Direct immunoenzyme double staining applicable for monoclonal antibodies

Summary A novel method for immunoenzymatic double staining was developed, using primary antibodies directly labeled with either horseradish peroxidase or alkaline phosphatase. The enzyme-antibody conjugates were applied simultaneously on sections of human tonsil. Intracytoplasmic antigens like immunoglobulins and light chains could easily be detected simultaneously in the same tissue section. With antibodies against cell surface antigens like IgM and T cell antigens areas containing B and T cells could be clearly distinghuished. This method opens the possibility to perform double staining with two monoclonal antibodies.     

Use of commercially available monoclonal antibodies for immunoenzyme double staining

Summary An immunoenzyme double-staining method for the simultaneous detection of two cellular epitopes, using commercially available mouse monoclonal antibodies, is described. The method employs a combination of the suppression of endogenous biotin and two successive indirect techniques with a blocking step in between. The first indirect method involves an unlabelled monoclonal antibody followed by an alkaline phosphatase-conjugated goat anti-mouse immunoglobulin. After a blocking step with normal mouse serum, the second indirect method is applied using a biotinylated monoclonal antibody followed by the visualization of this antibody by avidin-biotinylated peroxidase complex (ABC) or rabbit anti-biotin and peroxidase-conjugated swine anti-rabbit immunoglobulin in successive steps. Using these methods in combination with the introduction of dioctyl sodium sulphosuccinate and tetramethylbenzidine as chromogens for peroxidase activity, two cellular epitopes could be distinguished clearly in tissue sections by the green-and violet-stained peroxidase and alkaline phosphatase activities, respectively. The expression of two epitopes on the same cellular constituent is outlined by the coappearance of both enzyme activities as a bluish-purple colour. This method allows for the simultaneous identification, localization and enumeration of two cellular epitopes. These can serve as parameters for a number of pathological processes.     

The use of immunoenzyme methods in virology

Data on the development of theoretical basis of immunoenzyme (IE)method s (EMIT analysis and ELISA) and on using these methods in virology are presented. The problems arising in the use of most popular version of IE analysis. ELISA, are elucidated: the schemes for different variants of ELISA; the demands to adsorbent, enzyme and substrate for achieving the maximum of sensitivity of the technique; comparison of the advantages of different ELISA variants in identification of various antigens and antibodies. The data on using ELISA for indication and identification of viral antigens and virus specific antibodies are analyzed. The respiratory viruses have been used to demonstrate the possibilities of IE technique for rapid diagnostic of viral diseases and for studies in viral reproduction and structure.     

The use of commercial immunoenzyme kits for the diagnosis of toxoplasmosis

The results of comparative study of sera obtained from donors and from several groups of patients with suspected toxoplasmosis are presented. The study has been carried out with the use of commercial enzyme immunoassay (EIA) kits: Toxoplasma gondii IgG EIA and Toxoplasma gondii IgM EIA manufactured by Labsystems (Finland), Sevatest ELISA IgG Toxo Micro I manufactured by Sevac (Czechoslovakia). Statistical processing of the results has confirmed the identity of these kits. The necessity of using evaluation criteria (the separation point, the scale for the interpretation of results) when working with the Sevac kits is emphasized. Comparative evaluation of antibody profiles in the sera under test suggests that the titer less than 1:1600 should be regarded as the separation point for these kits. IgM antibodies to T. gondii have been found only in 22% of patients with high titers of IgG antibodies.     

The use of a dot immunoenzyme method for detection of viral antigens

The technique of dot immunoenzyme detection has been elaborated for viral antigens identification. The investigated samples (extracts of infected cells, fractions obtained during viruses and viral proteins purification) are placed in nitrocellulose filters, the free binding sites are blocked and then treated with specific immune serum. The formed antigen-antibody complexes are detected using antispecies immunoglobulins or protein A from Staphylococcus aureus, conjugated with horseradish peroxidase. The brown dots appear at samples location containing the viral antigens. Sensitivity of the technique is 1 ng of protein per sample as tested using adenoviral antigens.     

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.     

A one-step, low background coomassie staining procedure for polyacrylamide gels

Present Coomassie staining procedures require hours of destaining and/or have high backgrounds. This one-step staining procedure is easier, gives lower background with no loss in sensitivity, uses less chemicals, requires less time, and can be followed by silver stain if increased sensitivity is desired after analyzing the results.     

Multiple immunoenzyme staining: methods and visualizations for the observation with spectral imaging.

Several staining concepts and color combinations exist to perform successful double immunoenzyme staining on human tissue specimens. Most of these concepts are based on differences between both primary antibodies: animal species, mouse Ig isotype or IgG subclasses, conjugates, or concentrations. Traditionally, double immunoenzyme staining has used chromogens selected to provide maximum color contrast when observed with the unaided eye. Unfortunately, visually good color combinations always include at least one diffuse chromogen, because of the paucity of appropriate chromogen colors. This situation is drastically changed with the use of spectral imaging, where multicolor microscopy can be unmixed in individual images based on their spectral characteristics. Spectral unmixing can be performed even up to quadruple immunoenzyme staining. This work contains practical suggestions for immunoenzyme double staining procedures for some frequently encountered primary antibody combinations: rabbit-mouse, goat-mouse, mouse-mouse, and rabbit-rabbit. The suggested protocols are all suitable for a classical red-brown color combination plus blue nuclear counterstain that is composed of peroxidase activity (diaminobenzidine tetrahydrochloride), alkaline phosphatase activity (Liquid Permanent Red), and hematoxylin, respectively. Although the red and brown chromogens do not contrast very well visually, they both show a crisp localization and can be perfectly unmixed by spectral imaging.     

