Immunoenzyme analysis using paper disks

Filter paper discs have been used in the enzyme immunoassay (EIA) as solid phase instead of polystyrene plates. The use of paper discs has made it possible to achieve a multiple increase in the sensitivity of sandwich EIA, thus permitting the detection of Yersinia pestis capsular antigen at a concentration of 0.4 ng/ml. Paper discs can be used not only for the sorption of antigen and antibodies, but also for the affinity purification of preparations.     

The possibilities of using variants of immunoenzyme analysis for detecting L-form Salmonella typhi bacteria in biological fluids

The possibility of using, on principle, the enzyme immunoassay (EIA) in different modifications for the detection of S. typhi L-forms in biological fluids (blood, urine) was established. The inhibiting variant of EIA showed the highest sensitivity: 1 ng/ml. The direct sandwich variant permitted the quantitative determination of the antigen of S. typhi L-form in the widest range of 20-500 ng/ml. The indirect enzyme immunometric variant permitted the detection of S. typhi L-forms with a sensitivity of 10(5) colony-forming units per ml only in urine.     

Detection of Legionella pneumophila antibodies in the blood serum of pneumonia patients by an immunoenzyme method

The conditions of making the enzyme immunoassay (EIA) for the detection of antibodies to L. pneumophila have been optimized. The use of L. pneumophila purified serotypic antigen at a concentration of 0.25 micrograms/ml for the sensitization of polystyrene plates has been shown to increase the sensitivity and specificity of the assay. 220 patients with severe pneumonia have been examined. As revealed in this investigation, antibodies to L. pneumophila can be detected in 12.2% of cases. A high degree of correlation (94.4%) between the results of EIA and the indirect immunofluorescence test has been shown.     

An immunoenzyme method for demonstrating Coxiella burnetii antigens

Materials on the development of an enzyme immunoassay (EIA) system for the detection of the antigens of C. burnetii, the causative agent of Q rickettsiosis, are presented. The system is highly specific and effective with respect to both corpuscular antigens of phases 1 and 2 and soluble antigen (lipopolysaccharide). The sensitivity of this method varies within the range 5-100 ng/ml. The effectiveness of EIA as a quantitative (semiquantitative) control test used in the process of the production of Coxiella preparations has been demonstrated.     

Effectiveness of methods of isolating specific class-G rabbit antibodies for the immunoenzyme determination of human placental alkaline phosphatase

Four different chromatographic methods of IgG isolation from rabbit antisera to placental alkaline phosphatase (HPAP) have been compared. The antibodies were obtained by ion-exchange chromatography and affinity chromatography on protein-A-sepharose, on the sepharose with immobilized antigen. IgG samples were characterized by the content of specific antibodies to HPAP and checked in enzyme immunoassay (EIA). IgG purified on immobilized antigen were found to be the optimal both from the point of view of the specific antibodies content and EIA sensitivity, but satisfactory results could be also obtained with ion-exchange and protein-A-chromatography purified IgG. The last two isolation methods are simpler and provide 3-10 ng/ml sensitivity of HPAP detection, which is lower, as compared with the test employing affinity antibodies (1 ng/ml), but allows the detection of HPAP in serum samples.     

Development of enzyme immunoassay for the herbicide chlorsulfuron

Antibodies against the herbicide chlorsulfuron have been raised and characterized. Enzyme immunoassays (EIAs) for chlorsulfuron, involving labeled antigen or labeled antibodies, have been developed. The kinetics of antigen-antibody interactions in the EIA systems developed has been studied. Both systems exhibit equal sensitivity (1 ng/ml). The values of the coefficient of variation (CV), determined within the range of chlorsulfuron concentrations 1-100 ng/ml to be measured by the systems, are not in excess of 8%. The possibility of using glucose oxidase as a label in EIAs for chlorsulfuron has been demonstrated. Lack of cross-reactivity with a series of sulfonyl- and arylurea derivatives and triazines makes it possible to recommend the EIA systems developed for chlorsulfuron determination in the environment.     

