Use Of Enzyme Immunoassay Research Articles

Use of enzyme immunoassay for the rapid diagnosis of Chlamydia trachomatis endocervical infection in female adolescents

Enzyme immunoassay (EIA) has been proposed as an alternative to tissue culture for the detection of Chlamydia trachomatis in cervical specimens. The diagnostic efficacy of EIA was compared to tissue culture in 113 teenaged females attending an adolescent reproductive health program. Infection was diagnosed by tissue culture in 16% of subjects. Compared with tissue culture, EIA demonstrated a sensitivity of 100%, specificity of 88%, positive predictive value of 62%, and a negative predictive value of 100%. These data indicate that EIA is an acceptable alternative to tissue culture when screening for C. trachomatis endocervical infection in adolescent females.

Peroxidase-labelled monoclonal antibodies for use in enzyme immunoassay.

Desirable characteristics of enzyme-antibody conjugates for use in enzyme immunoassay are labelling uniformity, permanent availability and stability. The use of monoclonal antibodies (McABS) for preparation of enzyme conjugates, in place of polyclonal antibodies, ensures labelling uniformity and permanent availability. The problem of stability still exists. Monoclonal antibody-horseradish peroxidase (McAB-HRPO) conjugates produced in our laboratory showed variable stability. After extensive testing of McAB-HRPO conjugates it became obvious that sodium borohydride, used as a reducing agent, did not result in the production of stable conjugates without enzyme pretreatment with fluorodinitrobenzene (FDNB). Ascorbic acid or ethanolamine used as the reducing agent, resulted in McAB-HRPO conjugates which were stable for periods of ten months or more when stored filter sterilized at 4 degrees C.

Decreased level of nerve growth factor (NGF) and its messenger RNA in the aged rat brain

Trophic factors such as nerve growth factor (NGF) are thought to support survival, differentiation and maintenance of neurons. Recent results indicate that NGF produced in cortical and hippocampal areas is required for the function of cholinergic neurons in the basal forebrain. With the use of enzyme immunoassay and RNA blot hybridization we studied the NGF protein and NGF mRNA, respectively, in regions of the brain innervated by basal forebrain cholinergic neurons in adult and aged rats. Levels of NGF protein were decreased by 40% in hippocampus of aged (28 months) Fischer 344 rats compared with adults (6 months), whereas no alterations were observed in cerebral cortex. Moreover, a reduction by 50% in the NGF mRNA was found in samples of the aged forebrain (cerebral cortex, hippocampus, basal forebrain and hypothalamus) compared to the adult. NGF deficiencies may thus account for the loss of cholinergic neurons in the basal forebrain generally found to accompany normal aging and resulting in altered cognitive functions.

Use of enzyme immunoassay and nucleic acid hybridization for detecting Sindbis virus in infected mosquitoes

Aedes aegypti mosquitoes were inoculated intrathoracically with prototype Sindbis virus, held at 26.7°C for from 0–95 h and placed at −70°C. Individual mosquitoes were tested for virus by plaque assay in Vero cells, for viral RNA by nucleic acid hybridization using a cloned cDNA probe, and for viral protein by enzyme-linked immunosorbent assay. Virus was detected by plaque assay as early as 8 h after infection. Sindbis virus RNA was detected by nucleic acid hybridization 18 h after infection and by enzyme-linked immunosorbent assay 10 h after infection. The results of these comparisons suggest that both nucleic acid hybridization and enzyme-linked immunosorbent assay are applicable to direct detection of Sindbis virus in mosquitoes containing virus at levels usually found during arbovirus epidemics.

