Gene-coding assignments of rotavirus double-stranded RNA segments 10 and 11

The gene-coding assignments for genome segments 10 and 11 of a simian virus and two human rotaviruses were determined. For those viruses having a “long” RNA gel pattern (electropherotype), segments 10 and 11 encoded proteins NS₃ and O₄, respectively. The human virus with a “short” electropherotype had the opposite assignments and also differed in (enzyme-linked immunosorbent assay) serotype from the human virus with a long electropherotype.     

Development of an enzyme-linked immunosorbent assay of thromboxane B2 using a monoclonal antibody

An inhibition enzyme-linked immunosorbent assay (ELISA) was developed using a monoclonal antibody against thromboxane B₂ (TXB₂). As a specific antigen, the bovine serum albumin conjugate of TXB₂ was adsorbed onto polystyrene microtiter plates. The sensitivity of the monoclonal antibody was compared by means of three different enzyme conjugates, all commercially available. The detection limit with immunoglobulin conjugates of alkaline phosphatase and horseradish peroxidase was 0.04 ng of TXB₂ per sample. The use of horseradish peroxidase coupled with an avidin-biotin complex allowed a tenfold increase in sensitivity to 0.0045 ng of TXB₂ per sample. The suitability of the assay was checked with TXB₂-containing human serum and urine samples, which yielded unchanged standard curves. Recovery experiments had an accuracy of r=0.960 and r=0.987. Validity was confirmed by a good correlation between radioimmunoassay and ELISA (r=0.949). Results of an inhibition experiment with platelet-rich plasma in the presence and absence of ibuprofen demonstrated the practical applicability of this method.     

Detection of Legionella pneumophila serogroup 1 urinary antigen using an enhanced chemiluminescence ELISA

An enhanced chemiluminescence enzyme-linked immunosorbent assay has been developed for the detection of soluble antigen in the urine of patients with Legionnaires' disease (LD). In the assay antigen(s) in the urine samples are captured by a rabbit anti-L. pneumophila antibody coated onto microtitre strips. A fluorescein-isothiocyanate (FITC) conjugate of the same antibody is then added which binds to the captured antigen. Any immobilized FITC-labelled antibody is then detected with a horseradish peroxidase (HRP) conjugate of a monoclonal anti-FITC antibody. HRP activity is monitored after oxidation of luminol in the presence of H₂O₂ and iodophenol. The resulting luminescence is recorded using a camera luminometer. Urine specimens were available for testing from 31 patients with evidence of ongoing L. pneumophila serogroup 1 infection. A positive result was obtained in the cases of 12/12 specimens from culture-proven LD patients, and 16/19 specimens from patients with serological evidence of LD. Thus the sensitivity is estimated to be 28/31 (90%) The specificity was estimated using urine specimens from eight patients with non-L. pneumophila pneumonias of known aetiology. All eight specimens gave a negative result.     

Interaction of triiodothyronine-biotin conjugate with binding proteins in immunoassay systems

The study presents a new design of a test system for the detection of a thyroid hormone, free 3,3??,5-triiodo-L-thyronine (free T₃), by enzyme-linked immunosorbent assay (ELISA) and immunoradiometric assay (IRMA) in human blood serum. For this purpose, a low-molecular-weight bifunctional conjugate of T₃ with biotin (Bt) was synthesized. The conjugate T₃-Bt can be bound to biotin-binding proteins on a solid support via its biotin residue, and T₃ portion of the conjugate can interact with a monoclonal antibody against T₃ (anti-T₃-MAb) that is labeled with horseradish peroxidase or iodine-125 in ELISA and IRMA, respectively. The immunochemical interaction is hindered if the T₃ residue is involved in a preformed complex of T₃-Bt with avidin in a solid phase. Computer simulations revealed that the iodothyronine residue is shielded by a glycan component of avidin in such a complex. Binding of T₃-Bt to both T₃-free sites of anti-T₃-MAb and to avidin on a solid support was achieved by delayed introduction of T₃-Bt into avidin-coated plates that were preliminarily co-incubated with labeled anti-T₃-MAb and the analyzed T₃ sample.     

