Detection of one attomole of [Arg8]-vasopressin by novel noncompetitive enzyme immunoassay (hetero-two-site complex transfer enzyme immunoassay).

One attomole of [Arg8]-vasopressin (AVP) was detected by a novel noncompetitive enzyme immunoassay (hetero-two-site complex transfer enzyme immunoassay). AVP was indirectly biotinylated using N-hydroxysuccinimidobiotin and trapped onto an anti-AVP IgG-coated polystyrene ball. After washing, biotinylated AVP was eluted from the polystyrene ball with HCl and was reacted with 2,4-dinitrophenyl-fluorescein disulfide-bovine serum albumin-rabbit anti-AVP IgG conjugate.

Development of ultrasensitive enzyme immunoassay reviewed with emphasis on factors which limit the sensitivity.

Development of ultrasensitive enzyme immunoassays for antigens, haptens and antibodies is reviewed with emphasis on factors which limit the sensitivity. One of the most important conditions for ultrasensitive immunoassays is the use of non-competitive solid phase assay systems rather than competitive ones. Although non-competitive immunoassays are available for antigens and antibodies, there are only competitive immunoassays for hapten molecules which can not be bound simultaneously by two different antibodies.

Use of inactive beta-D-galactosidase for elimination of interference by anti-beta-D-galactosidase antibodies in immune complex transfer enzyme immunoassay for anti-thyroglobulin IgG in serum using beta-D-galactosidase from Escherichia coli as label.

A novel enzyme immunoassay (immune complex transfer enzyme immunoassay) for anti-thyroglobulin IgG using beta-D-galactosidase from Escherichia coli as label was reported previously. This immunoassay was highly sensitive in demonstrating anti-thyroglobulin IgG not only in all patients with Graves' disease and chronic thyroiditis but also in a large proportion of healthy subjects. However, the detection of anti-thyroglobulin IgG at low levels in some serum samples was difficult, probably due to the presence of anti-beta-D-galactosidase antibodies.

Time-resolved fluorometric sandwich immunoassay for human growth hormone in serum and urine.

A specific and sensitive time-resolved fluorometric sandwich immunoassay for human growth hormone (hGH) in serum and urine is described. A monoclonal anti-hGH IgG1 (5802)-coated polystyrene ball was incubated with serum or dialyzed urine and subsequently with europium ion-labeled monoclonal anti-hGH IgG1 (5801). Europium ion bound to the polystyrene ball was measured by time-resolved fluorometry.

Immune complex transfer enzyme immunoassay for (anti-human T-cell leukemia virus type I) IgG in serum using a synthetic peptide, Env gp46(188-209), as antigen.

An immune complex transfer enzyme immunoassay for (anti-human T-cell leukemia virus type I) IgG (anti-HTLV-I IgG) in serum using a chemically and safely synthesized peptide, env gp46(188-209), is described. Anti-HTLV-I IgG in test serum, which had been incubated with excess of inactive beta-D-galactosidase to eliminate interference by anti-beta-D-galactosidase antibodies, was reacted simultaneously with dinitrophenyl bovine serum albumin-env gp46(188-209) conjugate and env gp46(188-209)-beta-D-galactosidase conjugate. The complex formed of the three components was trapped onto polystyrene balls coated with affinity-purified (anti-dinitrophenyl group) IgG.

Novel and sensitive noncompetitive enzyme immunoassay (hetero-two-site enzyme immunoassay) for alpha-human atrial natriuretic peptide in plasma.

A novel and sensitive noncompetitive enzyme immunoassay (hetero-two-site enzyme immunoassay) for alpha-human atrial natriuretic peptide (alpha-hANP) in plasma, which uses only one monoclonal IgG for the ring structure of alpha-hANP, is described. Plasma was filtered through polysaccharide membrane to separate peptides from proteins. The plasma filtrate was incubated with N-hydroxysuccinimidobiotin to biotinylate alpha-hANP and subsequently with a polystyrene ball coated with monoclonal IgG for the ring structure of alpha-hANP to trap biotinylated alpha-hANP.

Immune complex transfer enzyme immunoassay for (anti-human T-cell leukemia virus type I) IgG in serum using a synthetic peptide, Cys-Gag p19(100-130), as antigen.

An immune complex transfer enzyme immunoassay for (anti-human T-cell leukemia virus type I) IgG (anti-HTLV-I IgG) in serum using a synthetic peptide, cys-gag p19(100-130), is described. Anti-HTLV-I IgG in test serum, which had been incubated with excess of inactive beta-D-galactosidase to eliminate interference by anti-beta-D-galactosidase antibodies, was reacted simultaneously with 2,4-dinitrophenyl bovine serum albumin-cys-gag p19(100-130) conjugate and cys-gag p19(100-130)-beta-D-galactosidase conjugate. The complex formed of the three components was trapped onto polystyrene balls coated with affinity-purified (anti-2,4-dinitrophenyl group) IgG.

Ultrasensitive enzyme immunoassay.