Evaluation of Enzyme Immunoassay as a Rapid Screening Test for Histoplasmosis and Blastomycosis

This study evaluates the usefulness of enzyme immunoassay (EIA) as a screening test for serum antibodies to Histoplasma capsulatum and Blastomycoses dermatitidis and compares the results using this assay with those using complement fixation (CF), immunodiffusion (ID), and radioimmunoassay (RIA), in 12 patients with active histoplasmosis, 12 with active blastomycosis, 28 with other acute or chronic lung diseases and 25 healthy blood donors from an endemic area; EIA was as sensitive as RIA and more sensitive than CF and ID in the 24 patients with active fungal infections. The specificity of EIA was equal to that of the other serologic tests. We conclude that EIA is a useful screening test for serum antibodies to H. capsulatum and B. dermatitidis that avoids some of the problems associated with other sensitive assays such as RIA. A negative EIA result is evidence against invasive histoplasmosis or blastomycosis and suggests an alternative diagnosis.

Long-term storage of peroxidase-labelled immunoglobulins for use in enzyme immunoassay

Optimal conditions for long-term storage of peroxidase-labelled immunoglubulins for use in enzyme immunoassays have been investigated with particular emphasis on the preservation of the immunological and enzyme activity. Immunoglobulin G preparations from different animal species were purified according to conventional methods and labelled with horseradish peroxidase. Conjugates were stored at different temperatures either in liquid form with 50% glycerol, after lyophilisation or as ammonium sulphate precipitates. In addition, the conjugates were also frozen and stated at −40°C. The immunological and enzymatic activity of the conjugates was evaluated at regular intervals throughout storage. The best results ere obtained with conjugates stored as ammonium sulphate precipitates at 4°C. Under these conditions, the complexes retained after 2 years of storage 92 and 91% of their enzymatic and immunological activity respectively and gave excellent reproducibility in ELISA. The conjugates exhibited a very low degree of self-aggregation. The ammonium sulphate did not interfere in the ELISA. Storage at elevated temperatures resulted in a greater decrease in enzymatic than immunological activity but the results obtained with the ammonium sulphate precipitates were clearly superior to those obtained with alternative storage procedures. The estimated half life of the whole enzymatic-immunological activity of conjugates at 4°C was 9 years. The stability of both activities at 25°C was also reasonably good for short periods. Conjugates stored for 40 days at 37°C lost 60% enzyme activity but retained unaltered their immunological activity and gave rise to high blanks in the ELISA, due to auto-aggregation.

Thermostable muscle antigens suitable for use in enzyme immunoassays for identification of meat from various species

AbstractA method for the extraction of thermostable muscle antigens for use in enzyme immunoassays is described. The method yields antigens devoid of contaminating proteins which reduce the adsorption of the antigens on to the plate. The effect of such proteins is stimulated by the addition of gelatin. Gelatin (5 mg ml−1) results in 100% inhibition of the antigen adsorption on to the plate.

Enzyme immunoassay of a plant phospholipid transfer protein

An antibody against the phospholipid transfer protein from maize seedlings has been raised in rabbits and purified by ion-exchange or affinity Chromatography. When checked by double-immunodiffusion, the antibody gave a single precipitin line with the pure protein or a crude extract. An immunoinhibition of the transfer activity was observed when the antibody was incubated with phospholipid transfer protein. This specific antibody was used to develop an enzyme immunoassay. The enzyme immunoassay measured transfer protein levels in the range 1–20 ng. The enzyme immunoassay was used to detect the transfer protein after high-performance liquid chromatography of a partially purified extract and to determine the amount of the transfer protein in crude extracts. The values found (2% of the postmicrosomal proteins or 4.1% of the total proteins precipitated by ammonium sulfate) indicate that the maize cytosol is rich in phospholipid transfer protein. This work, which describes for the first time in plants the use of enzyme immunoassay to quantify phospholipid transfer proteins, opens new perspectives in the study of the regulation of their biosynthesis.

Enzyme labeling of steroids by the N-succinimidyl ester method. Preparation of glucose oxidase-labeled antigen for use in enzyme immunoassay.