Enhanced chemiluminescence ELISA for Listeria specific antigen in cerebrospinal fluid using an FITC-anti-FITC system

An enzyme-linked immunosorbent assay (ELISA) is described for the detection of a soluble Listeria monocytogenes serogroup 4 antigen in cerebrospinal fluid samples (CSFs). In the ELISA an anti-Listeria monoclonal antibody, immobilized onto assay wells, was used to capture antigen from CSFs. the captured antigen was then reacted with a fluorescein isothiocyanate (FITC) conjugate of the same anti-Listeria antibody, which was detected with a horseradish peroxidase conjugate of a monoclonal antibody to FITC. The presence of antigen was detected by an enhanced chemiluminescence assay using a camera luminometer. Antigen was detected in the CSFs taken from five out of seven patients with culture proven L. monocytogenes serogroup 4 central nervous system infections, and in none of the CSFs taken from 25 other patients.     

An enzyme-linked immunosorbent assay for 6-keto PGF

An enzyme-linked immunosorbent assay for 6-keto prostaglandin F_1α, a stable metabolite of prostacyclin, has been developed. The assay allows quantitation of 6-keto PGF_1α in the range 1–200 pg/0.1 ml and shows very low cross reactivity to nine other prostaglandins. Dose dependent stimulation by thrombin of 6-keto PGF_1α formation in human endothelial cells in culture has been used to verify the assay. Quantitation by the enzyme linked immunosorbent assay agrees closely with determination by radioimmunoassay.     

Direct and indirect competitive monoclonal antibody-based ELISA of Aflatoxin B1 in groundnut

Direct and indirect competitive enzyme-linked immunosorbent assays were optimized for the determination of aflatoxin B₁ in groundnut utilizing a specific monoclonal antibody developed at the University of Strathclyde, UK. The monoclonal antibody was conjugated to horseradish peroxidase (HRP) for direct competitive assay, while a commercially available goat-antimouse IgG-HRP conjugate was employed for indirect competitive ELISA. Both ELISAs detected aflatoxin B₁ as low as 20 pg/well. Methanol-water-KCl (70 + 30 v/v, 0.5 %) extracts of groundnut were assayed by ELISA after diluting 1: 10 with PBS-Tween buffer or subjected to simple cleanup for 5:1 concentration prior to assay. The mean recoveries from groundnut spiked with 10 to 200/ig/kg of pure aflatoxin B₁ were >90% in either ELISA, but the toxin recoveries at concentrations of 1–5μg/kg were only 65–67 % when subjected to cleanup and concentration before assay. The mean within-assay, inter-assay, and sub-sample coefficients of variation by ELISA of aflatoxin B₁ in naturally contaminated groundnuts were, respectively, 8.9%, 11.1%, and 7.9% for direct competitive assay and 4.6%, 11.2%, and 8% for indirect competitive assay. Both ELISA methods are useful for routine analysis of aflatoxin B₁ in groundnuts.     

Enhanced chemiluminescent sandwich enzyme immunoassay for hen egg lysozyme

A sensitive chemiluminescent sandwich-type enzyme immunoassay for hen egg lysozyme was developed. The assay was performed on polystyrene microtitre plates using immobilized specific polyclonal rabbit antibody against lysozyme, a peroxidase conjugate and the H₂O₂/luminol-enhanced chemiluminescence detection reagent. The chemiluminescent signal was detected using either a microplate luminometer, or photographic film in a camera luminometer. The detection limit for lysozyme was 0.3 ng/mL, and this was three times lower than that obtained using a colorimetric method with H₂O₂ and o-phenylendiamine as substrates. Recovery of the assay was 97–112% and the relative standard deviation ranged from 3.6% to 10.3%. The immunoassay overcame interference from the food sample matrix when lysozyme, used as a bacteriostatic agent, was measured.     

Immunoenzyme analysis of human IgG in the kinetic mode

A kinetic sandwich enzyme-linked immunosorbent assay for the detection of human IgG (used as a model antigen) has been developed. Rabbit antihuman IgG has been used both for coating polystyrene microtitration plates and for the preparation of the conjugate of anti-human IgG with horse-radish peroxidase. The kinetics of the reaction of the antigen and the antibody-peroxidase conjugate with the reagents immobilized on polystyrene plates has been studied. The assay is optimized with respect to its sensitivity and the duration of intervals for every stage of the assay. The optimal time of the assay is about 10-15 minutes. The correlation between sensitivity and the duration of every stage of the assay has been established.     

