Antibody-based Enzyme-linked Immunosorbent Assay Research Articles

Detection of phialoconidia of Trichoderma harzianum in peat-bran by monoclonal antibody-based enzyme-linked immunosorbent assay

Murine monoclonal antibodies (MAbs) were raised against phialoconidia of a Trichoderma harzianum isolate T95. A hybridoma cell line, GH5, was raised that produced MAbs of the immunoglobulin class IgM that recognized, by enzyme-linked immunosorbent assay (ELISA), antigens from conidia of T95. They did not react with antigens from mycelium or chlamydospores of this isolate but recognized antigens from conidia of a number of other T. harzianum and T. viride isolates. Heat, protease and periodate treatment of T95 conidial antigens showed that GH5 MAbs recognized a protein epitope that formed part of a larger glycoprotein molecule. GH5 MAbs were used to develop an ELISA for the detection of phialoconidia of T. harzianum in peat-bran.

Determination of hydrolysed fumonisin B1 (HFB1) in corn by competitive direct enzyme-linked immunosorbent assay.

Fumonisin B1, a mycotoxin produced by certain Fusarium moulds, consists of two tricarballyic acid groups esterified to a 20-carbon backbone. Under alkaline conditions, or through metabolism, the aminopentol backbone, also known as hydrolysed Fumonisin B1 (HFB1) can be formed and is itself cytotoxic. Although the occurrence of HFB1 in corn-based foods is suspected, because of the ubiquitous nature of FB1 in corn, analytical methods for its detection are difficult. In the present report we describe a monoclonal antibody-based competitive direct enzyme-linked immunosorbent assay (CD-ELISA) for the rapid analysis of HFB1 in corn. The concentration required to inhibit enzyme conjugate binding by 50% (IC50) was 36 ng/ml. The limit of detection of HFB, by the CD-ELISA was 2ng/ml, when HFB1 was added in bovine serum albumin-phosphate buffered saline. The antibody also cross-reacted with the hydrolysis products of FB2, FB3, and FB4, having IC50 values of 331, 174, and 1700 ng/ml respectively. The antibody did not react with the intact fumonisins, sphingosine, sphinganine, or tricarballylic acid. Samples of corn spiked with HFB1 over the range of 5-1000 ng/g indicated the CD-ELISA has a limit of detection of 5 ng/g and an IC50 of 41 ng/g in the matrix. The CD-ELISA provides a sensitive and rapid tool for the analysis of corn-based foods for HFB1.

Development of an Enzyme-Linked Immunosorbent Assay for 3,5,6-Trichloro-2-pyridinol. 2. Assay Optimization and Application to Environmental Water Samples

A monoclonal antibody-based enzyme-linked immunosorbent assay (ELISA) for 3,5,6-trichloro-2-pyridinol (TCP), the major degradation product of chlorpyrifos, chlorpyrifos-methyl, and triclopyr pesticides, was optimized for the analysis of this hydrophilic compound in water. A direct ELISA format was chosen, and the concentration of immunoreagents was first selected to provide the highest sensitivity. Next, the influence of several physical (temperature, time) and chemical (pH, salt, detergent) conditions was studied. Under optimized conditions, the TCP concentration giving 50% reduction of the maximum ELISA signal (I 50 ) in the competitive standard curve was 0.62 nM (0.12 μg/L), and the assay was very specific for TCP. Preliminary evaluation of assay performance in water samples showed the absence of significant matrix effects for the waters tested, whenever the ionic strength of samples was approximately adjusted to that of standards. The assay provides a limit of detection of 0.04 μg/L and a working range of 0.09-0.91 μg/L TCP and allows for a precise and accurate determination of this pesticide degradation product in water at levels as low as 0.1 μg/L without sample cleanup.

