A rapid ELISA for screening aflatoxin B1 in animal feed and feed ingredients in Indonesia

To facilitate a sustainable aflatoxin management system in Indonesia, a simple, rapid and effective Enzyme Linked Immunosorbent Assay (ELISA) test for screening aflatoxin B₁ (AFB₁) in animal feed and feed ingredients was developed. Anti-AFB₁ polyclonal antibodies were produced against AFB₁-BSA and AFB₁-KLH immunogens. Using AFB₁ conjugated to horseradish peroxidase (HRP) as an enzyme marker in a direct competitive ELISA, an IC₅₀ of 0.85±0.15 µg/kg and a detection limit (IC₁₅) of 0.18±0.06 µg/kg of AFB₁ were achieved. The assay was highly specific to AFB₁ with very little cross reaction with other aflatoxin congeners (AFB₂ 0.9%, AFG₁ 3.1% and AFG₂ 1.2%) and metabolites. The ELISA was tolerant to methanol (up to 60%) and pH (pH 7.2-9.6) without significantly affecting the overall performance and was not affected by interferences from the animal feed and corn samples. Satisfactory recovery results were obtained from the spike and recovery study (77-97% recovery for 10-252 µg/kg). A pilot survey conducted on corn and animal feed samples collected from the local feed factories and poultry shops indicated that significant amounts of corn and animal feeds were contaminated by aflatoxin B₁ greater than the MRL (50 µg/kg).     

Monoclonal antibodies for fumonisins B1, B2 and B3

There has been only one report of fumonisin antibodies produced from a fumonisin-bovine serum albumin (BSA) immunogen in the past. Other, more exoteric, carriers, such as cholera toxin and keyhole limpet hemocyanin, have also been used. The resulting antibodies have a wide range of sensitivity and selectivity for the most common fumonisins B₁, B₂ and B₃. We report on fumonisin monoclonal antibodies produced in typical fashion from an immunogen consisting of FB₁ conjugated to BSA through its terminal amino group. These antibodies have excellent cross-reactivity with the three main fumonisins: FB₁, 100%, FB₂, 94%, and FB₃, 72%, adequate sensitivity with an IC₅₀ of 430 ng/mL for FB₁, and show excellent correlation between ELISA and total fumonisins as determined by HPLC, with a slope of 0.985 and an r² of 0.987.     

Penicillin-specific antibodies: Monoclonals versus polyclonals in ELISA and in an optical biosensor

Two penicillin-specific monoclonal antibodies mAb 19C9 and mAb 9H3 and the penicillin-specific polyclonal antibodies pAb K2 were evaluated for their use in a competitive ELISA and in the BIAcore™ optical biosensor. In the ELISA, an ampicillin-protein conjugate was used as a coating molecule. For the biosensor assay, ampicillin was immobilized on a CM5 chip. With both monoclonal antibodies and in both test systems, ampicillin, amoxicillin and benzylpenicillin were better recognized than oxacillin, cloxacillin and dicloxacillin. Because the reproducibility was better in the biosensor (CV = 1.6%) than in the ELISA (CV = 8.9%), the limit of detection for ampicillin in buffer solution using mAb 19C9 was lower in the biosensor (46 ng ml^-1) as compared to the ELISA (356 ng ml^-1). Ampicillin can thus be detected below the MRL (50 ng ml^-1) in the biosensor assay but not in the ELISA. Both the ELISA and biosensor assay using the polyclonal antibodies pAb K2 were more sensitive as compared to the assays with the monoclonals. The ELISA using pAb K2 allowed the detection of all tested penicillins below the MRL. In the biosensor assay, ampicillin was also detected below the MRL (IC50 = 10 ng ml^-1). In contrast to the binding of the monoclonals, no spontaneous dissociation was observed after injection of the polyclonal antibodies in the biosensor. Whereas the monoclonals were completely removed from the sensor surface using ampicillin in the buffer solution as regeneration solution, stronger conditions were necessary for the pAb binding.     

Immunochemical-based zilpaterol measurement and validation in urine and tissues

Because of abuse potential of the feed-additive zilpaterol, a need exists for rapid, sensitive and specific analyses. Polyclonal and monoclonal antibody-based enzyme-linked immunosorbent assays (ELISAs) were developed and their usefulness for agricultural applications explored. Immunobiosensor formats were developed for both monoclonal and polyclonal zilpaterol antibodies. Zilpaterol ELISA and immunobiosensor were tested by measuring tissue and urinary concentrations from sheep treated with zilpaterol for 10 days. The study demonstrated that sheep eliminated zilpaterol rapidly. A zilpaterol study in horses demonstrated that urinary zilpaterol in horses was initially much higher than in other species and that urinary zilpaterol depleted in a biphasic manner. Zilpaterol was detectable using either ELISA or ultra-high performance liquid chromatography-triple quadrupole-tandem mass spectrometry (UHPLC-TQ-MS/MS) after 21 days of withdrawal in horses. These studies demonstrated that the ELISA procedure was rapid and was in good agreement with instrumental methods while the biosensor method provided greater precision than the ELISA procedure.     