Two simple programs for the analysis of data from enzyme-linked immunosorbent (ELISA) assays on a programmable desk-top calculator

We have designed two programs for use with an inexpensive programmable calculator which rapidly and accurately convert raw data generated from enzyme-linked immunosorbent assays directly into antigen concentration. The first program computes and compares effective doses (ED₅₀)'s between a standard and each unknown sample assayed. The ED₅₀ from the unknown sample is then multiplied by a concentration factor which yields the unknown concentration. The second program linearizes the sigmoidal enzyme-linked immunosorbent assay titration curve using a logit-log transformation of the data in order to compute unknown concentration values. Both programs employ stringent limit conditions to decrease “nonsense” calculations. Data are then processed by a least-squares best-fit linear regression analysis.     

An enzyme-linked immunosorbent assay (ELISA) for measuring vitellogenin in English sole (Pleuronectes vetulus): development, validation and cross-reactivity with other pleuronectids

Vitellogenin (Vtg) is a yolk protein produced in the liver of oviparous animals in response to estrogen. Vitellogenesis is normally observed only in sexually mature females, but it can be induced in male and juvenile animals by exposure to exogenous estradiol (E₂) or substances that mimic estrogens. The abnormal production of Vtg by males can, therefore, be used as a biological indicator for exposure to xenoestrogens. In this study, an enzyme-linked immunosorbent assay (ELISA) for measuring Vtg in English sole (Pleuronectes vetulus) was developed and validated. Plasmatic Vtg was purified from E₂-injected male English sole using DEAE ion-exchange and Sepharose size-exclusion chromatography, and polyclonal antibodies against the purified Vtg protein were generated in rabbits. In this assay, a competition for the Vtg antibody was established between Vtg coated onto microtiter plate wells and free Vtg. Detection of adsorbed antigen–antibody complexes was achieved using a horseradish peroxidase conjugated anti-rabbit secondary antibody whose enzyme activity was revealed with 3,3′,5,5′-tetramethyl benzidine (TMB) substrate. Assay conditions provided a detectable Vtg range of 10–450 ng ml⁻¹ (85–20% of binding) of diluted sample. Plasma dilution curves from vitellogenic female and E₂-treated male English sole showed parallelism with the standard dilution curve. We are presently conducting field and laboratory studies to investigate estrogenic and anti-estrogenic activity resulting from exposure to contaminants.     

The conjugation of testosterone with horseradish peroxidase and a sensitive enzyme assay for the conjugate.

The formation of a horseradish peroxidase-testosterone conjugate for the enzyme-linked immunoassay of testosterone was investigated, using tritiated testosterone to follow the reaction. The formation of testosterone-3-(carboxymethyl) oxime-peroxidase by the mixed anhydride method was found to give a conjugate of high enzymatic activity and with three molecules of testosterone per molecule of peroxidase. The optimum conditions for the assay of peroxidase activity were studied and an assay capable of measuring 1 to 5 ng of the conjugate developed; the standard curve being virtually linear. The stability of the conjugate in solution and the effect of lyophilisation on enzymatic activity are also described. The peroxidase-testosterone conjugate was suitable for enzyme-linked immunoassay and the quantities measurable with the peroxidase assay covered the range necessary for a plasma testosterone assay. The stability of the conjugate was such that no particular precautions were necessary for its storage.     

Development of monoclonal antibody against isoquinoline alkaloid coptisine and its application for the screening of medicinal plants

In the development of immunoassay technique, the design of hapten containing a functional group suitable for protein conjugate is the key step for the preparation of antibodies against small molecules. Coptisine (MW 320), a bioactive constituent of Berberis and Coptis species, is small as an immunogen. In addition, coptisine has no reactive group in molecule for conjugating with a protein. To overcome this problem, 9-O-carboxymethyl-berberrubine was designed and conjugated with carrier protein. In order to confirm its immunogenicity, the ratio of hapten in the conjugate was determined by matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS). After immunization, hybridomas secreting antibodies against coptisine were produced by fusing splenocytes with mouse myeloma cell line, P3-X63-Ag8-653. Among hybridomas, the clone 2A1 secreting anti-coptisine monoclonal antibody (MAb) 2A1-9E-1 was obtained through the limited dilution method. The MAb-based enzyme-linked immunosorbent assay (ELISA) against coptisine was developed and characterized. The linear range of the assay in this ELISA method was extended from 1.56 to 25 μg ml⁻¹ possessing the detection limit of 1.56 μg ml⁻¹. The established ELISA using MAb 2A1-9E-1 was applied for the survey of isoquinoline alkaloids in various medicinal plants.     