Development of Enzyme-Linked Immunosorbent Assays for the Insecticide Chlorpyrifos. 2. Assay Optimization and Application to Environmental Waters

Monoclonal antibody-based enzyme-linked immunosorbent assays (ELISA) to chlorpyrifos were optimized for the analysis of this insecticide in environmental water samples. By optimizing the immunoreagent conditions and the composition of the competition buffer, assays with up to 1 order of magnitude better sensitivities were obtained. Reduction of the detergent (Tween 20) concentration provided most of the sensitivity improvement. Other factors studied, such as pH and ionic strength, behaved consistently with the nonpolar nature of the analyte and contributed in lesser extent to improve assay characteristics. Under optimized conditions, the chlorpyrifos concentration giving 50% reduction of maximum ELISA signal (I50) was in the 0.8−1.0 nM range, and the assays showed acceptable specificity for both chlorpyrifos and chlorpyrifos-methyl. Preliminary evaluation of assay performance in several water samples showed the absence of significant matrix effects for both formats evaluated. ELISAs provided a limit of de...

Enzyme-linked immunosorbent assay detection of pyrrolizidine alkaloids: immunogens based on quaternary pyrrolizidinium salts.

Polyclonal antibody-based enzyme-linked immunosorbent assays (ELISAs) were developed for the detection of retrorsine (1, 351 g/mol), monocrotaline (2, 325 g/mol), and retronecine (3, 155 g/mol) in the parts per billion (ppb) range. A set of three bifunctional linking arms (6-8) was synthesized. By N-alkylation of pyrrolizidine alkaloids (PAs) retrorsine, monocrotaline, and retronecine acetonide (9), six haptens (6.1, 6.2, 7.1, 7.2, 7.9, and 8.9) were synthesized and used to generate rabbit antisera. The resulting anti-retrorsine antiserum gave a 50% inhibition (I50) value of 0.9 +/- 0.2 ppb for retrorsine with detection limits of 0.5-10 ppb. The same ELISA system also detected isatidine (4, retrorsine N-oxide) dihydrate (403 g/mol) with an I50 of 1 ppb and senecionine (5,352 g/mol) with an I50 of 100 ppb. A second monocrotaline-based ELISA detected monocrotaline with an I50 of 36 +/- 9 ppb 2 with detection limits of 5-500 ppb and shows no cross-reactivity with 1 or 5; this ELISA demonstrates the potential for the substrate-specific detection method. A third retronecine-based ELISA detects 3 with an I50 of 3000 +/- 600 ppb (3 +/- 0.6 ppm) and detection limits of 600-10,000 ppb. None of these ELISAs cross-react with the structurally similar swainsonine (10) or lupinine (11) alkaloids. PAs were detected in extracts of Senecio vulgaris and Crotalaria retusa, but not in Lupinus spp., as a demonstration of the ELISA's usefulness.

Development of a Monoclonal Antibody-Based Immunoassay To Detect Furosemide in Cow's Milk

Furosemide is a potent diuretic drug used in both human and veterinary medicine. High-performance liquid chromatographic methods (HPLC) were developed to detect furosemide in blood and urine samples. Recently, immunoassay kits have appeared to measure furosemide; these were developed for the race horse industry where furosemide is used to treat epistaxis. In dairy cattle, furosemide is used for treatment of physiological parturient edema and there is a 48-h withdrawal period before milk from treated animals can be used. We report here the development of a monoclonal antibody-based enzyme-linked immunosorbent assay (ELISA) for detecting furosemide in milk. In addition, we report on the development of an HPLC method for detection of furosemide in milk that is an adaptation of published methods for its detection in serum and blood. Unlike the HPLC method, no sample preparation is necessary for the ELISA. Raw milk is added directly into the assay, or if needed, it is diluted with assay buffer. The immunoassay had a lower limit of quantification of 2 ppb and a lower limit of detection of approximately 0.5 ppb. Good correlations were observed between the HPLC and ELISA methods when samples with both incurred and spiked furosemide residues were analyzed. Keywords: Furosemide; immunoassay; residue detection; milk

