A monoclonal antibody-based enzyme-linked immunosorbent assay of aflatoxin B1 in peanut products

An improved enzyme-linked immunosorbent assay (ELISA) combined with monoclonal antibody (MAb) and one-step extraction method was established for the estimation of aflatoxin B₁ (AFB₁) in a peanut product. AFB₁ was converted to AFB₁-oxime, and then conjugated with bovine serum albumin (BSA). Spleen cells from mice immunized with AFB₁-BSA conjugates were fused with myeloma cells. After double selection with AFB₁-ovalbumin (OVA) and carbodiimide-modified OVA, five stable hybridoma cells secreting anti-AFB₁ MAbs (AF1, AF 2, AF 3, AF 4, and AF5) were cloned. Using these anti-AFB₁ MAbs, we developed the indirect competitive ELISA (cELISA) with alkaline phosphatase (ALP) — labeled sheep anti — mouse IgG as marker and the direct cELISA with AFBi-oxime horseradish peroxidase (POD) as marker. The minimum detectable limits of the indirect cELISA with AF 1, 2, 3, 4, and 5 were 5, 5, 5, 5, and 50 pg of standard AFB₁ per assay, respectively, and those of the direct cELISA with AF 1, 3, 4, and 5 were 2.5, 5, 25, and 100 pg of standard AFB₁ per assay, respectively. The cross reactivity of each toxins with these MAbs in the indirect cELISA was as follows: (a) AF 1 and AF 2 were reactive with AFB₂ as well as AFB₁, weakly with AFG₂ > AFG₁ > aflatoxicol II (COL II) > aflatoxicol I (COL I) and less weakly with other aflatoxins; (b) AF 3 and AF 4 were reactive with COL II as well as AFB₁, weakly with COLI > AFQ₁ and less weakly with others; (c) AF 5 was AFQ₁ as well as AFB₁ weakly with COL II > AFG₂ > COL I and less weakly with others. The 60% aqueous methanol extracts of oil-roasted blanched peanuts (“butter peanut”), naturally contaminated with AFB₁, were assayed by the direct cELISA without further purification. The direct cELISA with the most sensitive MAb AF 1 was allowed to determine 1 ng of AFB₁ per g samples.     

An enzyme-linked immunosorbent assay (ELISA) for the detection of antisperm antibodies in horse serum

An enzyme-linked immunosorbent assay (ELISA) was developed and evaluated to detect equine antisperm antibodies (ASA) in horse serum. Six maiden mares between 12 and 18 mo of age were immunized with stallion sperm cells (SC group, N=2), seminal plasma (SP group, N=2), or phosphate-buffered saline (PBS) as a control (C group, N=2). Horses received a second injection of the same antigen 2 wk after the first. Blood was collected weekly for 10 wk after initial immunization and again at Week 15. Serum ASA levels (IgG and IgA) were measured by ELISA using two assay systems, one containing stallion SC as the plate antigen and another containing SP.

In horses immunized with SC, peak IgG levels were detected by ELISA during Wk 2 and 3 after first injection using either plate antigen. The antibody levels persisted through Week 5 and then slowly declined until Week 15. Horses immunized with SP had IgG levels that did not differ from control horses using either ELISA plate antigen. The only significant elevation in serum IgA ASA occured during Week 5 after initial immunization and only in mares immunized with SC as detected by ELISA using SC as the plate antigen. Attachment of ASA to stallion spermatozoa was confirmed by an indirect immunofluorescence assay.     