Monoclonal Antibodies for the Mycotoxins Deoxynivalenol and 3-Acetyl-Deoxynivalenol

The mycotoxin deoxynivalenol (DON) is produced by the mold Fusarium graminearum and is found worldwide on cereal grains, in particular wheat and maize. Each year this compound, also known as 'vomitoxin' causes substantial losses to agricultural productivity. Three monoclonal antibodies were developed following the immunization of mice with a conjugate of DON and ovalbumin. One of these antibodies, produced by clone #22, was selected for the development of a competitive direct ELISA (CD-ELISA). This format consists of competition between a DON horseradish peroxidase conjugate (DON-HRP) and free DON for antibody attached to microwell plates. Color development in the assay was inhibited 50% (IC₅₀) by 18 ng DON/ml in phosphate-buffered saline (PBS). The antibody from this clone showed strong cross-reactivity to 3-acetyl deoxynivalenol (3-Ac-DON), with an IC₅₀ of 2.9 ng ml^-1 Cross-reactivity to 19 other trichothecene mycotoxins was low. The CD-ELISA was applied to wheat spiked with DON over the range 0.01-10 μg/g and extracted with a 10-fold excess of PBS. The midpoint for color development in the assay using this extraction was 0.27 μg DON/g wheat. Recoveries over the range 0.05-5 μg/g averaged 88.7% with a coefficient of variation of 10.9%. This assay is sufficiently sensitive and rapid to permit the screening of DON in wheat below the US Food and Drug Administration advisory level of 1 ppm in human food.     

Anti-mesotrione single chain antibody fragments derived using either phage display or via hybridoma technology: selection, characterization and stabilization

Single-chain antibody fragments (scAb), specific for the triketone herbicide mesotrione, have been expressed and purified from the bacterium Escherichia coli. ScAbs were identified following the cloning of variable heavy and light chains from anti-mesotrione monoclonal antibodies (mAbs) and from panning an immunized phage display library. Both the monoclonal and phage derived scAbs were produced from the same immunized mouse spleen. In competition ELISA, the mAb derived scAbs (35 and 36) showed similar specificity for mesotrione compared with the parental mAbs. MAb 35 was the most sensitive with a limit of detection (IC₂₀ value) within EC legislative limits for drinking water at 0.04 μg l^-1. The best phage scAb (H6) had sensitivities approximately 20× less than scAb 35. In competition ELISA, scAb 35 showed reduced sensitivity to sulcotrione, a structurally related triketone, with IC₅₀ values in the μM range. The introduction of an interdomain disulphide bond into scAb 35 resulted in greater stability in non-physiological environments. Immunoassay may provide a simple, cheap and rapid test for detection of mesotrione in a range of environments including soil and food.     

A rapid method for detection of PrP by surface plasmon resonance (SPR)

Surface plasmon resonance was used to develop a rapid, label-free and sensitive immunoassay for detection of Prion protein (PrP). Anti-PrP monoclonal antibodies immobilized on the biosensor surface were allowed to bind various concentrations of cellular prion protein (PrP^C), followed by a pulse with additional soluble anti-PrP polyclonal antibodies to intensify the signal. The interaction of antibody with antigen was monitored in real time. With this method, it was possible to detect PrP^C with a limit of 31.7 ng/ml in serum and 13.1 ng/ml in HEPES-saline, requiring 1 h for a single sample measurement. The assay also detected misfolded prion protein (PrP^Sc) in spiked serum and PrP^Sc in scrapie-infected mouse brains. This is a rapid and sensitive assay for the detection of PrP in serum that could be developed into a platform for a serum-based TSE test.     

Production of polyclonal and monoclonal antibodies against group A, B, and C capsular polysaccharides of Neisseria meningitidis and preparation of latex reagents.