Development of Improved Immunoassay and HPLC Methods for the Analysis of Chlorodiamino-s-triazine in Environmental Samples

An enzyme-linked immunosorbent assay (ELISA) for the analysis of chlorodiamino-s-triazine (CAAT) was developed using specific rabbit polyclonal antibodies. The assay was sensitive in the submicromolar range. The method was more sensitive and specific than previously reported mouse polyclonal antibody-based ELISAs. A competitive inhibition haptenated enzyme ELISA was optimized and validated which showed that the method was accurate, precise, and reproducible. A new HPLC method was developed to resolve polar s-triazine metabolites with improvements in run time and solvent use. The ELISA method was more sensitive than the HPLC method for detecting CAAT in soil and ground water samples. Solid-phase extraction was evaluated in an attempt to further increase the sensitivity of the ELISA method. However, nonspecific interferences were observed from various types of strong cation exchange columns from different manufacturers. This made it difficult to couple a solid-phase extraction technique with the ELISA. Keyw...

Anti-Thomsen-Friedenreich (T) antibody-based ELISA and its application to human breast carcinoma detection

An anti-Thomsen-Friedenreich (T) antibody-based enzyme-linked immunosorbent assay (ELISA) with high efficacy in human breast carcinoma detection is described. Immunoreactive T epitopes occur in ∼ 90% of all carcinomata; all humans have anti-T antibodies, naturally occurring anti-carcinoma antibodies, induced by their own intestinal flora. Carcinoma patients, but not control subjects, show alterations of serum anti-T hemagglutinin levels. Human anti-T antibodies are predominantly IgM. In the protocol presented here, anti-T IgM antibodies are quantitated by ELISA using Immulon 2 wells coated with human blood group O erythrocyte-derived T antigen as solid phase; in addition, total IgM in each serum is quantitated by ELISA in parallel with the anti-T IgM. Inter-assay coefficient of variation was 2% for both ELISAs. Although anti-T IgM values alone distinguish between carcinoma patients and control subjects, use of the quotient, Q Me, which also considers total IgM, increases this distinction. For a given serum, Q Me = (100 × (anti-T IgM) 2/total IgM). Sera of 242 subjects, 117 breast carcinoma patients, 36 benign breast disease patients and 89 healthy persons were analyzed. Q Me identified 88% of the breast carcinoma patients: it was positive in all six (100%) in situ, 11 13 (85%) Stage I, 48 58 (83%) Stage II and III and 38 40 (95%) Stage IV patients. Sera from 83% of the 36 benign breast disease patients were negative, i.e. within normal range; five of the six positive sera originated from patients with increased long-term risk of breast carcinoma, while sera from 11 other patients with increased carcinoma risk were negative. Overall, 90% of the 125 non-carcinoma control subjects were negative by both anti-T IgM and Q Me. In preliminary studies, the ELISA protocol detected 11 14 (79%) patients with carcinomata other than those of the breast. The identification of all six in situ breast carcinoma patients by Q Me points to its usefulness in carcinoma detection, especially early.

Hypersecretion of mucin in response to inflammatory mediators by guinea pig tracheal epithelial cells in vitro is blocked by inhibition of nitric oxide synthase.

Primary cultures of guinea pig tracheal epithelial cells in air/liquid interface were exposed to one of four agents associated with airway inflammation: the peptide histamine (100 microM), the lipid mediator platelet-activating factor (1 microM), the cytokine tumor necrosis factor-alpha (15 ng/ml; specific activity 2.86 x 10(7) U/mg), or enzymatically generated reactive oxygen species (purine [500 microM]+xanthine oxidase [20 mU/ml]). Effects of each of these substances on release of mucin by guinea pig tracheal epithelial (GPTE) cells were measured using a monoclonal antibody-based enzyme-linked immunosorbent assay (ELISA). Each secretagogue significantly enhanced release of mucin, but the stimulatory effect of each was inhibited by pre-(+)co-incubation of the cells with the competitive inhibitor of nitric oxide synthase, NG-monomethyl-L-arginine (L-NMA), but not by NG-monomethyl-D-arginine (D-NMA), the inactive stereoisomer that does not inhibit nitric oxide synthase. Neither L-NMA nor D-NMA affected mucin secretion by themselves. The results suggest that each of these inflammation-associated mediators provokes airway epithelial mucin secretion via a mechanism involving intracellular production of nitric oxide (NO) as a critical signaling molecule.