Detection in rabbit sera of blocking antibodies against staphylococcal fibronectin-binding protein by enzyme-linked immunosorbent assay

Abstract Antibody titres against fibronectin-binding protein (FnBP) of Staphylococcus aureus were determined in sera from rabbits immunized with staphylococcal whole cells or purified native fibronectin receptor. An ELISA technique for detection of antibody titres blocking the binding of soluble fibronectin to immobilized FnBP was developed. A recombinant staphylococcal FnBP fused to E. coli β-galactosidase (gal-FnBp) was used as the immobilized antigen in this test. Serum samples from two different rabbits immunized with native fibronectin receptor gave significant blocking titres, whereas the blocking titres of antisera against staphylococcal whole cells were about 4- to 5-fold lower. Using the gal-FnBP fusion protein, the sensitivity for detection of fibronectin by ELISA was also determined. The detection limit is around 5 ng. The findings are discussed with a view to developing an anti-staphylococcal adherence vaccine and quantitating fibronectin in solution.     

Quantitation of aflatoxin B1 and aflatoxin B1 antibody by an enzyme-linked immunosorbent microassay.

A specific microtest plate enzyme immunoassay has been developed for the rapid quantitation of aflatoxin B1 at levels as low as 25 pg per assay. Multiple-site injection of rabbits with an aflatoxin B1 carboxymethyloxime-bovine serum albumin conjugate was used for the production of hyperimmune sera. Dilutions of the purified antibody were air dried onto microplates previously treated with bovine serum albumin and glutaraldehyde and then incubated with an aflatoxin B1 carboxymethyloxime-horseradish peroxidase conjugate. The amount of enzyme bound to antibody was determined by monitoring the change in absorbance at 414 nm after the addition of a substrate solution consisting of hydrogen peroxide and 2,2'-azino-di-3-ethyl-benzthiazoline-6-sulfonate. Antibody titers determined in this manner closely correlated with those determined by radioimmunoassay. Competition assays as performed by incubation of different aflatoxin analogs with the peroxidase conjugate showed that aflatoxins B1 and B2 and aflatoxicol caused the most inhibition of conjugate binding to antibody. Aflatoxins G1 and G2 inhibited the conjugate binding to a lesser degree, whereas aflatoxins M1 and B2a had no effect of the assay.     

Development and evaluation of an enzyme-linked immunosorbent assay (ELISA) and dot immunobinding assay (DIBA) for the detection of Ganoderma infecting palms

The genus Ganoderma has a worldwide distribution causing root and stem rot of many plantation crops. A limiting factor in controlling the BSR disease is the lack of reliable diagnostic method(s) for early diagnosis. In this study, we developed polyclonal antiserum for Ganoderma mycelial and extracellular protein, and evaluated its efficacy with different plant samples collected from artificially inoculated coconut seedlings and Ganoderma infected field palms. We also tested the cross-reactivity with the soil-borne and saprophytic fungus collected from different parts of coconut palm. The antisera developed against the crude mycelial protein (CMP) and extracellular protein (ECP) showed a 1:1000 titre value for the detection of Ganoderma. The CMP antisera developed showed more cross-reaction when compared to ECP antisera of Ganoderma. In the DIBA test, at a 1:10 dilution of antigen, 1:1000 dilution of CMP and ECP antisera, 1:5000 dilution of secondary antibody gave clear distinctions in colour development between healthy and diseased samples. In the DIBA test, ECP antisera detected positive control (ECP of Ganoderma MTP and CRS-1 isolate), artificially inoculated roots, infected field roots, infected basal trunk and additionally lesions gave positive reactions which were not found in the CMP antisera tested. Therefore, both ELISA and DIBA tests may be useful for screening a large number of samples and help in the detection of infection at the earliest stage of disease development and this will certainly help to adopt suitable management strategies against Ganoderma disease in palm crops in advance.     