Polyclonal and monoclonal antibodies against capsular polysaccharides of Neisseria meningitidis serogroups A, B, and C were produced in order to develop immunological reagents allowing both the detection of soluble antigens during meningococcal meningitis and antigenic serogrouping of N. meningitidis cultures. The performance characteristics of monoclonal and polyclonal antibody latex reagents were compared. For the detection of soluble polysaccharide antigen, polyclonal antibody latex reagent was selected for N. meningitidis A and C. The latex reagent prepared with polyclonal antibodies against N. meningitidis B could not detect capsular polysaccharide even at 1 mg/ml. The monoclonal antibody B latex reagent which detected 100 ng of polysaccharide per ml was therefore chosen. For the serogroup identification of N. meningitidis, the use of a confirmatory test results in an overall specificity of 100% with polyclonal or monoclonal antibody latex reagents.     

A convenient enzyme-linked immunosorbent assay for testing whether monoclonal antibodies recognize the same antigenic site. Application to hybridomas specific for the beta 2-subunit of Escherichia coli tryptophan synthase

Seven hybridoma clones, producing antibodies directed against the β₂-subunit of Escherichia coli tryptophan synthase, have been obtained from mouse cells. To test whether the corresponding monoclonal antibodies recognize different epitopes on β₂, an ELISA double antibody binding system has been developed and is reported here. The antigen is first coated onto a microtitration plate. Two monoclonal antibodies are then added either separately or simultaneously, and the amount of bound antibody is quantitatively measured by use of immunoglobulin (rabbit anti-mouse IgG) linked to β-galactosidase. Additivity of the bound enzymatic activity is observed when the monoclonal antibodies bind to distinct epitopes.

Using this test, it is shown that, of the 7 anti-β₂ monoclonal antibodies obtained, 5 recognize the same antigenic site and the 2 others recognize a second site.     

Solid-phase competitive and sandwich-type erythro-immunoassays for human chorionic gonadotropin

A simple ‘1-step’ competitive erythro-immunoassay for human chorionic gonadotropin (hCG) employing V-shaped well microtitration plates coated with monoclonal anti-β-hCG antibody has been described. hCG of the test sample competes with the antigen-coupled sheep erythrocytes for binding to the antibody on the solid surface. The assay is able to detect up to 31.25 ng hCG/ml. A higher sensitivity enabling detection up to 0.25 ng hCG/ml is attained by the sandwich erythro-immunoassay using a chimera antibody prepared by coupling monoclonal anti--hCG antibody to an affinity-purified polyclonal antibody specific for sheep erythrocytes. This assay is amenable to the qualitative as well as quantitative use as described. The urinary components do not interfere in the assay. Results obtained by this assay on 47 human urine samples correlated well with the values obtained by ‘2-step’ sandwich enzyme immunoassay and radioimmunoassay.     

Real-time biospecific interaction analysis of antibody reactivity to peptides from the envelope glycoprotein, gp160, of HIV-1.

A new assay designed to quantitate antibody reactivity to specific peptides using biospecific interaction analysis (BIAcore) has been developed. Peptides of various lengths (15-40 amino acids) and isoelectric points (pI = 4.5-13) were covalently linked (immobilized) to a biosensor and interacted with polyclonal human sera. The immobilization procedure was highly reproducible, with bound peptides retaining high antibody reactivity. The assay was rapid, requiring only 25-30 min to immobilize the peptide and 2-8 min for each subsequent peptide/serum binding interaction. The same peptide surface has been used for up to 90 cycles of serum binding and regeneration with only a 0.3% decay in reactivity over cycle number. The quantitative BIAcore signal, measuring peptide/antibody binding interactions, was directly related to the antigen/antibody concentrations within the biosensor. The assay allowed interactants to be studied in their native form and without the need of additional secondary detection antibodies. Correlation between conventional peptide ELISA and BIAcore was obtained. The BIAcore linear range persisted over a series of eight two-fold dilutions. This extended linear dynamic response range is an improvement over conventional ELISA measurements. The sensitivity for monoclonal antibody detection is similar to conventional ELISAs and 4.9 ng/ml was readily detected.     