Application of a monoclonal antibody-based ELISA to the determination of carbaryl in apple and grape juices

Based on a high affinity monoclonal antibody, an enzyme-linked immunosorbent assay (ELISA) was developed for the quantification of carbaryl (1-naphthyl N-methylcarbamate). For standards, the detection limit of the ELISA was 0.065 ng ml −1, with a I 50 value of 0.706 ng ml −1. The ELISA was applied to the determination of carbaryl in apple and grape juices. The study of the influence of these matrices on the reliability of the assay indicated that the ELISA could determine carbaryl in fruit juices at the low ng ml −1 level simply by diluting the sample, without any cleanup or concentration step. Recovery and precision of the method were evaluated by spiking apple and grape juices with carbaryl in the 5–50 ng ml −1 range. No false negative or positive results were obtained. Coefficients of variation were lower than 13.0% in all cases, and mean recoveries were 100.0 and 92.2% for apple and grape juices, respectively. In addition of being precise and accurate, the method has proved to be simple and sensitive, with a quantification limit well below the maximum residue limits for carbaryl in apple and grape.

Development and Validation of a Monoclonal Antibody-Based Enzyme-Linked Immunosorbent Assay for Salinomycin in Chicken Liver Tissue

Salinomycin is one of the most widely used coccidiostats in U.S. agriculture. A rapid and accurate analytical method for this drug should provide producers and users with an effective management tool. The current chromatographic methods are sensitive but are labor-intensive. In addition, they require large amounts of expensive organic solvents for extraction and cleanup, which requires proper (and expensive) disposal. This paper reports the development of an enzyme-linked immunosorbent assay (ELISA) coupled to a simple aqueous extraction procedure for the analysis of salinomycin in chicken liver tissue. Recovery from spiked liver homogenates was quantitative in the range from 5.0 to 0.05 ppm. Analysis of chicken livers containing incurred residue by ELISA and high-performance liquid chromatography (HPLC) showed the results to be highly correlated (p < 0.0001). The ELISA method described here has a limit of quantitation of 50 ppb, which is more sensitive than the HPLC method.

Monitoring allergen exposure in asthma: new treatment strategies.

The development of monoclonal antibody-based enzyme-linked immunosorbent assay technology for measuring environmental allergen exposure has provided a benchmark for assessing the role of indoor allergens in causing asthma and other allergic diseases. Epidemiological studies from several parts of the world have shown that immunoglobulin E (IgE)-mediated sensitization to indoor allergens (mite, cat, dog and cockroach) is a risk factor for asthma attacks. A dose-response relationship between allergen exposure and sensitization has been demonstrated for mite allergens, and threshold values for exposure levels leading to sensitization or to exacerbations of symptoms have been defined. Comparative studies on airborne allergen levels have made it possible to determine the properties of aeroallergen particles, their concentration in indoor air, and the relationship to clinical symptoms. Together, these studies provide strong evidence that allergen exposure plays a causal role in the development of bronchial hyperreactivity and of the chronic inflammatory responses seen in patients with asthma. Logically, the primary preventive treatment should be allergen avoidance. Through knowledge of indoor allergen levels, both in dust and in the air, different avoidance strategies have been applied to the various indoor allergens, and there is increasing evidence of their clinical efficacy. Monitoring allergen levels in patients' houses should improve their understanding of the role of allergens in asthma and improve compliance with avoidance measures.