Ultrasensitive enzyme immunoassay methods not only for antigens but also for antibodies and haptens are reviewed. These methods are based on a noncompetitive type of assay using solid phase rather than a competitive one. One of the greatest obstacles limiting the sensitivity of noncompetitive immunoassay methods using solid phase is the nonspecific binding of labeled reactants to solid phase.

Detection of one milliattomole of ferritin by novel and ultrasensitive enzyme immunoassay.

A novel and ultrasensitive enzyme immunoassay (immune complex transfer two-site enzyme immunoassay) for ferritin is described. Ferritin was reacted simultaneously with affinity-purified dinitrophenyl biotinyl anti-ferritin IgG and affinity-purified anti-ferritin Fab'-beta-D-galactosidase conjugate. The complex formed of the three components was trapped onto affinity-purified (anti-dinitrophenyl group) IgG-coated polystyrene balls.

Immune complex transfer enzyme immunoassays for anti-angiotensin I IgG in serum using angiotensin I conjugates prepared by two different methods.

Anti-angiotensin I IgG in serum was measured by immune complex transfer enzyme immunoassays using angiotensin I conjugates prepared by two different methods. In the first method, angiotensin I was conjugated to dinitrophenyl bovine serum albumin and beta-D-galactosidase through covalent links. Anti-angiotensin I IgG in rabbit serum was reacted simultaneously with dinitrophenyl bovine serum albumin-angiotensin I conjugate and beta-D-galactosidase-angiotensin I conjugate, and the complex formed of the three components was trapped onto (anti-dinitrophenyl group) IgG-coated polystyrene balls.

Novel and sensitive noncompetitive (two-site) immunoassay for haptens with emphasis on peptides.

A novel and sensitive noncompetitive (two-site) enzyme immunoassay is described to measure attomole amounts of haptens, especially small peptides, which cannot be bound simultaneously by two different antibodies directed to the corresponding two different epitopes on the hapten molecules; consequently, they cannot be measured by the conventional two-site immunoassays. The principle of the assay is to label haptens to be measured with an appropriate substance, so that the hapten molecule can be bound simultaneously by both a binding substance molecule for the label, and anti-hapten antibody molecule, which allows two-site assay. Feasibility of the principle was demonstrated by using biotin as a label and angiotensin I, a 10 amino acid single chain peptide, as a model hapten.

Sensitive time-resolved fluorimetric immune-complex-transfer immunoassay for antithyroglobulin IgG in serum.

A sensitive time-resolved fluorimetric immune-complex-transfer immunoassay for antithyroglobulin IgG in serum is described. Antithyroglobulin IgG in test serum was reacted with dinitrophenyl europium ion-labeled thyroglobulin. The complex formed of antithyroglobulin IgG and dinitrophenyl europium ion-labeled thyroglobulin was trapped onto two polystyrene balls coated with affinity-purified rabbit antidinitrophenyl bovine serum albumin IgG.

Novel and sensitive noncompetitive (two-site) enzyme immunoassay for haptens with amino groups.

A novel and sensitive noncompetitive (two-site) enzyme immunoassay for haptens with amino groups is described. L-Thyroxine (T4) was used as a model hapten. T4 was indirectly biotinylated with glutathione as spacer between T4 and biotin molecules and trapped onto anti-(T4-bovine serum albumin) IgG-coated polystyrene balls.

Novel and sensitive enzyme immunoassay (immune-complex-transfer enzyme immunoassay) for alpha-fetoprotein from hepatocellular carcinoma.

Alpha-fetoprotein in sera from patients with hepatocellular carcinoma was fractionated into three peaks by affinity chromatography on a column of Lens culinaris agglutinin-Sepharose 4B. One peak (the first peak), which passed through the column without adsorption, was found in both healthy subjects and patients with liver cirrhosis and hepatocellular carcinoma. The second and third peaks were reactive with L.

Specific enzyme immunoassay for alpha-fetoprotein from hepatocellular carcinoma.

alpha-Fetoprotein in sera from healthy subjects and patients with liver cirrhosis and hepatocellular carcinoma was fractionated into three peaks by affinity chromatography on a column of Lens culinaris agglutinin-Sepharose 4B. One peak (the first peak) was found in all the serum samples, but the other two peaks (the second and third peaks) were found only in patients with hepatocellular carcinoma. For alpha-fetoprotein in the second and third peaks, a specific enzyme immunoassay was developed.

Development and applications of ultrasensitive enzyme immunoassays for antigens and antibodies.

Some enzymes can be measured at lower molar concentrations than the radioisotopes used as labels in radioimmunoassay. Therefore, enzyme immunoassay should be capable of higher sensitivity than radioimmunoassay, if appropriate techniques are used for enzyme-labelling of antibodies and formulation of assay protocols. Techniques required for enzyme immunoassay with attomole sensitivity have been developed, and successfully applied to the measurement of clinically important ligands at levels below those possible using radioimmunoassay.

Measurement of anti-thyroglobulin IgG in serum by novel and sensitive immune complex transfer enzyme immunoassay.