Quantification of human tissue transglutaminase by a luminescence sandwich enzyme-linked immunosorbent assay

Tissue transglutaminase (tTG) is a calcium-dependent enzyme that catalyzes crosslinking of peptidic glutamine residues with primary amines via isopeptide bonds and hydrolysis of ATP or GTP. The enzyme exerts a variety of functions at the cellular and tissue levels that may be disturbed in disease. Its role in pathoprocesses is poorly understood. For investigation of the involvement of tTG in disease, sensitive and specific assays should be available. We have developed the first sandwich enzyme-linked immunosorbent assay (ELISA) based on two monoclonal antibodies (mabs) against human tTG. tTG is captured by mab 3C10 and detected by biotinylated mab 10F3. After incubation with peroxidase-conjugated streptavidin, bound tTG is visualized by peroxidase reaction applying a luminescence substrate. The detection limit was 40 pg/ml. The assay was highly reproducible. Recovery of spiked tTG in crude samples was greater than 92%. The enzyme could be detected in cellular lysates and tissue homogenates of humans. The effect of typical effectors (retinoic acid and interferon-γ) on tTG expression could be demonstrated. A low signal was also obtained in mice samples, suggesting cross-reactivity of the mabs with murine tTG. The new sandwich ELISA may be successfully applied for investigation of physiological functions of tTG and of disorders associated with inadequate tTG expression.     

An enzyme-linked immunosorbent assay for the detection of cytoplasmic polyhedrosis virus

An enzyme-linked immunosorbent assay (ELISA) for the detection of Euxoa scandens cytoplasmic polyhedrosis virus (EsCPV) is described. Antisera to EsCPV, produced in rabbits and guinea pigs, are specific to EsCPVs when used in an indirect assay. This indirect assay approach permits the detection of homologous antigens at a concentration of about 1 μg/ml; however, this procedure is not suitable to test large numbers of unpurified specimens. For this type of analysis we used a double antibody sandwich assay which can detect 10 ng/ml of homologous antigen in unpurified material without nonspecific reactions. This assay is used to diagnose EsCPV infections in field and laboratory studies.     

A rapid detection of circulating antigens of Dirofilaria immitis in dogs by a dot enzyme-linked immunosorbent assay

Abstract This report describes a dot enzyme-linked immunosorbent assay (Dot-ELISA) for detecting circulating antigens in the sera of dogs infected with Dirofilaria immitis (D. immitis) . Circulating D. immitis antigens could be detected in 24 of 25 infected dogs. The remaining animal had two immature worms. However, non-infected dogs and dogs infected with other parasites were all negative, Few cross-reactions to different parasite antigens were observed. The advantages of the Dot-ELISA include; 1) there is no need for pretreatment and dilution of sera and samples could be immediately bound to nitrocellulose paper set into microfiltration apparatus, 2) this assay could be carried out within 2 h at room temperature, 3) the resulting enzyme-reaction could be measured by both visual observation and densitometric reading.     

An enzyme-linked immunosorbent assay for detection of pyrene and related polycyclic aromatic hydrocarbons

Polycyclic aromatic hydrocarbons (PAHs) can form DNA-binding compounds that show genotoxicity and carcinogenicity. Pyrene, as a PAH, was covalently linked to carrier protein bovine serum albumin and ovalbumin. A monoclonal antibody (McAb) was produced that showed high cross-reactivity values with chrysene (169.73%), benzo[a]pyrene (693.34%), benzo[a]anthracene (16.36%), and indeno[1,2,3-cd]pyrene (40.96%) and showed no significant cross-reactivity values with other homologues (<0.1%). A competitive enzyme-linked immunosorbent assay (ELISA) was developed for detection of pyrene and some homologues in water samples. The detection limit of the assay was 65.08 pg ml⁻¹. The average recoveries of PAHs from tap water, lake water, and mineral water were 99.13, 99.74, and 99.19%, respectively, indicating that matrices of water samples do not interfere with the assay. The results demonstrated that the developed ELISA seems to be a potential method for monitoring of pyrene and some homologous PAHs in water samples.     