Evaluation of a Monoclonal Antibody-Based Enzyme-Linked Immunosorbent Assay for the Diagnosis of Opisthorchis viverrini Infection in an Endemic Area

A monoclonal antibody-based enzyme-linked immunosorbent assay (MAb-ELISA) was evaluated for its potential in the diagnosis of opisthorchiasis in an area endemic for Opisthorchis viverrini infection. The method, based on the detection of the 89-kD O. viverrini metabolic antigen in the feces (coproantigen), was previously estimated to be sensitive enough to detect antigen excreted by a single mature fluke. In the present study, fecal specimens from 207 apparently healthy villagers in northeastern Thailand were analyzed in a double-blind test for the presence of O. viverrini eggs by microscopic examination and for antigen by MAb-ELISA. The microscopic examination was carefully done to minimize false-positive results due to eggs of Lecithodendriid trematodes. The specimens were divided into six groups based on the number of eggs per gram of feces, namely, egg negative, 1-500, 501-1,500, 1,501-3,000, 3,001-6,000, and more than 6,000. The results showed that the ELISA is sufficiently sensitive and specific for the diagnosis of O. viverrini infection. The slightly higher rate of coproantigen positive by the ELISA compared with microscopic examination may reflect lower specificity of the ELISA or its higher sensitivity over microscopic examination in detecting light infections. Different lines of evidence presented here support the latter explanation.

Determination of 8-hydroxydeoxyguanosine by an immunoaffinity chromatography-monoclonal antibody-based ELISA

The postulated importance of oxidative damage to DNA in aging and age-related degenerative pathologies such as cancer has prompted efforts to develop sensitive quantitation methods. 8-Hydroxy-2′-deoxyguanosine (8-OHdG) is a widely used marker for oxidative damage to DNA. To develop an immunoassay for quantitation of 8-OHdG, two monoclonal antibodies have been developed and characterized by competitive enzyme-linked immunosorbent assay (ELISA). Antibody 1F7 has 50% inhibition at 5 pmol 8-OHdG and 1 × 105 pmol dG, while antibody IF11 has 50% inhibition at 2.5 pmol 8-OHdG and 2000 pmol dG. Both antisera crossreact with guanosine and several structurally related derivatives, including 6-and 8-mercaptoguanosine, 8-bromoguanosine, 8-methylguanine, and 7-methylguanosine. Immunoaffinity columns were prepared with antibody 1F7, which exhibits higher selectivity than 1F11, to isolate 8-OHdG from DNA hydrolyzates followed by ELISA quantitation with antibody 1F11. This method allows the analysis of approximately one 8-OHdG/105 dG using 100μg DNA. To validate the assay, DNA extracted from human placental tissues were assayed by both ELISA and HPLC with electrochemical detection. Values by both methods correlated well ( r = 0.87, p < 0.001), but the levels determined by ELISA were approximately sixfold higher than those determined by HPLC. This may be due to oligonucleotides detected by the ELISA but not the HPLC method or crossreactivity with other damaged bases present in the immunoaffinity purified material. Placental samples from current smokers had significantly higher 8-OHdG by ELISA than those from nonsmokers ( p < 0.05). The method of immunoaffinity purification combined with ELISA quantitation has sufficient sensitivity for detecting 8-OHdG in human DNA samples. Although absolute values are higher than those determined by HPLC, the method provides a good alternative to the HPLC-EC method for monitoring relative oxidative damage in molecular epidemiological studies.