Development of Enzyme Immunoassays for the Herbicide Chlorsulfuron

Antibodies against the herbicide chlorsulfuron have been raised and characterized. Enzyme immunoassays (EIAs) for chlorsulfuron, involving labeled antigen or labeled antibodies, have been developed. The kinetics of antigen–antibody interactions in the EIA systems developed has been studied. Both systems exhibit equal sensitivity (1 ng/ml). The values of the coefficient of variation (CV), determined within the range of chlorsulfuron concentrations to be measured by the systems (1–100 ng/ml), are not in excess of 8%. The possibility of using glucose oxidase as a label in EIAs for chlorsulfuron has been demonstrated. Lack of cross-reactivity with a series of sulfonyl-/arylurea derivatives and triazines makes it possible to recommend the EIA systems developed for chlorsulfuron determination in the environment.     

A novel recombinant multiepitope protein as a hepatitis C diagnostic intermediate of high sensitivity and specificity

A novel recombinant multiepitope protein has been designed that consists of six linear, immunodominant, and phylogenetically conserved epitopes from hepatitis C virus. Five of these antigens (core, NS3, NS4I, NS4II, and NS5) are being used in many of the third-generation kits while sixth epitope (core3g) is an additional sequence from a newly identified Indian isolate. The genes for these epitopes have been joined together to code for a single multiepitope protein that has been evaluated for its diagnostic potential for the detection of anti-HCV antibodies in human plasma. Two separate synthetic genes have been designed, both encoding the same six epitopes in a single open reading frame along with spacers having additional amino acids to function as flexible (r-HCV-F-MEP) or rigid (r-HCV-R-MEP) linkers. High-level expression of hepatitis C multiepitope protein in Escherichia coli has been achieved. The protein has been purified using a single affinity step yielding >25 mg pure protein/liter culture and used as the coating antigen in anti-HCV EIA. The use of this multiepitope protein eliminates the requirement for multiple diagnostic intermediates for the development of anti-HCV diagnostic kit. The sensitivity and specificity of the HCV multiepitope protein was evaluated by Boston Biomedica Worldwide Performance Panels, HCV Seroconversion Panels and Viral Co-infection Panels, and was found to be comparable with commercially available anti-HCV EIA kits. This analysis indicated its unequivocal performance as capture antigen in anti-HCV EIA. The high epitope density, careful choice of epitopes and use of E. coli system for expression, coupled with simple purification protocol provides the potential for the development of an inexpensive diagnostic test with high degree of sensitivity and specificity.     

Highly sensitive enzyme immunoassay for beta-nerve growth factor (NGF): a tool for measurement of NGF level in rat serum

A sensitive two-site enzyme immunoassay (EIA) system was established for mouse beta nerve growth factor (NGF) isolated from mouse submaxillary gland. Our EIA system is based on the sandwiching of antigen between anti-mouse beta NGF antibody IgG coated on a polystyrene plate and biotinylated anti-mouse beta NGF antibody IgG. The bound antibody complex was quantified with streptavidin linked-beta-D-galactosidase (beta-D-galactosidase, EC 3.2.1.23). With this system NGF concentrations as low as 0.02 pg/well (corresponding to 8 x 10(-19) mol) could be measured reproducibly. The sensitivity of this EIA system permitted the quantification of endogenous immunoreactive beta NGF in rat serum. The mean level in serum of male rats (153.2 pg/ml) was found to be almost the same as that of female rats (127.6 pg/ml).     

The use of an immunoenzyme method for the rapid diagnosis of legionellosis

An enzyme immunoassay (EIA) system for the detection of L. pneumophila antigen in clinical material (sputum, urine, bronchial washings) has been developed. The use of EIA permits the detection of L. pneumophila antigen in the urine of 75-80% of patients during the first week of the disease. The specificity and sensitivity of EIA makes it possible to recommend this method for the rapid diagnosis of L. pneumophila infection.     

Preparation and characterization of monoclonal antibodies to the cholera toxin

Monoclonal antibodies to cholera toxin were obtained. They do not cross-react with the termolabile toxin (LT) of Escherichia coli, ricin, diphtherial toxin, staphylococcus enterotoxins of SEA, SEB, SEI, SEG, or the lethal factor and protective antigen of the anthrax toxin. Pairs of antibodies for the quantitative measurement of the cholera toxin in sandwich enzyme immunoassay (EIA) were selected. The detection limit of the toxin is 0.2 ng/ml for plate EIA and 0.44 ng/ml for microchip EIA. The presence of milk, broth, or surface water in the toxin samples does not reduce the sensitivity of EIA.     

