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At least 19 recordsLinked to original sources
PubMed1979

Specificity of immunoassays. II. Heterogeneity of specificity of antibodies in antisera used for steroid immunoassay and the selective blocking of less specific antibodies, including a new method for the measurement of immunoassay specificity.

Practical aspects of the measurement of the specificity of immunoassay are reviewed. Antibody heterogeneity in an antiserum makes a pragmatic rather than a theoretical approach necessary. A new method for the measurement of immunoassay specificity is described. This method is based on the errors caused by the cross-reacting antigens and is directly relevant to the validity of results obtained by immunoassay methods. The effect of selectively blocking the least specific antibodies in antisera raised against steroid haptens is tested. The practical consequences of these considerations are tested using steroid radioimmunoassay and enzyme-immunoassay.

Androgens↗
PubMed1979

Luminescence immunoassay (LIA): a solid-phase immunoassay monitored by chemiluminescence.

A luminescence immunoassay (LIA) has been developed utilizing the chemiluminescent luminol reaction with heme as catalyst. Rabbit antibody against human serum albumin was quantitated in antigen coated plastic tubes using commercially available goat anti-rabbit IgG conjugated to horseradish peroxidase which was the source of heme. The measurable range of antibody is considerably wider by LIA than by enzyme immunoassays. The time to develop and measure activity is short and constant which makes LIA suitable for automation. In its present form, LIA is slightly less sensitive but has better day-to-day reproducibility than corresponding enzymes immunoassays.

Immunoassay↗
PubMed1976

Specificity of immunoassays. I. Effect of plasma proteins on the specificity of steroid immunoassay.

The theory of the measurement of the specificity of antigen binding by antibodies is reviewed. The specificity of steroid immunoassay has been investigated using a new solid phase radioimmunoassay system. The effects of plasma proteins on the specificity of the immunoassays have been tested. The methods used and the conclusions reached have been tested with homogeneous antibodies in order to simplify the interpretation of the results obtained. A new criterium of antibody homogeneity is proposed. The importance of correcting all results accurately for "non-specific binding" is emphasised. A new method for plotting radioimmunoassay standard curves is presented.

Aldosterone↗
PubMed1976

Enzyme-linked immunoassay: conjugation of the Fab' fragment of rabbit IgG with beta-D-galactosidase from E. coli and its use for immunoassay.

1. A method for the conjugation of the Fab' fragment of rabbit IgG with beta-D-galactosidase from Escherichia coli is described. The method consists of two main steps: treatment of the Fab' fragments containing sulfhydryl groups with excess N,N'-o-phenylenedimaleimide, to introduce maleimide residues into the fragments, and then incubation of the dimaleimide-treated Fab' fragments with beta-D-galactosidase, which also contains sulfhydryl groups, to form the rabbit Fab'-beta-D-galactosidase complex. More than 90% of the enzyme used can be converted to the Fab'-enzyme complex, and the complex is readily separated from free Fab' fragments by chromatography on a Sepharose 6B column. 2. The application of the rabbit Fab'-beta-D-galactosidase complex for immunoassay of macromolecular antigens is shown by measuring human IgG by the sandwich method. The rabbit (anti-human IgG) IgG-coupled Sepharose 4B is incubated with human IgG and then with the rabbit (anti-human IgG) Fab'-enzyme complex, and the enzyme activity bound to the Sepharose is measured. In this way it is possible to determine as little as 0.3 fmoles of human IgG.

Animals↗
PubMed1979

Specificity of immunoassays. III. Use of two antisera of differing specificities to improve the specificity of steroid immunoassay.

The specificity of immunoassays can be improved by using a second antiserum to bind substances that cross-react. Both theory and practice show that the effectiveness of the procedure is dependent of the complex interplay of the concentrations of the two antibodies, the concentrations of the three antigens involved (the labelled tracer, the antigen whose concentration is to be measured and the substance that cross reacts) and the affinities of the antibodies for these antigens. Measured cross-reactions can frequently be reduced to zero in the most important concentration ranges thereby enabling one to perform assays upon unpurified materials which other wise would not be possible. Limitations of the method are discussed.