Comparative Detection of Fumonisin by HPLC, ELISA, and Immunocytochemical Localization in Fusarium Cultures

Fumonisins are a group of mycotoxins that are elaborated by Fusarium moniliforme and Fusarium proliferatum and that have recently been associated with animal and human disease. In this study, the time course of fumonisin B1 (FB1) production in corn was monitored in five Fusarium cultures using high-performance liquid chromatography (HPLC), enzyme-linked immunosorbent assay (ELISA), and in situ localization by an enzyme-linked immunocytochemical technique (ELICT). Using HPLC on culture extracts prepared with 50% (vol/vol) acetonitrile in water, FB1 was detectable at 3 days with maximal FB1 (ranging from 230 to 3,000 ppm) occurring between 14 and 28 days. Although there was a positive correlation between FB1 detected by HPLC and ELISA, the latter consistently yielded higher results than HPLC. Maximal FB1 “equivalents” detected by ELISA ranged from 12,000 to 35,000 ppm. Following fixation of Fusarium from cultures, ELICT revealed the presence of large deposits indicative of fumonisin or fumonisin-like cross-reacting compounds in mycelia, microconidia, and microconidia. Prior to fixation, these compounds were extractable in 50% (vol/vol) acetonitrile in water. ELICT results qualitatively correlated with HPLC and ELISA over the time course of the cultures. Taken together, the results suggest that (a) ELISA or ELICT could be used for qualitative screening of FB1-producing cultures, and (b) in addition to FB1, the monoclonal antibody-based ELISA detected one or more compounds that structurally resemble FB1 and occur concurrently with FB1.

Humoral immune response against human cytomegalovirus (HCMV)-specific proteins after HCMV infection in lung transplantation as detected with recombinant and naturally occurring proteins

The humoral immune response to four intracellularly located cytomegalovirus (CMV) proteins was studied in 15 lung transplant recipients experiencing active CMV infections. Five patients had primary infections, and 10 had secondary infections. Antibodies of the immunoglobulin M (IgM) and IgG classes were measured in an enzyme-linked immunosorbent assay (ELISA) system in which procaryotically expressed recombinant proteins were used as a substrate and also in a monoclonal antibody-based capture ELISA which uses naturally occurring proteins as a substrate. The proteins investigated were the lower matrix protein pp65 (ppUL83), the major DNA-binding protein p52 (ppUL44), and the two immediate early proteins IE1 and IE2 (different splicing products of UL123). Higher levels of antibodies were found to pp65 and especially to p52 than to the immediate early antigens. Antibody levels detected in the recombinant protein-based ELISAs were generally lower than antibody responses detected with the matching antigen capture ELISA. Moreover, some patients appeared to have antibodies mainly to epitopes present on naturally occurring proteins. The antibody responses detected in both assays were related to the viral load during infection as assessed by the CMV antigenemia test, which is a quantitative marker for CMV load. It was found that although epitopes on naturally occurring proteins induce higher antibody responses and responses in more patients, antibodies directed to epitopes present on the recombinant proteins were inversely related to the viral load during a CMV infection. Therefore, antibodies to epitopes on the recombinant proteins might be more clinically relevant in this group of lung transplant recipients.

Development and application of a monoclonal antibody against Thiothrix spp

Historically, methods used to identify Thiothrix spp. in environmental samples have been inadequate because isolation and identification procedures are time-consuming and often fail to separate Thiothrix spp. from other filamentous microorganisms. We described a monoclonal antibody-based enzyme-linked immunosorbent assay (ELISA) procedure which was used to identify Thiothrix spp. in wastewater, artesian springs, groundwater, and underwater subterranean samples. The ELISA utilized monoclonal antibody T3511 to a species-specific carbohydrate epitope of Thiothrix spp. No cross-reactions were observed among non-Thiothrix strains consisting of 12 species and nine genera. In field trials, the ELISA identified 100% of 20 biochemically and cytologically confirmed Thiothrix spp.-containing samples with no false positives. Indirect immunofluorescent microscopy utilizing T3511 was effective for wastewater samples but not for those from natural spring water because of background fluorescence in the latter. In addition, electron micrographs of Thiothrix spp. labeled with T3511-biotin-anti-mouse antibody-gold showed that epitope T3511 was intracellular both in laboratory strains and environmental isolates. The minimum level of detection of the ELISA was 0.10 microgram/ml.