Multicenter comparison of rapid lateral flow stool antigen immunoassay and stool antigen enzyme immunoassay for the diagnosis of Helicobacter pylori infection in children

BACKGROUND AND AIM: The stool antigen enzyme immunoassay (EIA) methods are widely used for diagnosing Helicobacter pylori infection. Recently, a novel, rapid stool antigen test, the lateral flow immunoassay (LFI) method, has been developed. The primary purpose of this study was to compare the EIA method with the LFI method for the diagnosis of H. pylori infection in children. MATERIALS AND METHODS: Stool specimens from children being evaluated for H. pylori infection were also examined using the LFI (ImmunoCard STAT! HpSA) and EIA methods (Premier Platinum HpSA). The sensitivity, specificity and accuracy of the test were based on the 13C-labeled urea breath test. RESULTS: One hundred and eighty-two children and adolescents, 3-17 years of age (mean 9.2 years), were studied. In addition, 29 patients who received eradication therapy were re-evaluated 2 or 3 months post-treatment. The 13C-labeled urea breath test was positive in 64 patients (35.2%). The sensitivity, specificity and accuracy of the LFI method were 90.6% (95% CI = 80.7-96.5%), 95.8% (92.1-99.4%), and 94.0% (90.5-97.4%), respectively and for the EIA method, sensitivity, specificity and accuracy were 96.8% (95% CI, 89.0-99.6%) and 99.2% (97.5-100%), and 98.3% (96.5-100%), respectively. There were no significant differences in results among the age groups 3-5, 6-10 and 11-17 years. As for the assessment of H. pylori eradication, the results of the LFI and EIA methods agreed with those of 13C-urea breath test in 27/29 and 29/29 patients, respectively. CONCLUSIONS: The LFI stool antigen method showed a good sensitivity, specificity and accuracy for diagnosing H. pylori infection in children. This novel method may be useful in clinical practice as an office-based test because it is rapid, reliable and easy to perform.     

The use of microdot immunoenzyme analysis with visual detection for the determination of tularemia antibodies

The possibility of using the micropoint enzyme immunoassay (EIA) on a nitrocellulose membrane with the visual evaluation of results for the detection of tularemia IgG antibodies in hamadryas baboons at the postvaccinal period has been studied. The sensitivity of this assay has been compared with that of the passive hemagglutination (PHA) test, the microagglutination (MA) test and EIA with the spectrophotometric evaluation of results in plates. As shown in this study, EIA in the above-mentioned modification can be successfully used for the detection of tularemia antibodies in the blood serum. The sensitivity of micropoint EIA has proved to be not inferior to that of EIA in plates, while exceeding the sensitivity of the PHA test 10- to 20-fold and the sensitivity of the MA test 10- to 1,000-fold. This method is simple, reliable, highly sensitive, economic and requires no special equipment, which makes it highly promising for the diagnosis of tularemia and the evaluation of humoral immunity at the postvaccinal period.     

Annotations on the measurement of liver ferritin, especially in the rat.

A radial immunodiffusion method (RID) to measure liver ferritin protein has been reevaluated. The RID has been compared with an electroimmunoassay (EIA) and with an enzyme-linked immunoassay (ELISA). The RID and ELISA provided similar results; the RID measured more ferritin protein in liver homogenates as compared with the EIA. After incubation of liver homogenate with deoxycholate (30 mg/ml) a slight increase of the ferritin concentration is detectable.     

Immunoenzymatic detection of specific brain antigens as a criterion of the permeation of blood-brain barrier in rats with acute cerebral ischemia

EIA detection system for the measurement of alpha 2 M globulin and GFAP antigen has been developed. The limit of the sensibility was only 1 ng/ml for alpha 2M and 0.8 ng/ml for GFAP. The system was used for the studies of the penetration through the blood-brain barrier in rats with experimental acute brain ischemia. The measurement of alpha 2M and GFAP antigens by EIA technique 16-20 hours after the occlusion of the carotid artery has revealed disturbances in the blood-brain barrier permeability for specific brain proteins. The method is recommended for indirect evaluation of the blood-brain barrier functional disorders.     

