An enzyme-linked immunoassay of testosterone

An enzyme-linked immunoassay of testosterone is described. The conjugation of testosterone with high specific activity horseradish peroxidase and a highly sensitive assay for this enzyme having previously been studied, here we describe the immobilization of the anti-testosterone antibody and the development of assay conditions permitting the determination of 50 pg to 1.5 ng testosterone in one working day. In this work attention has been paid to keeping the assay method as simple as possible. The method is discussed in terms of other enzyme-linked immunoassays for steroids.     

Enzyme-Linked Immunosorbent Assays for Detection of Xylem-Limited Bacteria: Use of Trinder Reagent

Quantitation and detection of xylem-limited bacteria with an enzyme-linked immunosorbent assay with a peroxidase conjugate is described. The use of the Trinder reagent (4-aminoantipyrine) allows the determination of extremely small quantities of peroxidase with no precipitate formation or inactivation of the enzyme by H₂O₂. Comparison of the enzyme-linked immunosorbent assay method with microscopic and histochemical tests for the presence of the phony peach disease bacterium in 9-year-old “June Gold” peach trees gave comparable results. The peroxidase conjugate with the Trinder reagent is more sensitive than the alkaline phosphatase conjugate typically used for enzyme-linked immunosorbent assay quantitation.     

Enzyme immunoassay of testosterone in plasma, with use of polyethylene glycol to separate antibody-bound and free hormone.

We describe an enzyme immunoassay for testosterone in which we use a testosterone-3-(carboxymethyl)oxime horseradish peroxidase conjugate as the label and an antiserum, raised in rabbits, to testosterone-3-(carboxymethyl)oxime-bovine serum albumin conjugate. Polyethylene glycol (Carbowax 6000) is used to separate antibody-bound and free steroid. The assay has a sensitivity of 12 pg/assay tube and satisfies the usual criteria of specificity, precision and accuracy. The results agree well with those obtained with a comparable radioimmunoassay.     

Immunoenzyme method of sequential saturation for determining antigens using the model of insulin

The method for the determination of insulin by means of the enzyme immunoassay, based on the use of insulin-peroxidase conjugates, has been developed. In this assay the scheme of the successive saturation of the active sites of antibodies is used. The antigenic properties of two conjugates differing in the method of their preparation are compared. The conjugates were obtained by the covalent binding of peroxidase, oxidized in its carbohydrate component, with insulin (conjugate 1) or hexamethylene-diamine-modified insulin (conjugate 2). The conjugates represented a mixture of oligomers differing in their molecular weight. Conjugate 1 possessed higher affinity to antibodies and higher enzymatic activity than conjugate 2. The method for evaluating the quality of antisera to insulin used in the assay has been proposed. The time of the insulin assay is 5-16 hours, the limit of insulin detection is 5 microU/ml, the variation factor is 3-12%.     

Preparation and Characterization of Ligand-Modified Labelled Liposomes for Solid Phase Immunoassays

Abstract

Small unilamellar vesicles conjugated with an enzyme label and with specific ligands for biological molecules may prove to be useful as signal enhancement vehicles in the development of enzyme-linked immunoadsorbent assays and other detection applications. Bifunctional vesicles have been prepared by covalently attaching horseradish peroxidase (HRP) and monoclonal antibodies to the outside of the lipid bilayer. The reaction conditions were optimized to obtain 7-12 antibody molecules and 100-200 HRP molecules per vesicle. The enzyme retained 70-80% of its specific activity after immobilization with no apparent change in vesicle stability. These bifunctional vesicles were used in a noncompetitive immunoassay for D-Dimer, a fibrin dimer formed at the early stages of thrombogenesis. The assay results using vesicles led to a detection limit for D-Dimer in human plasma which was five times lower than what was achieved using a conventional enzyme-antibody conjugate assay. HRP labelled (bifunctional) liposomes can also be used in competitive assays for the detection of small ligands in bulk solution. HRP and biotin-conjugated vesicles were prepared and used in competitive assays for biotin in free solution. The lowest detection limit for biotin using vesicles as the signal generation mechanism was found to be a factor of 10 lower than what could be observed with a traditional biotin-HRP conjugate. A model has been developed for the competition between a small ligand in solution and a large ligand-conjugated vesicle for binding sites on a solid surface.     