Androstenedione↗
PubMed1979

Enzyme immunoassay for feline oncornavirus-associated cell membrane antigen (FOCMA) and detection of FOCMA in cell extract by enzyme immunoassay inhibition test.

An enzyme immunoassay (EIA) for FOCMA has been developed. The assay uses alkaline phosphatase-conjugated rabbit anti-cat IgG as the second antibody and p-nitrophenyl phosphate as the substrate for the enzyme to detect cat FOCMA antibody bound to the target cells. In comparison with the indirect immunofluorescence (IIF) test, which was originally used for FOCMA assay, our results showed a good correlation between the two methods. The EIA gives a more objective measure of FOCMA reactivity than does IIF. FOCMA was successfully extracted from FOCMA-positive cell membranes by 0.5% Triton X-100 and further fractionated by ammonium sulfate. The FOCMA activity was assayed by IIF and EIA inhibition test. Most of the FOCMA activity was found in the fractions precipitated by 30% and 50% ammonium sulfate saturation.

Animals↗
PubMed1979

Steric hindrance enzyme immunoassay (SHEIA); a novel method in enzyme immunoassay.

We have developed a new method for separation of antibody bound and unbound enzyme conjugates. The technique as applied to the assay of choriomammotropin involves the use of beta-D-galactosylamine bound to agarose to separate the unbound choriomammotropin-beta-galactosidase conjugates for antibody bound conjugates. When beta-galactosidase was conjugated with choriomammotropin using the N-hydroxy-succinamide ester of m-maleimidobenzoic acid the affinity of the enzyme conjugate to beta-D-galactosylamine attached to agarose diminished markedly following incubation with antibody. In a typical enzyme immunoassay of choriomammotropin, 5 microliter of swelled affinity gel per tube was required to precipitate unbound enzyme following one hour gentle shaking at room temperature. Choriomammotropin antibody was used at titer of 1:1,000. The standard curve for the assay was adjusted to cover a range of 0-10 mg/l with maximum sensitivity between 1-4 mg/l.

Animals↗
PubMed1992

Monoclonal antibody-based enzyme-linked immunoassays for the measurement of palytoxin in biological samples.

Mouse monoclonal and rabbit polyclonal antibodies were produced against conjugates of keyhole limpet hemocyanin and chemically defined palytoxin haptens. Palytoxin haptens were produced by derivatization of the primary amino group with sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate or succinimidyl 3-(2-pyridyldithio)propionate. Selected antibodies were used to develop five palytoxin-specific enzyme-linked immunoassay formats for the quantitation of palytoxin in biological matrices, including crude extracts of Palythoa tuberculosa. The formats developed include an indirect competitive inhibition enzyme-linked immunoassay, two types of direct competitive inhibition enzyme-linked immunoassays, and both indirect and direct sandwich enzyme-linked immunosorbent assays. The sandwich enzyme-linked immunosorbent assays are capable of detecting as little as 10 pg palytoxin per test, but may be subject to matrix interference. The direct competitive inhibition enzyme-linked immunoassays detect as little as 30 pg palytoxin per test with a total assay time of only 4 hr. The enzyme-linked immunoassays do not cross-react with the other marine toxins tested, but do cross-react with certain non-toxic, treated preparations of palytoxin. The enzyme-linked immunoassays were used to quantitate palytoxin in P. tuberculosa extracts and to monitor toxin isolation. These enzyme-linked immunoassay systems can substitute for the mouse bioassay of palytoxin, providing a rapid, sensitive, and accurate means of toxin detection.

Acrylamides↗
PubMedDate not supplied

Enzyme immunoassay for cortisol in serum using cortisol 21-amine.

Cortisol 21-amine (21-amino-11beta,17-dihydroxy-4-pregnene-3,20-dione) was prepared and an enzyme immunoassay for cortisol in serum was established using cortisol 21-amine conjugated with alkaline phosphatase. The minimal amount of cortisol detected was 1ng/tube and the measurable range was from 1 to 80 microgram/d1, using 10 mu 1 of serum sample. This enzyme immunoassay satisfied the standard criteria of dilution, accuracy and precision. The values correlated well with those obtained by radioimmunoassay. This enzyme immunoassay is applicable to the routine determination of serum cortisol in any clinical laboratory. Cortisol 21-amine was found to be a useful derivative for preparing cortisol-enzyme conjugate in enzyme immunoassay.

