacity for amatoxin by 30% (ratio 1.4:1). The purified amatoxin-specific IgG was assayed for its therapeutic efficiency in mice poisoned with alpha-amanitin, but was unable to neutralize the toxic effects of the mushroom toxin. On the contrary, equimolar amounts of the amatoxin-specific immunoglobulins enhanced the toxicity of alpha-amanitin in the mouse by a factor 2.

Production of antibodies to amanitins as the basis for their radioimmunoassay.

The production of antibodies against amanitins is described. By means of these antibodies, a radioimmunoassay was developed which allows detection of as little as 0.5 ng of amanitins in 1 ml of serum. By this method, the clearance of alpha-amanitin from the blood of poisoned mice was measured.