Direct enzyme immunoassay in microtitration plate and test strip format for the detection of saxitoxin in shellfish

Saxitoxin was coupled to horseradish peroxidase via a novel adaptation of the periodate reaction. Based on polyclonal antibodies against saxitoxin, this conjugate was used for the development of two formats of direct enzyme immunoassay (EIA)–a microtitration enzyme-linked immunosorbent assay (ELISA) and a test strip EIA. The detection of saxitoxin without instrumentation by visual evaluation of the test strip EIA is described. The detection limits for saxitoxin were 7 pg/ml (0·35 pg/assay) in the ELISA and 200 pg/ml in the test strip EIA using visual evaluation. Employing a simple procedure of sample preparation, both ELISA and test strip EIA were applied to the analysis of shellfish. The detection limits for saxitoxin in shellfish tissue of the ELISA and the test strip assay were 3 and 4 ng/g, respectively.     

A highly sensitive enzyme immunoassay of anti-insulin antibodies in guinea pig serum

A highly sensitive enzyme immunoassay of anti-insulin antibodies in guinea pig serum is described. Guinea pig anti-insulin serum was diluted to various extents with nonspecific guinea pig serum and incubated with insulin. After incubation, free insulin was separated from insulin-anti-insulin antibody complex by treatment with dextran-charcoal. Anti-insulin antibodies in the complex were dissociated from insulin by incubation with 0.23 M HCl and inactivated. The amount of dissociated insulin was measured by sandwich enzyme immunoassay using anti-insulin IgG-coated polystyrene balls and affinity-purified anti-insulin Fab'-horseradish peroxidase conjugate. The detection limit of anti-insulin antibodies in guinea pig serum was 6.7 pg/assay or 150 ng/liter of serum. The present enzyme immunoassay was 10,000-fold more sensitive than the previously described enzyme immunoassay, in which insulin-coated polystyrene balls were incubated with diluted guinea pig anti-insulin serum and subsequently with rabbit (anti-guinea pig IgG) Fab'-horseradish peroxidase conjugate.     

Optimization of the use of horseradish peroxidase and its antibodies in immunoenzyme analysis

The steady-state kinetics of peroxidation of 8 aromatic amines was studied. p-Phenylenediamine, o-dianisidine (o-DA) and 3,5,3',5'-tetramethylbenzidine were found to be optimal substrates of horse-radish peroxidase. The kinetics of oxidation of these substrates by horseradish peroxidase modified with three molecules of Strophanthin K was studied as well. Within the temperature range from 37 to 53 degrees C the inactivation rate constants were determined for peroxidase and its conjugate with Strophanthin K. The effect of sugars and polyols on thermal stability of the conjugate peroxidase-Strophanthin K was investigated. A comparative kinetic study was performed of oxidation of o-DA and its conjugate with dextran. The results obtained made a basis for an enzyme immunoassay of cardiac glycosides during their isolation from plant raw material.     

Enzymatic recycling-based amperometric immunosensor for the ultrasensitive detection of okadaic acid in shellfish

Electrochemical immunosensors based on a competitive indirect enzyme-linked immunosorbent assay (ciELISA) and an enzymatic recycling system were developed for the detection of okadaic acid (OA). OA-ovalbumin (OA-OVA) conjugate was immobilised on screen-printed electrodes (SPEs) and competition of a newly generated monoclonal antibody (MAb) for free and immobilised OA was subsequently performed. Secondary antibodies labelled with alkaline phosphatase (ALP) or horseradish peroxidase (HRP) were used for signal generation. Experimental parameters were firstly optimised by colorimetric ELISA on microtiter wells and on SPEs. The ELISA system was then tested by amperometry at +300 mV vs. Ag/AgCl (detection of p-aminophenol produced by the reaction of p-aminophenyl phosphate with ALP) or -200 mV vs. Ag/AgCl (detection of 5-methyl-phenazinium methyl sulfate, redox mediator in the HRP bioelectrocatalysis). The limits of detection (LODs) with standard solutions were 1.07 and 1.98 microgL(-1) when using ALP and HRP labels, respectively. An electrochemical signal amplification system based on diaphorase (DI) recycling was integrated into the ALP-based immunosensor, decreasing the LOD to 0.03 microgL(-1) and enlarging the working range by two orders of magnitude. Preliminary results with mussel and oyster extracts were obtained and compared with the colorimetric immunoassay, the colorimetric protein phosphatase inhibition assay (PPIA) and LC-MS/MS.     