Immunogenicity of immunoliposomes

Multilamellar immunoliposomes were prepared from dipalmitoylphosphatidylcholine (DPPC), cholesterol (CH), sphingomyelin (SPH) and biotinylated dipalmitoylphosphatidylethanolamine (PEB) in the molar ratio of 1:1:1:0.1 with surface linked avidin-biotinylated sheep (anti-mouse IgG) IgG (AV-sIgG_B) or GK1.5 monoclonal rat (anti-mouse L3T4 antigen) IgG (AV-GK1.5_B). The ability of these immunoliposomes to induce antibody responses against AV, sIgG or GK1.5 was determined. GK1.5_B and sIgG_B elicited a low-level antibody response (5–10 μg/ml serum) after i.v. immunization and boosting. Liposomes (1 μmol) containing GK1.5_B or sIgG_B were more effective than free GK1.5_B or sIgG_B in eliciting antibodies (20–30 and 100–120 μg/ml serum, respectively). Liposomal AV mixed with either sIgG or GK1.5 gave antibody levels comparable to immunization with free GK1.5_B or sIgG_B. Liposomes with surface AV-sIgG_B or AV-GK1.5_B elicited antibodies against AV and high levels against GK1.5 or sIgG. Immunoliposomes possessing surface AV-sIgG_B or AV-GK1.5_B were eliminated from the circulation of normal mice relatively slowly (T_1/2 15.5 and 30 min): in contrast, liposomal AV-sIgG_B or AV-GK1.5_B was rapidly eliminated from the circulation of immunized mice (T_1/2 4.5 and 4.0 min). These results demonstrate that liposomes with surface IgG (immunoliposomes) are immunogenic, and that repeated administration elicits anti-IgG antibodies that result in a significant reduction in blood circulation residence times.     

Development of polyclonal ELISA for the N-methylcarbamate insecticide bendiocarb

A polyclonal antibody-based enzyme-linked immunosorbent assay (ELISA) method was developed for the N-methylcarbamate insecticide bendiocarb (2,2-dimethyl-1,3-benzodioxol-4-yl methylcarbamate). Two novel haptens having dimethylbenzodioxyl and dimethylbenzofuranyl groups connected to oxyacetyl-γ-aminobutanoic acid and oxyacetyl-β-alanine spacer arm respectively were synthesised. The first hapten was conjugated to carrier proteins to make antigens that were used to raise polyclonal antibodies in rabbits. The antibodies specifically recognised bendiocarb and its metabolite 2,2-dimethyl-1,3-benzodiox-4-ol with an IC₅₀ value of 9 ppb (ng ml^-1). The assay was standardised using the competitive ELISA format at 0.0625 µg antibody concentration and at 1/10k pesticide-HRP dilution. Matrix effect studies were carried out in four vegetable and cereal food samples. Matrix effect elimination in cabbage, cauliflower and rice was achieved by simple dilution of the extract. Five different approaches were attempted to achieve matrix clean up in paddy rice. C-18 column and gel permeation column chromatography (GPC) helped in the matrix removal. The spike and recovery studies for all the four food samples gave a recovery in the range of 75-95%, thus indicating the efficiency of the matrix elimination procedures developed.     

Development of enrofloxacin ELISA using a monoclonal antibody tolerating an organic solvent with broad cross-reactivity to other newquinolones

Antibacterial reagents, especially quinolones, are widely used in animals and humans, and have caused serious problems to human health because of their residual contaminants in food. In order to screen for different kinds of newquinolones at the same time, a sensitive and specific enzyme-linked immunoassay (ELISA) has been developed. The anti-enrofloxacin monoclonal antibody was selected because of its ability to react with structurally related newquinolones in organic solvent. The antibody has 100% cross-reactivity with norfloxacin, ciprofloxacin and other newquinolones at 50% inhibition of control values IC₅₀, but not with nitroflazone, sulphadimethoxine. The lowest detection limit of this ELISA was 0.7 ng/ml (ppb) when enrofloxacin was used as the calibrator. Eel extracts were spiked with enrofloxacin and the average recoveries at 10, 50, 100 ng/ml were 98, 102 and 91%, respectively. The proposed ELISA is a useful method for the practical microquantitation of various newquinolones in biological and environmental specimens.     

Production and characterization of monoclonal antibodies to staphylococcal enterotoxins: Use in immunodetection and immunopurification

Four cell lines producing monoclonal antibodies were obtained by fusion of NSI myeloma cells with splenocytes of BALB/C mice immunized with only 1 μg of each staphylococcal enterotoxin A, B, C₁ and D by a modified technique of intrasplenic boosting. This procedure was considerably more efficient than the more commonly used intravenous boosting. The antibodies EC-A₁, EC-B₁, EC-C₁ and EC-D₁, all of the IgG₁ subclass, have high affinities for the corresponding enterotoxins A, B, C₁ and D, with dissociation constants of 1.4, 2.8, 1.4 and 1.5nM respectively; in addition EC-B₁ showed a high affinity (2.1 nM) for enterotoxin C₁. All these antibodies recognize, by immunoblotting, the homologous purified enterotoxins as well as enterotoxins from the bacterial culture supernatants. A rapid indirect double sandwich ELISA using a pair of antibody preparations was developed, where monospecific monoclonal antibodies were used to coat plastic plates and polyspecific rabbit antibodies were used to detect the enterotoxins under field conditions. These antibodies which are capable of immunoadsorbing the enterotoxins from staphylococcal culture filtrates and from natural fluids such as milk, were used to immunopurify enterotoxins A, C, and D. The homogeneity and integrity of the affinity purified toxins A, C₁ and D was verified by direct automated Edman degradation and yielded single amino terminal sequences which were moderately homologous to those published previously for B and C₁ enterotoxins     