Enzyme labeling of a steroid with alkaline phosphatase by the N-succinimidyl ester method was investigated. The activated ester of 4-hydroxytestosterone 4-hemiglutarate was treated with alkaline phosphatase to give a labeled antigen. Various molar ratios of steroid to enzyme, ranging from 5 to 200, were employed. The loss of enzymic activity was less than 20% under the coupling conditions used. Satisfactory immunoreactivities with an anti-testosterone antiserum in the enzyme immunoassay procedure were obtained with the labeled antigens prepared at molar ratios higher than 20. The effect of steroid/enzyme molar ratio in the labeling on the sensitivity of the testosterone assay was then examined. It was found that the sensitivity of the assay is significantly influenced by the molar ratio, and a higher ratio results in a decrease in assay is significantly influenced by the molar ratio, and a higher ratio results in a decrease in assay sensitivity. A doserespared at a molar ratio of 30. The active ester method proved to be useful for the preparation of alkaline phosphatase-labeled antigens as well as for β-galactosidase and horseradish peroxidase labelings, because of its simplicity and excellent reproducibility.

Prostate-Specific Antigen in Prostate Cancer

Prostate-specific antigen (PA) has been evaluated clinically as a tumor marker of prostate cancer with the use of enzyme immunoassay (EIA). For serodetection of prostate cancer, PA was assayed in a total of 1,109 sera. From mean +/- 3 S.D. of normal controls, upper cut-off values in males were decided as 2.5 and 1.2 ng/ml in Americans and Japanese, respectively. Serum PA values in prostate cancer patients were positive in 78% of Americans and 62% of Japanese. However, in benign prostatic hypertrophy (BPH) cases, a high false positive rate of 41% was observed in Americans. Simultaneous assays of serum PA and PAP showed high sensitivity and specificity in the detection of prostate cancer. This antigen could be used, as well as PAP, for monitoring prostate cancer patients. Furthermore, serum PA levels prior to treatment may express to some degree the malignant potential of the cancer. These results suggest that PA may be useful as a tumor marker of prostate cancer.

Use of enzyme immunoassay in a serological survey of leptospirosis in sheep.

A total of 731 serums, all from Merino rams from 20 farms, were tested for antibodies against Leptospira interrogans serovars hardjo, pomona and tarassovi using the microscopic agglutination test (MAT). The enzyme immunoassay (EIA) technique was used to test all serums for IgM and IgG antibodies to serovar hardjo. In the MAT, reactions to serovar hardjo were most common with 236 rams (32.3%) reacting at 1/100 or greater. Only 1.9% of serums reacted against serovar tarassovi and 1.1% against serovar pomona. The percentage of sheep with positive MAT reactions to serovar hardjo ranged from 0 0 to 94.9 between farms. When using EIA, 46 (6.2%) of the serums were positive for IgM antibody and 246 (33.6%) were positive for IgG antibody. Correlation of the EIA for detection of IgG antibody with the MAT was good. The EIA detection of IgG antibody was considered to be a good alternative test to the MAT for epidemiological studies in sheep.

Enzyme-Linked Immunoassays for the Detection of Microbial Antigens and Their Antibodies

Publisher SummaryAntibodies could be labeled with enzymes for use in histochemical staining procedures by enzyme-immunoassay (EIA). The use of EIA is an extension of previously used serological tests, using enzyme-labeled antibody or antigen to determine antibody content. Direct detection of antigen by EIA represents a more dramatic departure from previous methods based on culture. Also, the method has enabled detection of infectious agents that are difficult to cultivate, such as hepatitis A virus and rotavirus, or agents that cannot be cultivated, such as hepatitis B. The use of EIA tests for detection of microbial antigens provides an alternative to culture as a means for direct identification of a specific microbial agent. It also provides a means to detect microbial agents which have not been successfully propagated. The detection of circulating antigen or detection of antigen in other body fluids by EIA is more difficult than detection of antibody because of the sensitivity required, and because of interfering substances in specimens such as feces and respiratory secretions. For this reason, very few antigen detection assays have the sensitivity and specificity required to be used as a primary diagnostic test. The number of tests that have been developed, however, is impressive and because of the possibilities for rapid, specific diagnosis, the interest in antigen detection by EIA remains high.

