Preparation of conjugated antigens based on zearalenone carboxymethyloxime and their use in immunoenzyme analysis

Zearalenone-6'-carboxymethyloxime was synthesized, and its conjugates with albumins and gelatin were prepared. Polyclonal rabbit antibodies against the conjugate with bovine serum albumin were shown to be highly specific to zearalenone and to have a lower cross-reactivity toward its structural analogues (alpha-zearalenol--28%, beta-zearalenol--6%, zearalanone--12%, and alpha-zearalanol--5%). The sensitivity of enzyme immunoassay using gelatin-based immobilized conjugates for determination of zearalenone in solutions was 1 ng/ml, and this allowed us to determine this substance in feed at a threshold concentration of 200 micrograms/kg.     

Preparation of oligosaccharide-polyacrylamide conjugates and their use as antigens in enzyme immunoassay (EIA)

Oligosaccharides derived fromSalmonella lipopolysaccharides or from human milk were converted to theirN-acetyl-N-(4-acrylamidophenyl)-1-amino-1-deoxyalditol derivatives. These derivatives were copolymerized with acrylamide to give linear, water-soluble polymers, which were used as coating antigens in EIA assays.     

Elaboration of enzyme immunoassay based on recombinant antigens and intended for diagnostics of syphilis

Enzyme immunoassay (EIA) using recombinant antigens for the detection of Treponema pallidum-specific antibodies in sera of syphilis patients was developed. Four low-molar-mass Treponema antigens (Tp15, Tp17, TmpA, Tp47) were investigated; 17- and 47-kDa proteins were demonstrated as immunodominant as they permitted to obtain the most sensitive EIA. Using a mixture of these proteins a 3rd-generation-EIA kit Dia-Syph was constructed, its sensitivity being 99.4% during tests of 165 sera of syphilitic patients. No false result was obtained on the commercial panel PSS01 (BBI, USA). The specificity of the elaborated test system (99.7%) was determined on 295 sera.     

Preparation of tritium-labeled hyaluronic acid oligomers and their use in enzyme studies

Tritium-labeled oligosaccharides can be prepared from hyaluronic acid by the Wilzbach technique with greater ease than usual by use of the specificity of hyaluronidase in the course of purification. Labeled oligomers of hyalobiuronic acid were isolated and shown to be radiochemically homogeneous, and are used in studies of complex enzymic kinetics. The techniques used may have general use in preparing generally labeled oligosaccharides.     

Preparation of magnetic latexes and their use for the immunodetection of microbial antigens

The possibility of detecting antigens of plague, tularemia, and brucellosis microbes with magnetic latex (ML)-based test systems has been demonstrated. MLs were prepared from latexes (polyacroleine microspheres, 1.2–1.8 ± 0.1 μm) by exposing the particles to a 25–35%-solution of ferrous sulfate for 0.5 h and then to a 15–25%-aqueous solution of ammonia for 0.5 h in a 100°C water bath and dehydrating after each operation. The possibility of preparing magnetic latex immunosorbents (MLIS) by ligand immobilization on ML and using them in magnetic latex ELISA (ML-ELISA) for the detection of microbial antigens was demonstrated. The detection limit in ML-ELISA equaled 10²–10³ microbial cells in 1 ml (cells/ml). Relative experimental error was not higher than 8%.     

Type-specific enzyme immunoassay for detection of pneumococcal capsular polysaccharide antigens in nasopharyngeal specimens

We developed a new competitive EIA method for the demonstration of pneumococcal capsular polysaccharides from respiratory samples. The pediatric types 4, 6B, 9V, 14, 18C, 19F and 23F were selected for this study, because these capsular polysaccharides were included in the first heptavalent pneumococcal conjugate vaccines, which were used in the Finnish Otitis Media Vaccine Trial. Sensitivity of the EIA tests for purified polysaccharide antigens varied between 5 and 100 ng/ml, depending on the type. The assays performed well in 100 nasopharyngeal samples (NPS) samples processed through an enrichment culture, with an almost 100% sensitivity compared with routine culture. The method appeared type-specific, except that EIA for 6B capsule also detected 6A. The method is applicable for type-specific identification of pneumococcus in carriage studies.     

Novel poly-substituted aryl acridinium esters and their use in immunoassay

A new series of stable acridinium ester conjugates have been developed for use as non-isotopic labels in immunoassay. They have proved to be a flexible alternative to radioimmunoassay. We present data showing the successful development of immunoassays in sandwich, competitive and receptor formats. In addition, hydrophilic acridinium ester analogues have been synthesized, encapsulated in liposomes, and utilized as labels in immunoassay. The potential of this technology is discussed.     