Amines↗
PubMed1979

Enzyme immunoassay for detecting Brucella antibodies in cow's milk.

An enzyme immunoassay (EIA) was developed for detecting Brucella antibodies in milk of cows infected with Brucella abortus. The enzyme immunoassay using heat-killed cells of B. abortus strain 19 was of comparable sensitivity to the Brucella ring test in detecting antibodies in milk of a reference positive control cow experimentally infected with B. abortus strain 2308 and in milk of 16 naturally infected cows from which B. abortus was isolated. No detectable enzyme immunoassay reactions were present in milk of 11 noninfected controls. The enzyme immunoassay is a procedure which can be readily automated so that screening tests for brucellosis could be conducted at a reference laboratory where uniform conditions can be maintained.

Animals↗
PubMed1978

Quantitative fluorescent immunoassay of antibodies to, and surface antigens of, Actinomyces viscosus.

Optimal conditions for a fluorescence immunoassay of antibodies to, and surface antigens of, Actinomyces viscosus ATCC 19246 are described. In the standard fluorescence immunoassay, 10(8) colony-forming units of A. viscosus reacted with an antibody preparation, were washed, and then were treated with an excess of fluorescein-conjugated goat anti-rabbit immunoglobulin G. After another set of washes, fluorescence was determined in a spectofluorometer; in most cases excitation was at 485 nm, with emission measured at 525 nm. These conditions minimized interference from light scatter and stray light. Under appropriate conditions, antibodies to A. viscosus could be readily determined, with the fluorescence of the specific antibody-treated cells more than five times the fluorescence of controls treated with normal rabbit serum. Organisms coated with specific antibody could be detected at levels approaching 10(5) colony-forming units per ml. The standard fluorescence immunoassay procedure was readily adapted to the measurement of either particulate or soluble surface antigens of A. viscosus by competition of the antigen with a fixed amount of antibody in the standard assay system; the competition resulted in an antigen dose-dependent inhibition of fluorescence. The fluorescent immunoassay system thus appears to be a general one that could be applied to other microbial systems as well.

Actinomyces↗
PubMed1979

Substrate-labeled fluorescent immunoassay for phenytoin in human serum.

A homogeneous substrate-labeled fluorescent immunoassay has been applied to the measurement of phenytoin concentrations in human serum. We coupled a fluorogenic enzyme substrate, galactosyl-umbelliferone, covalently to a derivative of phenytoin. Under assay conditions, this drug-substrate conjugate was nonfluorescent but became fluorescent upon hydrolysis catalyzed by bacterial beta-galactosidase. When antibody to phenytoin is bound to the drug-substrate conjugate, it is inactive as an enzyme substrate. Addition of phenytoin to competitive-binding reactions relieves the inactivation, and the resulting fluorescence is proportional to the phenytoin concentration. We validated the fluorescent immunoassay by comparing values for phenytoin obtained with this technique to those obtained by gas chromatography and by enzyme immunoassay (EMIT). All three methods correlated well. The major metabolite of phenytoin, 5-(p-hydroxyphenyl)-5-phenylhydantoin, and other drugs at concentrations expected in serum had no effect on the assay. The fluorescent immunoassay is rapid and simple to perform and requires only 2 microL of serum sample per test.

Chromatography, Gas↗
PubMed1975

Fibrinogen-fibrin degradation products: hemagglutination inhibition immunoassay in plasma.

Immunoassay for fibrinogen and/or fibrin degradation products (FDP) is generally in the clot and hence assay of serum may not reveal the true concentration of FDP in blood. We have developed a hemagglutination inhibition immunoassay for FDP in human plasma. D fragment appears to possess an antigenic determinant, called D-neoantigen, not found in native fibrinogen. Rabbit antiserum produced against D fragment was absorbed with immunosorbent columns coupled with fibrinogen and normal human serum, respectively, so that it contained only those antibodies directed against the neoantigenic determinant of D fragment. In this immunoassay, sheep erythrocytes (SRBC) were stabilized with glutaraldehyde and subsequently sensitized with D fragment by means of tannic acid. Hemagglutination of absorbed anti-D-neoantigen serum against SRBC sensitized with D fragment was titered to be 1:256. The hemagglutination was inhibited by D fragment but not by fibrinogen; the sensitivity of detecting D fragment was 8 mug/ml. Human plasma from normal subjects did not inhibit. This appears to be the first report of a hemagglutination inhibition immunoassay for FDP in plasma.