Enzyme labeling of steroids by the N-succinimidyl ester method. Preparation of horseradish peroxidase-labeled antigen for use in enzyme immunoassay.

Enzyme labeling of a steroid with horseradish peroxidase by the N-succinimidyl ester method has been investigated in comparison with that with β-galactosidase. The reaction of the activated ester of 4-hydroxytestosterone 4-hemiglutarate with horseradish peroxidase provided a labeled antigen showing high immunoreactivity with an anti-testosterone antiserum in the enzyme immunoassay procedure. The effect of steroid/enzyme molar ratio, ranging from 2 to 60, in the labeling on the assay sensitivity was then examined. It was found that, in contrast to the case of β-galactosidase, the sensitivity of the assay using horseradish peroxidase-labeled antigen is not significantly influenced by the molar ratio. Dose-response curves with high sensitivities could be obtained by the use of these labeled antigens at an appropriate dilution of the antiserum. The active ester method proved to be useful for the preparation of enzymelabeled antigens because of its simplicity and excellent reproducibility.

Enzyme labeling of steroids by the N-succinimidyl ester method. Preparation of alkaline phosphatase-labeled antigen for use in enzyme immunoassay.

Enzyme labeling of a steroid with alkaline phosphatase by the N-succinimidyl ester method was investigated. The activated ester of 4-hydroxytestosterone 4-hemiglutarate was treated with alkaline phosphatase to give a labeled antigen. Various molar ratios of steroid to enzyme, ranging from 5 to 200, were employed. The loss of enzymic activity was less than 20% under the coupling conditions used. Satisfactory immunoreactivities with an anti-testosterone antiserum in the enzyme immunoassay procedure were obtained with the labeled antigens prepared at molar ratios higher than 20. The effect of steroid/enzyme molar ratio in the labeling on the sensitivity of the testosterone assay was then examined. It was found that the sensitivity of the assay is significantly influenced by the molar ratio, and a higher ratio results in a decrease in assay is significantly influenced by the molar ratio, and a higher ratio results in a decrease in assay sensitivity. A doserespared at a molar ratio of 30. The active ester method proved to be useful for the preparation of alkaline phosphatase-labeled antigens as well as for β-galactosidase and horseradish peroxidase labelings, because of its simplicity and excellent reproducibility.

The use of enzyme immunoassay in snake venom research: Theakston, R. D. G. (Liverpool School of Tropical Medicine, Pembroke Place, Liverpool L3 5QA, U.K.)

The use of enzyme immunoassay in snake venom research: Theakston, R. D. G. (Liverpool School of Tropical Medicine, Pembroke Place, Liverpool L3 5QA, U.K.)

Comparison of three conjugation procedures for the formation of tracers for use in enzyme immunoassays

One of the requirements for enzyme immunoassay is the formation of a labelled component of the assay system which can be either the ligand or the binder. Three methods for conjugating alkaline phosphate to human placental lactogen were investigated, involving water-soluble carbodiimide, glutaraldehyde, and the periodate oxidation technique. Of the 3, the periodate oxidation technique proved superior giving a conjugate with only slight changes in the Michaelis constant ( K m) and maximum velocity ( V max) which could be used for an enzyme immunoassay for human placental lactogen in human plasma.

Preparation of polyamine antibody and its use in enzyme immunoassay of spermine and spermidine with β- D-galactosidase as a label

An enzyme immunoassay for polyamines is described which uses β-galactosidase labeled spermine and antiserum raised in rabbits against spermine-bovine serum albumin synthesized by coupling spermine to mercaptosuccinylated bovine serum albumin with a bifunctional cross-linker, N-(γ-maleimidobutyryloxy)-succinimide. The lower limit of detection by this assay, which involves a double antibody technique for the separation of antibody-bound and free antigen, was 1 ng of spermine per tube. The anti-spermine serum showed 88% cross-reaction with spermidine but only 0.13% with putrescine, 0.08% with 1,3-diaminopropane, and 0.04% with cadaverine. The method has been used to measure serum polyamine levels in healthy subjects and cancer patients, in whom mean concentrations of 58.1 ng/ml and 94.8 ng/ml (as spermine), were respectively noted. This enzyme immunoassay is specific, accurate and easy to perform, and appears suitable for routine clinical use.