Validation of a hypoxia-inducible factor-1 alpha specimen collection procedure and quantitative enzyme-linked immunosorbent assay in solid tumor tissues

Hypoxia-inducible factor-1 alpha (HIF-1α) is an important marker of hypoxia in human tumors and has been implicated in tumor progression. Drugs targeting HIF-1α are being developed, but the ability to measure drug-induced changes in HIF-1α is limited by the lability of the protein in normoxia. Our goal was to devise methods for specimen collection and processing that preserve HIF-1α in solid tumor tissues and to develop and validate a two-site chemiluminescent quantitative enzyme-linked immunosorbent assay (ELISA) for HIF-1α. We tested various strategies for HIF-1α stabilization in solid tumors, including nitrogen gas-purged lysis buffer, the addition of proteasome inhibitors or the prolyl hydroxylase inhibitor 2-hydroxyglutarate, and bead homogenization. Degassing and the addition of 2-hydroxyglutarate to the collection buffer significantly increased HIF-1α recovery, whereas bead homogenization in sealed tubes improved HIF-1α recovery and reduced sample variability. Validation of the ELISA demonstrated intra- and inter-assay variability of less than 15% and accuracy of 99.8 ± 8.3% as assessed by spike recovery. Inter-laboratory reproducibility was also demonstrated (R² = 0.999). Careful sample handling techniques allow us to quantitatively detect HIF-1α in samples as small as 2.5 μg of total protein extract, and this method is currently being applied to analyze tumor biopsy specimens in early-phase clinical trials.     

Capillary-based enzyme-linked immunosorbent assay for highly sensitive detection of thrombin-cleaved osteopontin in plasma

In this study, a highly sensitive capillary-based enzyme-linked immunosorbent assay (ELISA) has been developed for the analysis of picomolar levels of thrombin-cleaved osteopontin (trOPN), a potential biomarker for ischemic stroke, in human plasma. Using a square capillary coated with 8.5 μg/ml anti-human trOPN capture antibody for ELISA, the linear range obtained was 2 to 16 pM trOPN antigen. This concentration range was in the detection window of trOPN antigen in plasma samples. Compared with the conventional microplate-based ELISA, the current capillary technique significantly reduced the amounts of reagent from milliliter to microliter, reduced the analysis time from 8 to 3 h, and had a better sensitivity and detection limit performance from approximately 50 pM down to 2 pM of trOPN antigen. These results indicate that this capillary-based immunoassay is a potential tool for biomarker detection and may be useful in clinical trials and medical diagnostic applications.     

Preferential detection of pro-carboxypeptidase R by enzyme-linked immunosorbent assay

We generated two monoclonal antibodies (mAbs), 2A16 and 10G1, against pro-carboxypeptidase R (proCPR), also known as thrombin activatable fibrinolysis inhibitor (TAFI). By use of these mAbs, we developed a sandwich enzyme-linked immunosorbent assay (ELISA) system to detect proCPR. Since the amount of the antigen detectable by the ELISA was essentially the same in fresh plasma and serum incubated at 37 C for 1 hr, we concluded that the ELISA system detected not only proCPR, but also inactivated CPR generated from proCPR. However, an appreciable amount of proCPR remained unactivated in serum. For extensive activation of proCPR in plasma, thrombin and thrombomodulin complexes (TTM) can be used together with CaCl2. Following extensive conversion of proCPR to CPR by T-TM and CaCl2, converting plasma to serum (T-TM serum), antigenicity became undetectable by ELISA. Further analysis revealed that 2A16 reacts only with proCPR although 10G1 reacts with proCPR, active CPR and inactivated CPR. Therefore, we concluded that the ELISA system preferentially detects proCPR and not CPR. Our sandwich ELISA system utilizing 2A16 and 10G1 provides a suitable method for detecting proCPR and can be used to determine levels of proCPR in plasma samples from patients.     