Preparation of some immobilized linked enzyme systems and their use in the automated determination of disaccharides

1. Glucose oxidase (EC 1.1.3.4), amyloglucosidase (EC 3.2.1.3), invertase (EC 3.2.1.26) and beta-galactosidase (EC 3.2.1.23) were covalently attached via glutaraldehyde to the inside surface of nylon tube. 2. The linked enzyme system, comprising invertase immobilized within a nylon tube acting in series with glucose oxidase immobilized in a similar way, was used for the automated determination of sucrose. 3. The linked enzyme system, comprising beta-galactosidase immobilized within a nylon tube acting in series with glucose oxidase immobilized in a similar way, was used for the automated determination of lactose. 4. The linked enzyme system, comprising amyloglucosidase immobilized within a nylon tube acting in series with glucose oxidase immobilized in a similar way, was used for the automated determination of maltose. 5. Mixtures of glucose oxidase and amyloglucosidase were immobilized within the same piece of nylon tube and used for the automated determination of maltose. 6. Mixtures of glucose oxidase and invertase were immobilized within the same piece of nylon tube and used for the automated determination of sucrose.     

Evaluation of a malaria antibody enzyme immunoassay for use in blood screening

Transfusion-transmitted malaria is rare, but it may produce severe problem in the safety of blood transfusion due to the lack of reliable procedure to evaluate donors potentially exposed to malaria. Here, we evaluated a new enzyme-linked immunosorbent assay malaria antibody test (ELISA malaria antibody test, DiaMed, Switzerland) to detect antibodies to Plasmodium vivax (the indigenous malaria) in the blood samples in the Republic of Korea (ROK). Blood samples of four groups were obtained and analyzed; 100 samples from P.vivax infected patients, 35 from recovery patients, 366 from normal healthy individuals, and 325 from domestic travelers of non-endemic areas residents to risky areas of ROK. P.vivax antibody levels by ELISA were then compared to the results from microscopic examination and polymerase chain reaction (PCR) test. As a result, the ELISA malaria antibody test had a clinical sensitivity of 53.0% and a clinical specificity of 94.0% for P.vivax. Twenty out of 325 domestic travelers (6.2%) were reactive and 28 cases (8.6%) were doubtful. Of the reactive and doubtful cases, only two were confirmed as acute malaria by both microscopy and PCR test. Thus we found that the ELISA malaria antibody test was insufficiently sensitive for blood screening of P.vivax in ROK.     

Preparation and use of magnetic sorbents for studying microorganism antigens

In this work simple techniques for obtaining polyacrylamide sorbents with magnetic properties are described. These techniques have permitted obtaining block and microgranulated sorbents with the immobilization of antibodies from plague antiserum in the cellular gel structure for the specific sorption of killed and live Yersinia pestis cells and their first fraction; pig brain gangliosides have also been incorporated into the gel structure with a view to the sorption of cholera toxin from the filtrate of Vibrio cholerae culture. The magnetic properties of sorbents, obtained by the copolymerization of powdered magnetic ferric oxides in gel, have made it possible to increase the effectiveness of specific sorption due to mixing and rapid separation in different magnetic fields, as well as to facilitate and accelerate manipulations with the sorbent at all stages. The capacity of different types of sorbents and the time of sorption have been determined.     

Comparison of excretory/secretory and circulating antigens of Toxoplasma gondii by enzyme immunoassay and immunoblotting.

, , , , , and 1992. Comparison of excretory/secretory and circulating antigens of Toxoplasma gondii by enzyme immunoassay and immunoblotting. International Journal for Parasitology 22: 1083–1088. Toxoplasma gondii trophozoites (RH strain) were cultured in embryonic fibroblasts in order to study the kinetics of production of excretory/secretory antigens, and the results were compared to the production of circulating antigens in an in vivo mouse model. By capture-ELISA, excretory/secretory antigens were first detected on the fourth day of culture whereas circulating antigens were first detected 1 day after infection. Similar concentrations of antigens were detected in both models as evidenced by comparable absorbance values. By immunoblotting, the excretory/secretory antigens were also detected later compared to circulating antigens (day 4 vs day 1). Seven major polypeptides were detected in both antigen preparations, six of them having the same molecular mass (110, 75,48, 30, 24 and 22 kDa).     

Synthesis, characterization, and evaluation of ferrocene-theophylline conjugates for use in electrochemical enzyme immunoassay

A series of 8-(ferrocenylalkyl)theophylline conjugates were synthesized for evaluation in a homogeneous, competitive electrochemical immunoassay for theophylline with amperometric detection of the ferrocene label at +320 mV. The electrical signal was amplified via redox cycling with the glucose oxidase/glucose system. The resulting catalytic current was strongly inhibited upon binding of the conjugates to anti-theophylline antibodies such that a large excess of theophylline was required to achieve complete reversal leading to an assay with poor sensitivity in the clinical range. A study of the nonspecific interaction of the antibodies with various ferrocene derivatives indicated that this was reduced when a charged functional group was present on the metallocene ring. Consequently, a conjugate was synthesized with a quaternary ammonium group which when incorporated into the assay resulted in improved sensitivity.     