A simple enzyme-linked immunosorbent assay for testosterone

A new, enzyme-linked, immunosorbent assay for testosterone is described. The assay uses horseradish peroxidase coupled to testosterone as the tracer and offers the same sensitivity and reliability but greater convenience than radioimmunoassay. The assay is also simpler and more rapidly completed than previously described assays for testosterone.     

Enzymatic labeling of a single chain variable fragment of an antibody with alkaline phosphatase by microbial transglutaminase

Functional cross-linking of a single chain Fv fragment of anti-hen egg-white lysozyme antibody (scFv) and alkaline phosphatase (AP) was explored using microbial transglutaminase (MTG) from Streptomyces mobaraensis. A specific peptidyl linker for MTG was genetically fused to the N-terminus of each protein and the resultant proteins were obtained separately by bacterial expression. The recombinant peptide-tagged scFv and AP were site-specifically cross-linked by MTG through the extra peptidyl linkers in vitro, which mainly yielded the heterodimer (i.e., scFv-AP conjugate). The enzymatic cross-linking reaction had little influence on either the antigen-binding ability of the scFv moiety or the enzymatic activity of the AP moiety of the conjugate, allowing use within an enzyme-linked immunosorbent assay. The results obtained suggest that the enzymatic approach with MTG facilitates the posttranslational construction of functional fusion proteins.     

Resolution of optical isomers by chiral high-performance liquid chromatography: separation of dihydrodiols and tetrahydrodiols of benzo[a]pyrene and benz[a]anthracene

A competitive enzyme-linked immunoassay (CELIA) for human serum angiotensin-1-converting enzyme (ACE) was developed. The sensitivity was amplified by using a secondary antibody and an avidin biotin-conjugated horseradish peroxidase complex as the enzyme-labeled reagent. This configuration was compared to three other configurations for an indirect CELIA, and was found to be the most sensitive. A sensitivity of 39 ng/ml ACE was achieved with intraassay and interassay coefficients of variation of 6.3 and 8%, respectively. CELIA will detect ACE in human serum without interference from either pharmacological or endogenous ACE inhibitors. In normal human volunteers, ACE values obtained using CELIA correlated well with values obtained by enzymatic assay.     

Development of a plasma panel test for detection of human myocardial proteins by capillary immunoassay

A chemiluminescence immunoassay for the detection of four heart marker proteins: myoglobin, creatine kinase mb [CKmb], troponin I [TnI], and fatty acid-binding protein [FABP], was designed. The immunoassay was based on enzyme-linked immunosorbent assay [ELISA] and antibodies immobilized in glass capillaries pre-treated with 3-aminopropyltriethoxysilane. The protein bound to the antibody was detected by using an anti-protein-horseradish peroxidase [HRP] conjugate. The reaction of the HRP with luminal and hydrogen peroxide-based substrate generated the chemiluminescence and a photodiode detector was used to measure the light intensity. The same assay protocol was used to detect all four proteins. Ultrasound waves were used to improve the silanization of glass and the antibody immobilization process. The optimization of the duration and intensity of the ultrasound was performed for the myoglobin assay. Ultrasound improved the silanization procedure and the capillaries gave an approximately 2.5 times greater ELISA response. Ultrasound also improved the sensitivity by approximately 100% when monoclonal antibody was immobilized on a glass capillary. Calibration curves corresponding to analyte concentrations ranging from 2.4 to 2400 ng/ml in plasma samples were recorded. The detection limits were in the region of 1.2 myoglobin, 0.6 CKmb, 5.6 TnI, and 4 ng/ml FABP in plasma with a coefficient of variation of 3-9.9%.     

Generation of monoclonal antibodies to human acylphosphatase (muscular isoenzyme) and application in solid-phase immunoassay

Monoclonal antibodies to human acylphosphatase (muscle isoenzyme) were generated by an improved hybridoma technique. Immunization consisted of four antigen administrations in an overall period of 15 weeks. After cell fusion and repeated subcloning of positive lines, seven monoclonal antibodies with good affinity and specificity were selected. These antibodies were characterized for their affinity constant and immunoreactivity. The latter was determined using peptides generated by CNBr cleavage of the antigen. One of the selected antibodies had an affinity constant such that it could be used to develop a competitive enzyme-linked immunosorbent assay. In our test, the antigen that was coated on the matrix, and the free one, competed for the antibody-horseradish peroxidase conjugate. No cross-reactivity with the erythrocyte iso-enzyme was found, and the test showed a limit in sensitivity of 0.32 ng/ml of antigen. We expect that the enzyme immunoassay could be useful for clinical application.     