Animals↗
PubMed1977

Enzyme immunoassay of serum thyroxine with the "Autochemist" multichannel analyzer.

We have modified the homogeneous enzyme immunoassay of thyroxine and have adapted it to the AutoChemist. Both enzyme immunoassay and radioimmunnoassay procedures were performed on 2016 patients' sera on which thyroxine determinations had been ordered. We find the automated enzyme immunoassay to be precise and accurate, and for the first time it permits total automation of an immunoassay on a multichannel biochemical-profiling instrument. More importantly, it represents the first time that a thyroid-function test has been included in a multitest biochemical screening profile performed on a single automated instrument.

Autoanalysis↗
PubMed1979

A systems approach to fluorescent immunoassay: general principles and representative applications.

We have developed an automated system for the immunoassay of subnanogram quantities of clinically interesting compounds by molecular fluorescence. The system includes all the necessary reagents and an automated fluorometer. The microprocessor-based instrument consists of a measurement and data-processing module and an automated sampling unit. With use of 10 pmol/L amounts of fluorescent dyes such as fluorescein, measurements with precision and accuracy of 1--3% are attained. In a competitive-binding fluorescence immunoassay, antigen labeled with a fluorescent dye competes with antigen in the sample or standard for a limited amount of antibody immobilized on a polyacrylamide bead 2--5 micrometers in diameter. After separating antibody-bound from free tracer, we measure the amount of fluorescence bound to the beads. In representative example assays, correlation of fluorescence immunoassay (y) with a reference radioimmunoassay (x) of thyroxine was y = 1.01x + 13 nmol/L, r = 0.98. Correlation of fluorescence immunoassay (y) with a reference radioimmunoassay (x) of triiodothyronine was y = 0.99x + 0.004 nmol/L, r = 0.96.

Autoanalysis↗
PubMed1979

[Determination of thyroxine in serum by a heterogeneous enzyme immunoassay: results of a joint trial].

This paper describes the evaluation of a heterologous enzyme immunoassay for the determination of total thyroxine in serum by a group of seven clinical chemical laboratories. The test follows the principles of the enzyme linked immunosorbent assay (ELISA) and uses peroxidase as a marker. The evaluation of analytical reliability yielded the following results within the analytical range from 39 unto 322 nmol/l: 1. Within-batch precision ranged from 3.1 unto 10.4% (coefficient of variation) with single analyses. 2. Between-batch precision ranged from 3.7 unto 20.4% with single analyses. 3. Between-laboratories precision ranged from 5.4 unto 6.8%. 4. Pure thyroxine, added to serum or thyroxine-free serum, gave recoveries between 93 and 120%. 5. Analysis of control sera gave results essentially comparable to the assigned values based upon radioimmunoassays. 6. Analysis of 288 clinical sera gave slightly higher results by the enzyme immunoassay than by the analogous radioimmunoassay from the same manufacturer. 7. Comparison with other methods of analysis (radioimmunoassays, competitive protein ligand assays, hormonal iodine assay) yielded partly comparable, partly higher results. 8. Comparison with the homogenous enzyme immunoassay (EMIT) led to comparable results. 9. Interference due to hyperlipemia or hemolysis was not observed. 10. There might be an interference in hyperbilirubinaemic sera, due to an as yet unknown factor. With respect to practicability the ELISA-test compares favourably with the analogous solid phase radioimmunoassay. The main differences are the absence of radioactive material and a longer shelf-live of reagents. Following the manual procedure the time taken to perform the enzyme immunoassay is slightly longer than for the analogous radioimmunoassay.

Enzyme-Linked Immunosorbent Assay↗