Estimation of Der p and Der f I quantities in the reference preparations of Dermatophagoides mite extracts

A monoclonal antibody-based enzyme-linked immunosorbent assay (MoAb-ELISA) was developed to measure the major Dermatophagoides mite allergens, Der p I and Der f I. The assay was highly species-specific and sensitive. Using this assay system, the absolute mass unit of Der p I and Der f I in the reference preparations of the extracts was estimated. The primary standards used were the purified Der p I and Der f I preparations. The reference preparations of the D. pteronyssinus and D. farinae extracts (92-Dp and 92-Df), which had been prepared from the same amount of mite bodies of both species, were found to contain the same levels of the Der I allergens, 10.1 micrograms/ml of Der p I and 10.0 micrograms/ml of Der f I, respectively. A histamine release assay with leucocytes from mite-allergic donors showed that the total allergenic potency of 92-Dp and 92-Df was comparable. This results indicates that the estimated Der I levels in these extracts seem to be valid, at least, in the balance between the two species, although further comparisons of the absolute quantities by several different laboratories are needed. The Der I levels in the WHO/IUIS international reference preparation of D. pteronyssinus and the CBER standard mite extracts, E4-Dp and E5-Df, were also estimated using this assay system. They were found to contain 4.4 micrograms/vial and 13.3 micrograms/ml of Der p I and 9.5 micrograms/ml of Der f I, respectively.

Effects of spice oil treatment of rice on moulding and mycotoxin contamination

Moulding and mycotoxin production have been studied during storage of rice at 85 and 90% relative humidity (r.h.) following treatment with cinnamon and clove oils. The clove oil-treated rice was artificially inoculated with Aspergillus flavus. Aflatoxin B 1 and ochratoxin A were determined by monoclonal antibody-based enzyme-linked immunosorbent assay. Both moulding and mycotoxin were inhibited by 9 μl cinnamon oil g −1 but, with smaller doses, fungi could be isolated during the early part of storage, but not subsequently. Thus, following treatment with 3 or 6 μl cinnamon oil g −1, fungi were isolated from direct plated grains after 30 days but not after 45 days. Fungi were also isolated from washings of grain up to 30 days after treatment with 3 or 6 μl g −1, although fewer than from untreated grain. Propagules could also be detected up to 15 days after treatment with 9 μl g −1. Clove oil was much less effective than cinnamon oil in preventing moulding. After treatment with 8 μl g −1, the proportion of seeds infected with fungi and numbers of propagules in washings decreased only after 60 days' storage and with 4 μ g −1 after 90 days at both relative humidities. However, aflatoxin contamination could not be detected 90, 60 and 60 days after treatment with, respectively, 2, 4 and 8 μl g −1 at 85% r.h., although ochratoxin A was present in all treatments. At 90% r.h. aflatoxin was absent only after 90 days from treatment with 4 and 8 μl g −1. No T2 toxin was detected.

Development of a monoclonal antibody-based enzyme-linked immunosorbent assay for the detection of live propagules of Trichoderma harzianum in a peat-bran medium

Mouse monoclonal antibodies (MAbs) and mouse and rabbit (polyclonal) antisera were raised against a Trichoderma harzianum isolate T95. A cell line, Th.HD3 3, raised from splenocytes from a mouse immunized with lyophilized mycelium and Quil A adjuvant, produced MAbs of the immunoglobulin sub-class IgG 2a that were specific recognizing, by ELISA, antigens from a number of Trichoderma species and species from the related genus Hypocrea but not from a range of other soil-borne fungi. These MAbs were used to develop an enzyme-linked immunosorbent assay (ELISA) for the detection of live propagules of Trichoderma harzianum in a peat-bran medium. Heat, protease and periodate treatment of the T95 antigen and binding on Western blots indicate that the Th.HD3 3 MAbs recognized a protein epitope of ca M r 18,500 which forms part of a larger glycoprotein molecule.