The preparation of puromycin antibody and its use in enzyme immunoassay for the quantification using beta-D-galactosidase as a label.

An antibody specific for puromycin (PU) was prepared by immunization of rabbits with a PU conjugate of bovine serum albumin, which was newly synthesized by coupling PU to mercaptosuccinylated bovine serum albumin via a cross-linker, N-(gamma-maleimidobutyryloxy)succinimide. Enzyme labeling of PU was performed using beta-D-galactosidase [EC 3.2.1.23] via N-(m-maleimidobenzoyloxy) succinimide. An ultrasensitive and specific enzyme immunoassay for PU was developed utilizing these reagents by a double antibody technique. The standard curve of the assay was linear in the range of 2 pg to 100 pg, and the lower limit of detection was 28.2 pm (2 pg/tube); so the enzyme immunoassay was found to be approximately 326,000 times more sensitive than a microbiological assay. Further, the enzyme immunoassay is free from interference by any purine or pyrimidine analogs, or by other drugs commonly used for the inhibition of protein synthesis. Using this assay, drug levels were easily determined in rat tissue following PU administration. Since PU is extensively available as an inhibitor of protein synthesis, the enzyme immunoassay should provide useful tool for developing biochemical and toxicity studies of PU.

An electrochemical assay system for peroxidase and peroxidase-couplers reactions based on a fluoride ion-selective electrode.

A sensitive and accurate potentiometric reaction-rate method for the assay of peroxidase (EC 1.11.1.7) and peroxidase-coupled reactions is described. The technique is based on the peroxidase-catalyzed rupture of the covalent C--F bond in certain organo-fluoro compounds in the presence of H2O2 and the measurement of liberated F- by a fluoride ion-selective electrode. In a study screening 26 organo-fluoro compounds, 12 were susceptible to C--F bond rupture. However, analytically useful reaction kinetics were observed only with 4-fluoroaniline, 5-fluor-2-methylaniline, 4-fluorophenol, 2,3,5,6-tetrafluorophenol, pentafluorophenol, and 3-fluoro-DL-tyrosine. With this assay system, peroxidase activity as low as 25 mU/L could be precisely measured within 2 min. The reaction was coupled to glucose oxidase and cholesterol oxidase for the determination of glucose and cholesterol. Glucose oxidase activity in solution (to 2 U/L) was measured to demonstrate the feasibility of the assay for oxidase enzymes. Only a few microliters of sample is required and most determinations can be completed within minutes. The assay system offers broad applications and is especially attractive for use in enzyme immunoassays.

9] Methods for the preparation of enzyme-antibody conjugates for use in enzyme immunoassay

Publisher Summary This chapter describes the methods for the preparation of enzyme–antibody conjugates for use in enzyme immunoassay. Enzyme-linked immunoadsorbent assay type assays are becoming established as the method of choice for screening for antigens derived from, and antibodies directed against, microorganisms. The effect of the cross-linking reaction on the antibody and enzyme activity can be determined by measuring both the activities before and after the conjugation reaction. Methods that have been used to measure the antibody activity include: radial immunodiffusion, reverse-rocket immunoelectrophoresis, and radioimmunoassay. The efficiency of the coupling reaction can be determined by separating the conjugated from the nonconjugated material and measuring the proportion of antibody and enzyme activity in the conjugate. The one-step glutaraldehyde method is a widely used and technically undemanding procedure and many enzyme immunoassays have been successfully developed, using labels prepared by it. Nonlabeled antibody should be removed from the conjugate, as it lowers the sensitivity of the assay. It is found that for those assays, where the enzyme activity is measured on the solid phase, removal of free enzyme from the conjugate is not essential.