Evaluation of an enzyme-linked immunosorbent assay for detection of Mycosphaerella pinodes in pea seeds

A polyclonal antiserum was raised against soluble mycelial extracts of Mycosphaerella pinodes aiming at pathogen detection in infected pea seeds by ELISA. When tested against the homologous antigen, it allowed the detection of 5 ng fungal soluble protein ml^-1 buffer, by double-antibody sandwich ELISA (DAS-ELISA). Positive reactions were obtained with isolates of M. pinodes of wide geographical origins but also with all tested isolates of Ascochyta pisi and Phoma medicaginis var. pinodella, two closely related pathogens forming with the target organism the Ascochyta complex. Out of the 11 other genera of pea seed-borne fungi tested, only two (Alternaria sp. and Stemphylium sp.) cross-reacted strongly by both antigen-coated plate (ACP-ELISA) and DAS-ELISA. Cross-absorption of the crude antiserum could not lead to a species-specific antiserum; however, a combination of P. medicaginis var. pinodella and Stemphylium sp. antigens resulted in an antiserum preferentially recognising A. pisi and M. pinodes. The cross-absorbed antiserum detected 50 and 500 ng of fungal protein ml^-1 buffer and healthy seed extracts respectively. DAS-ELISA proved suitable for the detection and quantification of M. pinodes in infected pea seeds tested singly.     

Toxoplasma gondii: Recombinant GRA5 antigen for detection of immunoglobulin G antibodies using enzyme-linked immunosorbent assay

In this study, for the first time, the evaluation of Toxoplasma gondii full-length recombinant GRA5 antigen for the serodiagnosis of human toxoplasmosis is shown. The recombinant GRA5 antigen as a fusion protein containing His-tag at both terminals was obtained using an Escherichia coli expression system. The usefulness of rGRA5 for the diagnosis of toxoplasmosis in an ELISA was tested on a total of 189 sera from patients with different stages of the infection and 31 sera from sero-negative individuals, obtained during routine diagnostic tests. Anti-GRA5 IgG antibodies were detected in 70.9% of all seropositive serum samples. This result was comparable to ELISA using a Toxoplasma lysate antigen (TLA) and six combinations of recombinant antigens. The sensitivity of IgG ELISA calculated from all positive serum samples was similar for TLA (94.2%), rMAG1 + rSAG1 + rGRA5 (92.6%), rGRA2 + rSAG1 + rGRA5 (93.1%) and rROP1 + rSAG1 + rGRA5 (94.2%) cocktails, whereas the sensitivity of cocktails without rGRA5 antigens was lower giving 82.0%, 86.2% and 87.8%, respectively. Thus, the present study showed that the full-length rGRA5 is suitable for use as a component of an antigen cocktail for the detection of anti-T. gondii IgG antibodies.     

Measurement of human granulocyte-macrophage colony-stimulating factor (GM-CSF) by enzyme-linked immunosorbent assay

Summary An IgG monoclonal antibody against recombinant human granulocyte-macrophage colony-stimulating factor (GM-CSF), designated HGMI, was produced by fusion of immune mouse splenocytes with HAT-sensitive murine myeloma cells. A sandwich enzyme-linked immunosorbent assay (ELISA) for measurement of human GM-CSF was developed using this HGMI and a polyclonal antibody against GM-CSF raised in a rabbit. GM-CSF in culture supernatants of phytohemagglutinin (PHA)- or concanavalin A (Con A)-stimulated peripheral blood mononuclear cells (PBMC) were measured by this ELISA system and the conventional CFU-GM colony formation method. The data indicated that the ELISA was highly efficient and sensitive for the detection of as little as 50 pg/ml recombinant GM-CSF. The CFU-GM colony assay may be influenced by other cytokines which can enhance or suppress colony formation, and ELISA for GM-CSF is more useful for kinetic studies of precise levels of production from PBMC.     

Serological response to chlamydial infection in sheep, studied by enzyme-linked immunosorbent assay and immunoblotting

Abstract Two enzyme-linked immunosorbent assays (ELISA) using highly purified elementary bodies (EB) of Chlamydia psittaci A/22 strain (ovine) or 6BC strain (psittacine) were set up for the detection of antichlamydial antibodies in sheep. No significant differences were observed between the two ELISAs, whereas these tests proved to be more sensitive than complement fixation test and showed a good correlation ( r = 0.75) with immunofluorescence assay. The periodate treatment of chlamydial antigens modified the results of serological responses studied by ELISA, depending on the sera. The average reduction of ELISA values by periodate was 28%, ranging between 5% and 65%. The immunoblot analysis of sheep sera showed high cross reactivity between the polypeptides of A/22 and 6BC strains. However, some differences were observed. The major outer membrane protein (MOMP) of 6BC strain was recognized at different molecular weight position (40 000 kDa) in comparison with the MOMP of A/22 strain (38 000 kDa). In addition, a clear band of 97 000 kDa was detected by all sheep sera tested with A/22 strain. This band was undetectable in the blots performed with 6BC strain.     