Plasminogen activator-anti-human fibrinogen conjugate

A covalent conjugate between the plasminogen activator urokinase and polyclonal rabbit anti-human fibrinogen has been formed using the heterobifunctional coupling reagent N-succinimidyl 3-(2-pyridyldithio) propionate. The resultant urokinase-anti-human fibrinogen conjugate was separated from unreacted material by gel filtration. The conjugate exhibited amidase activity against the small chromogenic substrate pyroglutamyl-glycyl-arginine-p-nitroanilide as well as plasminogen activator activity in an assay employing plasminogen and the plasmin substrate D-valyl-leucyl-lysine-p-nitroanilide. Retention of antibody specificity for fibrinogen was demonstrated using an enzyme linked immunoassay procedure. The conjugate was found to have greater stability in human plasma than unconjugated urokinase.     

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.     

A highly specific heterologous enzyme-linked immunosorbent assay for measuring testosterone in plasma using antibody-coated immunoassay plates or polypropylene tubes.

A highly specific and sensitive enzyme-linked immunosorbent assay, using a heterologous combination of antiserum raised against testosterone-3-(O-carboxymethyl) oxime-bovine serum albumin and penicillinase-labeled testosterone-11 beta-carboxymethyl ether, was developed for measuring testosterone in human plasma. Immunoassay plates (96 wells) provided a sensitivity of 2.5 pg/well. This was achieved by maintaining the molar ratios of steroid to enzyme between 10 and 20. The assay was very specific for testosterone and did not show any cross-reaction with the related C19 steroids tested. Replacement of immunoassay plates with the locally available polypropylene tubes raised the detection limits to 25 pg/tube, but improved the range of doses of testosterone that could be measured up to 10,000 pg. The antiserum to testosterone derivative was linked to both immunoassay plates and polypropylene tubes through immunochemical bridges. Comparison of testosterone values of 52 plasma specimens obtained by both solid phase methods with those of radioimmunoassay showed good correlation.     

Peroxidase refolding in the presence of specific antibodies

A panel of eight monoclonal antibodies raised against horseradish root peroxidase has been assembled and characterized. Affinity constants were determined for all antibodies, and their specificity for various structural forms of the enzyme (native peroxidase, apoperoxidase, and denatured peroxidase) were assessed by competitive enzyme immunoassay. The effects of the antibodies on the process of refolding of peroxidase after its denaturing with 6.5 M guanidine hydrochloride were studied spectrophotometrically, by the restoration of the enzymatic activity in the reaction of 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonate). The yield of the active enzyme in the course of the refolding was increased 1.5 to 1.7 times in the presence of antibody H1. Effects of the antibodies constituting the panel on the activity of native peroxidase and the stability of its dilute solutions were analyzed.     

An enzyme-linked lectin assay for alpha1,3-galactosyltransferase

UDP-Gal:Galbeta1-4GlcNAc alpha1,3-galactosyltransferase (alpha3GalT) is responsible for the synthesis of carbohydrate xenoantigen Galalpha1-3Galbeta1-4GlcNAc. In this work a convenient and sensitive assay system for quantification of alpha3GalT activity by enzyme-linked lectin assay (ELLA) with colorimetric detection is described. Microtiter plate wells whose surface had been coated with the polyacrylamide conjugate of the disaccharide Galbeta1-4GlcNAc (acceptor) are incubated with alpha3GalT in the presence of "cold" UDP-Gal as glycosyl donor. Formation of product by enzymatic extension of the glycan chain is detected by the biotinylated plant lectin Viscum album agglutinin. The standard curve for correct quantification of alpha3GalT activity is completed after running standard assays with no (background) or known quantities of enzyme activity. Product formation detected in this manner is proportional to enzyme activity and the concentrations of the acceptor and the glycosyl-donor UDP-Gal. In accordance with the known specificity of alpha3GalT, no enzymatic conversion of Le(x) into GalalphaLe(x) was observed using this assay. Human alphaGal antibodies were isolated using a disaccharide-exposing affinity adsorbent and their specificity was studied. Relative to the application of these natural immunoglobulins as product-detecting tool, the ELLA proved to be more sensitive.     

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.