Core alpha1,3-fucose is a key part of the epitope recognized by antibodies reacting against plant N-linked oligosaccharides and is present in a wide variety of plant extracts

Carbohydrates have been suggested to account for some IgE cross- reactions between various plant, insect, and mollusk extracts, while some IgG antibodies have been successfully raised against plant glycoproteins. A rat monoclonal antibody raised against elderberry abscission tissue (YZ1/2.23) and rabbit polyclonal antiserum against horseradish peroxidase were screened for reactivity in enzyme-linked immunosorbent assay against a range of plant glycoproteins and extracts as well as neoglycoproteins, bee venom phospholipase, and several animal glycoproteins. Of the oligosaccharides tested, Man3XylFucGlcNAc2(MMXF3) derived from horseradish peroxidase was the most potent inhibitor of the reactivity of both YZ1/2.23 and anti- horseradish peroxidase to native horseradish peroxidase glycoprotein. The reactivity of YZ1/2. 23 and anti-horseradish peroxidase against Sophora japonica lectin was most inhibited by a neoglycoconjugate of bromelain glycopeptide cross-linked to bovine serum albumin, while the defucosylated form of this conjugate was inactive as an inhibitor. A wide range of plant extracts was found to react against YZ1/2.23 and anti-horseradish peroxidase, with particularly high reactivities recorded for grass pollen and nut extracts. All these reactivities were inhibitable with the bromelain glycopeptide/bovine serum albumin conjugate. Bee venom phospholipase and whole bee venom reacted weakly with YZ1/2.23 but more strongly with anti-horseradish peroxidase in a manner inhibitable with the bromelain glycopeptide/bovine serum albumin conjugate, while hemocyanin from Helix pomatia reacted poorly with YZ1/2.23 but did react with anti-horseradish peroxidase. It is concluded that the alpha1, 3-fucose residue linked to the chitobiose core of plant glycoproteins is the most important residue in the epitope recognized by the two antibodies studied, but that the polyclonal anti-horseradish peroxidase antiserum also contains antibody populations that recognize the xylose linked to the core mannose of many plant and gastropod N-linked oligosaccharides.      

New Approaches with Different Types of Circulating Cathodic Antigen for the Diagnosis of Patients with Low Schistosoma mansoni Load

Background

Schistosomiasis mansoni is a debilitating and sometimes fatal disease. Accurate diagnosis plays a key role in patient management and infection control. However, currently available parasitological methods are laborious and lack sensitivity. The selection of target antigen candidates has turned out to be a promising tool for the development of more sensitive diagnostic methods. In our previous investigations, the use of crude antigens led to false-positive results. Recently, focus has been given to highly purified Schistosoma mansoni antigens, especially to circulating antigens.

Method

Thus, our main goal was to test different types of circulating cathodic antigen glycoprotein (CCA), as “crude antigen,” the protein chain of recombinant CCA and two individual peptides. These schistosome proteins/peptides were tested in a new diagnostic method employing immunomagnetic separation based on the improvement of antigen–antibody binding.

Principal Findings

Use of recombinant CCA as a diagnostic antigen allowed us to develop a diagnostic assay with high sensitivity and specificity with no false-negative results. Interestingly, the “crude antigen” worked as a good marker for control of cure after praziquantel treatment.

Conclusions/Significance

Our new diagnostic method was superior to enzyme-linked immunosorbent assay in diagnosing low endemicity patients.     