Detection of Colletotrichum falcatum causing red rot of sugarcane by enzyme-linked immunosorbent assay

Red rot disease of sugarcane caused by Colletotrichum falcatum Went is one of the most destructive diseases of sugarcane (Saccharum officinarum L.) worldwide. The pathogen spreads primarily through infected sugarcane setts and hence the use of disease-free setts is essential to prevent the disease. In order to develop immunological method for detection of C. falcatum, two proteins with molecular weights of 27 kDa and 45 kDa were purified from the mycelium of C. falcatum race Cf 05 and used as antigen source to raise polyclonal antibodies in NewZealand white rabbit. The developed polyclonal antibodies were tested for detection of C. falcatum by enzyme-linked immunosorbent assay (ELISA) and immunoblot analysis. The polyclonal antibodies specifically detected C. falcatum in extracts from infected plants, both in immunoblot and ELISA. The ELISA results showed that the developed polyclonal antibodies were highly specific to C.falcatum. The developed antibodies were very sensitive and could detect C.falcatum proteins even at a dilution of 1:50,000. Higher ELISA absorbance values were recorded even at an antigen dilution of 1:500. In western blot analysis, protein bands with molecular weights of 27 kDa and 45 kDa reacting to antisera raised against 27 kDa and 45 kDa mycelial proteins of C. falcatum, respectively, were detected in protein samples from red rot infected canes. The high specific reactivity and sensitivity of the antisera indicate its potential suitability for ELISA-based detection of C. falcatum.     

An enzyme-linked immunosorbent assay to measure insulin receptor dephosphorylation by PTP1B

Considerable effort exists within drug discovery to develop novel compounds to improve the underlying metabolic defects in type 2 diabetes. One approach is focused on inhibition of the tyrosine phosphatase, PTP1B, an important negative regulator of both insulin and leptin signaling. Historically, tyrosine phosphatase assays have used either small organic phosphates or, alternatively, phosphorylated peptides from the target proteins themselves. In characterizing inhibitors of PTP1B, measuring turnover of small organic phosphates is limited to evaluation of compounds that bind the active site itself. Peptide substrates allow identification of additional subsets of inhibitors (e.g., those that bind the second aryl-phosphate site), but assays of peptide turnover often involve detection steps that then limit full kinetic evaluation of inhibitors. Here we use a polyclonal antibody specific for the phosphorylated insulin receptor to allow much more sensitive detection of peptide phosphorylation. This kinetically robust enzyme-linked immunosorbent assay (ELISA) gives k(cat) and K(m) values for a phosphorylated insulin receptor peptide consistent with values determined by a continuous fluorescence-based assay. Furthermore, IC50 values determined for well-behaved active site inhibitors agree well with values determined for p-nitrophenyl phosphate cleavage. This assay permits full characterization of a larger subset of inhibitors as drug candidates for this promising target.     

Development of recombinant antibody-based enzyme-linked immunosorbent assay (ELISA) for the detection of skatole

The occurrence of boar taint and the European Commission recommendation to discontinue the surgical castration of pigs by the year 2018 creates an urgent need for new analytical methods that are simple, affordable, and suitable for field testing. We describe the generation and engineering of a skatole-specific antibody derived from a synthetic antibody library and the development of ELISA for its detection. The immunoassay is capable of detecting skatole with IC50 of 222 μg L⁻¹, which is within the analytical threshold level suggested for skatole, and with low cross-reactivity interference from other indolic compounds.