Anti-thyroglobulin IgG in human serum was measured by a novel and sensitive immune complex transfer enzyme immunoassay. Anti-thyroglobulin IgG in human serum was reacted with dinitrophenyl thyroglobulin, and the complex formed between human anti-thyroglobulin IgG and dinitrophenyl thyroglobulin was trapped onto an affinity-purified rabbit (anti-dinitrophenyl bovine serum albumin) IgG-coated polystyrene ball. The polystyrene ball was washed to eliminate most nonspecific IgG in test serum, and the complex was eluted from the polystyrene ball, to which nonspecific IgG had been adsorbed, with dinitrophenyl-L-lysine and transferred to a clean polystyrene ball coated with rabbit anti-thyroglobulin IgG.

Novel and sensitive noncompetitive enzyme immunoassay for kassinin in rat plasma.

A novel and sensitive noncompetitive enzyme immunoassay for kassinin is described. Kassinin was biotinylated using sulfosuccinimidyl-6-(biotinamido)hexanoate. The biotinylated kassinin was trapped on anti-kassinin IgG-coated polystyrene balls and, after washing to eliminate other biotinylated substances, was eluted with HCl.

Novel and sensitive noncompetitive enzyme immunoassay for peptides.

A novel and sensitive noncompetitive enzyme immunoassay for peptides is described. Peptides were biotinylated using sulfosuccinimidyl-6-(biotinamido)hexanoate and were trapped onto anti-peptide IgG-coated polystyrene balls. After washing the polystyrene balls to eliminate other biotinylated substances, the biotinylated peptides were eluted with HC1 and were reacted with anti-peptide Fab'-peroxidase conjugate.

Development and applications of sensitive enzyme immunoassay for antibodies: a review.

The sensitivity of the conventional enzyme immunoassay methods is seriously limited by the presence of nonspecific immunoglobulins at high concentrations in test serum. In order to overcome this difficulty, various methods have been developed, and the detection limit of serum antibodies has been lowered to 50-100 ng/l, which is 300,000-fold lower than that by the conventional enzyme immunoassay method using antigen-coated solid phases and anti-immunoglobulin antibody-enzyme conjugates. As a result, anti-insulin antibodies have been demonstrated in most of patients treated with insulin for 0.

More sensitive and simpler immune complex transfer enzyme immunoassay for antithyroglobulin IgG in serum.

A more sensitive and simpler immune complex transfer enzyme immunoassay for antithyroglobulin IgG in serum and its use for the measurement of antithyroglobulin IgG in healthy subjects and patients with thyroid diseases are described. Antithyroglobulin IgG in test serum was reacted simultaneously with dinitrophenylthyroglobulin and thyroglobulin-beta-D-galactosidase conjugate. The complex formed of antithyroglobulin IgG, dinitrophenyl thyroglobulin, and thyroglobulin-beta-D-galactosidase conjugate was trapped onto two polystyrene balls coated with affinity-purified rabbit (antidinitrophenyl bovine serum albumin) IgG.

Existence of anti-thyroglobulin IgG in healthy subjects.

An autoantibody, anti-thyroglobulin IgG, was detected in a large proportion of healthy subjects. Sera were collected from 232 healthy subjects aged 7-83 yr, who had no apparent symptoms with normal serum levels of thyroid-stimulating hormone, confirming the absence of Graves' disease and chronic thyroiditis. Anti-thyroglobulin IgG in serum was measured by a novel enzyme immunoassay, the principle of which has been shown to provide 3,000 to 10,000-fold higher sensitivity than the conventional methods.

Enzyme immunoassay for alpha-human atrial natriuretic polypeptide--direct measurement of plasma level.

A sandwich enzyme immunoassay for alpha-human atrial natriuretic polypeptide (alpha-hANP) was developed. Polystyrene balls were coated with monoclonal IgG1 specific for the N-terminal half of the ring structure of alpha-hANP, and rabbit Fab' specific for the C-terminal (17-28) of alpha-hANP was conjugated to horseradish peroxidase. The polystyrene ball was incubated with alpha-hANP standards or plasma and subsequently with the conjugate.

Sensitive sandwich enzyme immunoassay of human growth hormone (hGH) in serum and urine using monoclonal antibody-coated polystyrene balls.

A sensitive sandwich enzyme immunoassay for human growth hormone (hGH) using monoclonal antibody is described. A monoclonal anti-hGH IgG-coated polystyrene ball was incubated with hGH and subsequently with affinity-purified rabbit anti-hGH Fab'-horseradish peroxidase conjugate. Peroxidase activity bound to the polystyrene ball was assayed by fluorimetry using 3-(4-hydroxyphenyl) propionic acid as a substrate.

Development and clinical application of sensitive enzyme immunoassay for macromolecular antigens--a review.

Radioimmunoassay has been a powerful tool to measure haptens and antigens which are important for the investigation and diagnosis of diseases, especially endocrine disorders. However, the use of radioisotopes in radioimmunoassay suffers from serious disadvantages. Radioisotope-labeled reagents are unstable and hazardous to health.