A direct competitive enzyme-linked immunosorbent assay by antibody coated for diethyl phthalate analysis

A direct competitive enzyme-linked immunosorbent assay (ELISA) has been developed for detection of diethyl phthalate (DEP). Protein-hapten conjugate was synthesized to produce polyclonal antibodies against DEP. Experimental parameters were optimized, including immunoreaction conditions, the dilution ratio of horseradish peroxidase (HRP)-antigen conjugate, time of the antibody coated, effect of pH, and ionic strength. The limit of detection was 0.096 ng/ml, and the linear range was 0.1-3500 ng/ml with a regression coefficient (R²) of 0.9957. Recoveries were between 96.4 and 106.2%. The cross-reactivities of the anti-DEP antibody to six structurally related phthalate esters were less than 9%. The method was successfully applied to the determination of DEP in tap water, river water (Yangtze River), and leachate from plastic drinking bottles. This immunoassay was highly specific, sensitive, rapid, simple, and suitable for DEP monitoring. The results obtained were compared with those obtained using the high-performance liquid chromatography method.     

Design and synthesis of a photocleavable biotin-linker for the photoisolation of ligand–receptor complexes based on the photolysis of 8-quinolinyl sulfonates in aqueous solution

The ability of avidin (Avn) to form strong complex with biotin (Btn) is frequently used in the detection and isolation of biomolecules in biochemical, analytical, and medicinal research. The fact that the binding is nealy irreversible, however, constitutes a drawback in term of the isolation and purification of intact biomolecules. We recently found that 8-quinolinyl esters of aromatic or aliphatic sulfonic acids undergo photolysis when irradiated at 300–330 nm in aqueous solution at neutral pH. In this work, a biotin–dopamine (BD) conjugate containing a photocleavable 8-quinolinyl benzenesulfonate (QB) linker, BDQB, was designed and synthesized for use in the efficient recovery of dopamine–protein (e.g., antibody) complexes from an Avn–Btn system. The complexation of BDQB with a primary anti-dopamine antibody (anti-dopamine IgG₁ from mouse) on an Avn-coated plate was confirmed by an enzyme-linked immunosorbent assay (ELISA) utilizing a secondary antibody (anti-IgG₁ antibody) conjugated with horseradish peroxidase (HRP). Upon the photoirradiation (at 313 nm) of the BDQB–IgG₁ complex, the release of dopamine–IgG₁ complex was confirmed by ELISA. Characterization of the resulting photoreleased dopamine–anti-dopamine IgG₁ complex was performed by SDS–PAGE and Western blot.     

Enzyme-linked lectin assay (ELLA): Use of alkaline phosphatase-conjugated Griffonia simplicifolia B4 isolectin for the detection of α- -galactopyranosyl end groups

Alkaline phosphatase has been coupled to Griffonia simplicifolia I B₄ isolectin using a one-step glutaraldehyde conjugation procedure. This enzyme-lectin conjugate (AP-GS I-B₄) has been used to specifically detect plastic-bound natural and synthetic glycoproteins bearing α- -galactopyranosyl end groups. The extent of reactivity of the AP-GS I-B₄ with the glycoproteins appears to be proportional to the number of terminal galactosyl residues present. Furthermore, this assay, termed ELLA (enzyme-linked lectin assay), is specifically inhibitable by low-molecular-weight sugars containing terminal α- -galactosyl groups. The ELLA reactions may be assayed rapidly and objectively by the use of commercially available ELISA-plate readers using standard filters.     

应用异型双功能交联剂制备酶-抗体结合物

酶标免疫测定法(ELISA)中最关键的化合物是酶-抗体结合物,将酶和抗体交联起来需用交联剂。本文作者使用了N-琥珀酰亚胺基3-(2-吡啶基二硫)丙酸酯(简称SPDP)将辣根过氧化物酶(HRP)和兔抗小鼠IgG(兔IgG)交联起来。我们试验了SPDP/HRP,SPDP/IgG和HRP/IgG的不同比例,以期获得活性高的酶-抗体结合物。此外还研究了从结合物中去除自由HRP和自由IgG的方法。用SDS-PAGE及硝酸纤维膜电泳转移法证明本法制备的结合物不含HRP及IgG的自身聚合物。用ELISA法鉴定结合物制品时,一般稀释度可达到1:10,000以上,有的可达到1:20,000(当结合物浓度A_(280nm)=1.0,底物显色A_(492nm)=1.0时)。