Functional properties of proteins immobilized on albumin microspheres

Bovine serum albumin (BSA) microspheres with an average diameter of 12.5 micron were prepared by crosslinking of BSA molecules with glutaraldehyde in the presence of polymethylmethacrylate dissolved in chloroform-toluene. Trypsin and anti-human IgG antibody were immobilized onto their surfaces by the glutaraldehyde-activation method. The catalytic activity and storage stability of the immobilized trypsin were satisfactorily high. The enzyme immunoassay (EIA) method using BSA-microspheres as a solid phase has a high sensitivity (the minimum concentration of detectable antigen in the sample: 0.2 ng/ml) and a wide concentration range (final concentration 0.027-3000 ng/ml) for the detection of human IgG.     

Development and validation of an enzyme immunoassay for progesterone in bovine milk or blood plasma

We have developed and validated a sensitive, simple and direct (i.e. without extraction) enzyme immunoassay (EIA) system for the measurement of progesterone in bovine milk and blood plasma. Progesterone (P) has been analysed by a microtiterplate EIA, employing polyclonal antibodies against P-7α-carboxyethylthioether-BSA as the antigen. The enzyme used as a label is horseradish peroxidase (HRP) and the chromogen is tetramethylbenzidine (TMB). Sensitivity of the EIA has been greatly improved by introduction of a heterologous tracer, in which progesterone is coupled to HRP at the 6β position. Compared to the radioimmunoassay (RIA) in which the same antiserum has been used, the sensitivity is 20 times greater. The detection limit of the assay is 0.4 pg per well. The working range of the standard curve is 0–20 pg per well (i.e. 0–40 ng per ml), and 50% reduction of the initial binding is obtained with 2.5-5.0 pg. Results can be obtained either by spectrophotometric measurement at 450 nm, or by naked eye. Total time needed for the assay of 40 replicate samples is approximately 3 h. Comparison of the EIA system with a previously validated RIA system gave a regression line EIA = 0.85 RIA + 2.11 (r = 0.93, n = 400 milk samples). Application of the milk-progesterone EIA to pregnancy testing (n = 66) gave an accuracy of 79.6% for positive diagnoses and 100% for negative diagnoses.     

Enzyme immunoassay system for detection of staphylococcal enterotoxin,type C

An enzyme immunoassay (EIA) system for detection of staphylococcal enterotoxin, type C, has been developed. The sensitivity of the system is 1 ng/ml. The optimum EIA parameters have been worked out. The absence of false positive results with heterologous toxins confirms the specificity of the assay system. The possibility of the detection of staphylococcal enterotoxin, type C, in staphylococci isolated from different sources has been shown.     

Immunobiological properties of monoclonal antibodies to Mycoplasma hominis antigens

Approaches to obtaining stable mouse hybridomas synthesizing monoclonal antibodies (McAb) to M. hominis key antigens were developed. 4 clones capable of the stable synthesis of McAb of different IgG classes were obtained. Clones A3/2 and A5/D produced antibodies to the thermostable determinant to with a mol. wt. of 80-120 kD, sensitive to sodium periodate and resistant to potassium proteinase. Clone H9/B2 synthesized McAb which interacted with potassium proteinase-sensitive M. hominis thermolabile determinant with a mol. wt. of 80 kD. McAb of clone A3/2, labeled with fluorescein isothiocyanate and horse-radish peroxidase, specifically reacted with M. hominis antigens in the immunofluorescence test and the immunoenzyme assay (EIA). The sensitivity of EIA was 0.25 ng/ml of antigen protein. These data may serve as prerequisites for the development of diagnostic test systems aimed at the detection of M. hominis antigens in different clinical substances.     

Soluble metabolic antigens of Trichinella spiralis: their isolation and characteristics]

Cultivation of Trichinella muscular larvae, purified by centrifugation in 20 ... 50% saccharose density gradient, in protein--free nutrient media at a dosage of 3.5-.10(3) lar./ml in the presence of insulin has made it possible to obtain a soluble antigen of Trichinella. It has been shown by means of electrophoresis in polyacrylamide gel that the soluble (secretory-excretory) antigen has three protein fractions while the somatic Trichinella antigen has 18 fractions. It has been shown that the soluble (secretory-excretory) antigen can be used for immobilization of erythrocytes on the surface that enables the sensitivity and specificity of serological methods for diagnosis of trichinellosis to be increased.