A time-resolved immunofluorometric assay for quantification of the bovine collectin conglutinin

A high capacity time-resolved immunofluorometric assay (TRIFMA) for the bovine collectin conglutinin was developed. The TRIFMA was constructed as a non-competitive sandwich assay based on polyclonal antibodies as the capture reagent and a novel monoclonal antibody raised against conglutinin as the detection reagent and was set up to run on an automatic analyzer designed for the TRIFMA detection system. Polyclonal antibodies immobilized on microtiter plate wells were incubated overnight at 4 degrees C with diluted plasma samples, including quality controls (QC) and dilutions of a plasma with known conglutinin concentration. Conglutinin was sandwiched between the capture antibodies and the monoclonal antibody and the detection optimised with biotin-labelled secondary antibodies and streptavidin-Eu(3+). Plates were washed four times between each step and finally incubated with enhancement solution before measuring the fluorescence. The assay detection limit was 0.34 ng/ml and the working range 0.80 ng/ml-0.20 microg/ml. Intra-plate and inter-plate coefficients of variation (CV) were in the range of 5.0-8.3% and 6.2-7.2%, respectively, at concentrations of 3.4 and 150 ng/ml. Recovery was 90.9+/-2.4% and 98.8+/-2.5% when samples were spiked with 20 ng/ml and 100 ng/ml purified bovine conglutinin (BK). No circadian rhythm (24-h variation) in conglutinin plasma levels was observed across animals, indicating that the plasma levels were not influenced by, e.g. feeding. Samples could be stored at -20 degrees Celsius and were not sensitive to repeated freezing and thawing. In conclusion, the developed TRIFMA for bovine conglutinin is specific and reliable over a measurement range covering most situations.     

一种新型冠状病毒假病毒的制备及初步应用

目的 制备一种新型冠状病毒(SARS-CoV-2)假病毒,并将其应用于抗体中和能力检测和抗体广谱性评估.方法 整合2种近期出现的SARS-CoV-2变异病毒株(20A.EU1和B1.1.7)刺突(spike,S)蛋白的突变序列、以及对胞浆区肽段19个氨基酸进行局部缺失突变,构建突变型S蛋白表达质粒,转染293T细胞进行...     

Development of concanavalin A-enzyme immunosorbent assay for glycated haptoglobin using polyclonal and monoclonal antibodies.

Two-site lectin-haptoglobin-enzyme immunosorbent assay (L-Hp-ELISA), is described. Haptoglobin binding to Concanavalin A, immobilized to polystyrene microtiter plate, was estimated by anti-haptoglobin polyclonal and monoclonal antibodies conjugated with horse-radish peroxidase. The range of haptoglobin binding to Concanavalin A, measured by the L-Hp-ELISA was 25 to 300 ng/ml using polyclonal, and 50 to 600 ng/ml using monoclonal anti-haptoglobin antibodies, respectively.     

Plasma androstenedione and oestrone levels in the climacteric syndrome

Plasma androstenedione (A) and oestrone (E₁) levels were measured by radioimmunoassay in a group of 78 healthy women who had undergone a natural menopause. Of this total, 23 were symptomless (Group 1), 39 presented with a moderate climacteric syndrome (Group 2) and 16 had a severe climacteric syndrome (Group 3). The average body weight was found to be significantly higher in Groups 2 (P < 0.01) and 3 (P < 0.05), than in Group 1, but the age distribution and number of years since the menopause were similar in all three groups. Nevertheless, significantly lower levels of A (0.75± 0.06 ng/ml, P < 0.01, in Group 2; 0.24 ± 0.05 ng/ml, P < 0.001, in Group 3) and E₁ (20.80 ± 2.18 pg/ml, P < 0.05, in Group 2; 12.22 ± 1.65 pg/ml, P < 0.001, in Group 3) were observed in the women with climacteric symptoms than in those with no symptoms (A = 1.08 ± 0.08 ng/ml, E₁ = 27.73 ± 2.22 pg/ml in Group 1).

Since, after the menopause, the concentrations of A and E₁ in the plasma represent the most important source of oestrogens, these results suggest that climacteric symptoms are related to oestrogen deficiency which is secondary to low A production.