The use of the microdot immunoenzyme method for determining antibodies to Mycobacterium tuberculosis antigens

The microdot enzyme immunoassay (EIA) has been used for the determination of antibodies to M. tuberculosis protein fractions, crude antigenic preparations, PPD and old tuberculin in tuberculosis patients and healthy persons. Purified protein fractions have been found to possess the highest sensitivity and specificity in microdot EIA. The determination of antibodies to these fractions has permitted the differentiation of persons infected with M. tuberculosis from healthy ones. The use of M. tuberculosis protein fractions permits the determination of IgA and IgC in the sera of tuberculosis patients.     

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.     

The use of immunoenzyme analysis and immunoblotting for the serological characterization of mycobacterial antigens]

The enzyme immunoassay and immunoblotting were used for the study of the serological activity of different mycobacterial antigens and the spectrum of antibodies to them in patients with different forms of tuberculosis and healthy persons. Antibodies in patients' sera were shown to bind antigens with different molecular weight. The level and spectrum of antibodies to purified protein fraction I made it possible to differentiate between patients with various forms of tuberculosis and healthy persons.     

The use of immunoenzyme analysis in the laboratory diagnosis of plague]

The possibility of using the enzyme immunoassay (EIA) for the early diagnosis of pneumonic plague was studied in experiments on monkeys. EIA was shown to be more effective than the passive hemagglutination test. The diagnostic value of blood serum samples was found to be higher than that of nasopharyngeal mucus samples taken from the sick animals. The conclusion on the suitability of EIA for the early laboratory diagnosis of this disease was made.     

The use of direct immunoenzyme analysis with genus-specific peroxidase conjugate in leptospirosis

For the first time anti-Patoc I peroxidase conjugate was obtained from the serum of a hyperimmunized guinea pig by the method of Nakane and Kawaoi (1974) in the modification of Mathiesen et al. (1978). In the experimental study of 37 Leptospira strains belonging to 19 serological groups, carried out with the use of direct EIA techniques, anti-Patoc I peroxidase conjugate was found to be genus-specific and highly active. The possibility of detecting Leptospira antigens of 7 serological groups in the blood and in suspensions of the liver and kidneys of golden hamsters by direct EIA with the use of the above-mentioned conjugate was established. The use of direct EIA with anti-Patoc I peroxidase conjugate for the diagnosis of leptospirosis is recommended.     

A humanized immunoenzyme with enhanced activity for glucuronide prodrug activation in the tumor microenvironment

Antibody-directed enzyme prodrug therapy (ADEPT) utilizing β-glucuronidase is a promising method to enhance the therapeutic index of cancer chemotherapy. In this approach, an immunoenzyme (antibody-β-glucuronidase fusion protein) is employed to selectively activate anticancer glucuronide prodrugs in the tumor microenvironment. A major roadblock to the clinical translation of this therapeutic strategy, however, is the low enzymatic activity and strong immunogenicity of the current generation of immunoenzymes. To overcome this problem, we fused a humanized single-chain antibody (scFv) of mAb CC49 to S2, a human β-glucuronidase (hβG) variant that displays enhanced catalytic activity for prodrug hydrolysis. Here, we show that hcc49-S2 displayed 100-fold greater binding avidity than hcc49 scFv, possessed greater enzymatic activity than wild-type hβG, and more effectively killed antigen-positive cancer cells exposed to an anticancer glucuronide prodrug as compared to an analogous hβG immunoenzyme. Treatment of tumor-bearing mice with hcc49-S2 followed by prodrug significantly delayed tumor growth as compared to hcc49-hβG. Our study shows that hcc49-S2 is a promising targeted enzyme for cancer treatment and demonstrates that enhancement of human enzyme catalytic activity is a powerful approach to improve immunoenzyme efficacy.     

The use of a layering technique for enhancing stability of lyophilized reagents.

An enzyme-mediated assay has been developed for the measurement of salicylate using salicylate monooxygenase purified from Pseudomonas cepacia ATCC 29351. Two assay formulations were produced, based on either a multiple-reagent or a single-reagent formulation, to allow sufficient flexibility for automated use. The multiple-reagent formulation was especially suited to diagnostic laboratories performing infrequent manual salicylate estimation where stability of the reconstituted reagent is of paramount importance. This was achieved by preparing the enzyme and color reagents in separate vials, so keeping the enzyme at a stable pH. For more frequent assay use where a reconstituted reagent shelf life was less important, the single-reagent system offers advantages of convenience. However, the working reagent required a pH of 10.0 upon reconstitution. Although the enzyme was sufficiently active at this pH to give a reliable assay, its storage stability was poor at pH 10.0, preventing lyophilization of the reagent at a pH suitable for immediate use on reconstitution. This incompatibility was overcome by use of a layering technique. The enzyme was separated from the buffering solution in the same vial by freezing the buffering solution and then overlayering with the enzyme reagent prior to a second freezing cycle and subsequent freeze drying.     

The immunoenzyme analysis of ceruloplasmin

A highly specific and sensitive enzyme immunoassay (EIA) system, suitable for the qualitative analysis of ceruloplasmin, has been developed. The possibility of its use for the examination of children with mononucleosis and pseudotuberculosis has been studied. An increase in the concentration of ceruloplasmin has been more pronounced in infectious mononucleosis (0.506 +/- 0.026 g/l) and pseudotuberculosis (0.421 +/- 0.157 g/l). The results of EIA coincided with the data obtained by radial immunodiffusion.