A method for the preparation of steroid-protein antigens for the use in immunoassay of steroids

A general method for the preparation of steroid-protein conjugates coupled through carbon 6 of Δ⁴ steroids was demonstrated using 6β Br-testosterone acetate and 6β Br-progesterone covalently bound to sulfhydryl-group-enriched bovine serum albumin. The antigenicity of the conjugates and the specificity of the antibodies were evaluated. With anti-progesterone the assay accuracy was determined to be 10.3%, the intra-assay precision was 8.7% and the inter-assay precision was 12.6%. The material was used to assay the progesterone content of normal human male and female serum as well as the changes in this hormone following corpora lutea induction in constant-estrus androgenized female rats.     

Tandem conjugation of enzyme and antibody on silica nanoparticle for enzyme immunoassay

We present a new type of enzyme-antibody conjugate that simplifies the labeling procedure and increases the sensitivity of enzyme-linked immunosorbent assay (ELISA). The conjugates were prepared through layer-by-layer immobilization of enzyme and antibody on a silica nanoparticle scaffold. A maximal amount of enzyme was immobilized on the nanoparticle, followed by antibody linkage through Dextran 500. The conjugate could be easily purified from unreacted reagents by simple centrifugations. In comparison with the conventional antibody-enzyme conjugate used in ELISA, which often has one or two enzyme molecules per antibody, the new type of conjugate contained more enzyme molecules per antibody and provided a much higher signal and increased sensitivity. When used in an ELISA detection of the hepatitis B surface antigen (HBsAg), the detection limit was three times lower than that of the commercially available ELISA kit.     

The use if Francisella tularensis lipopolysaccharide in the dot solid phase enzyme immunoassay

To determine antitularemia antibodies in the sera of humans and animale, the possibility of using dot immunoassay with the use of F. tularensis lipopolysaccharide (LPS) as antigen-containing preparation was ascertained. Experiments demonstrated that this method made it possible to determine specific antitularemia antibodies in the sera of sick and immunized humans and animals. Investigetions carried out with the use of heterologous antisera to F. novicida, F. novicida-like and F. philomiragia, as well as Brucellf abortus, Vibrio cholerae and Yersinia enterocolitica, revealed that F. tularensis S-LPS was highly specific. The results obtained in this investigation are indicative of good prospects of using F. tularensis LPS in dot blotting for the laboratory diagnostics of tularemia in humans.     

Fluorescence immunoassay system based on the use of a pH-sensitive phase-separating polymer

Poly(N-isopropylacrylamide-co-methacrylic acid) [P(NIPAAm-co-MAA)], a linear water-soluble pH-sensitive phase-separating polymer, was synthesized and used as a novel separation carrier for the reactants in immunoassay. This polymer precipitates out of water below a critical pH 5.8 at 37 degrees C and redissolves when the pH of solution is above 6.2. The characteristic of this polymer makes it possible to carry out the immunochemical steps of an immunoassay in a true solution and then to quickly separate the resulting product from the reaction mixture. The above approach was applied to determination of alpha-fetoprotein with the competitive immunoassay format. Compared with traditional ELISA using the same reactants, the proposed method was much faster (the assay time decreased from 100-120 to 30 min) and showed similar sensitivity, i.e., 0.04 ng/mL. In addition, a sandwich immunoassay method for the determination of hepatitis B surface antigen was also studied, and the results showed that the pH phase-separating immunoassay could be carried out through a sandwich or a competitive method. This general technique may also be used for a wide variety of separation processes in addition to immunoassay, in which a specific component is to be isolated for analysis, recovery, or disposal.     

A malachite green procedure for orthophosphate determination and its use in alkaline phosphatase-based enzyme immunoassay

An improved procedure for phosphate determination based on a highly colored complex of phosphomolybdate and malachite green is described. All necessary reagents are combined in one concentrated solution, making the assay sensitive and convenient. The procedure is based on the finding that the dye is easily soluble and stable in the presence of 6 N acid. The addition of Tween 20 is required to stabilize the dye-phosphomolybdate complex at phosphate concentrations above 10 microM. The time of color development at 25 degrees C is about 3 min. The procedure was adopted to measure alkaline phosphate activity in heterogeneous enzyme immunoassay with rho-nitrophenyl phosphate and pyrophosphate as substrates. In both cases, a 4-fold increase in sensitivity in terms of absorbance readings was obtained compared to the standard method based on rho-nitrophenol measurement. In visual analysis, the gain in sensitivity was as high as 20-fold, due to contrast color change (yellow to greenish blue).