ABSTRACT
This unit provides a series of techniques that dramatically improve the sensitivity of direct and indirect in situ detection. TSA is a peroxidase-based signal amplification system which is compatible with all in situ hybridization as well as immunocytochemical detection systems. The assay is performed on a glass slide and combines the use of fluorescent probes in either direct or indirect format using either an enzyme-labeled streptavidin or antibody. Protocols are provided for brightfield and fluorescent detection of single DNA targets, DNA or RNA in cultured cells, multitarget detection for DNA/RNA and DNA and RNA. In addition a protocol is included for the TSA plus system, a DNP-based detection system using horseradish peroxidase and alkaline phosphatase.
Subject(s)
Cytogenetics , In Situ Hybridization/methods , Tyramine/analysis , Animals , Catalysis , DNA/analysis , Fluorescent Dyes/chemistry , Humans , Immunohistochemistry/methods , Peroxidases/chemistry , RNA/analysis , Streptavidin/chemistryABSTRACT
The peroxidase-mediated deposition of hapten- and fluorochrome-labeled tyramides has recently been shown to increase the sensitivity of immunofluorescence and fluorescence in situ hybridization techniques. We have evaluated a number of red, green, and blue fluorescent tyramides for detection of antigens in tissue sections and cytospin preparations and for the detection of hapten- and horseradish peroxidase-labeled probes hybridized in situ to cells and chromosomes. With few exceptions, all fluorescent tyramide-based methods provided a considerable increase in sensitivity compared to conventional immunofluorescence and FISH methods.
Subject(s)
Fluorescent Dyes/chemistry , Immunohistochemistry/methods , In Situ Hybridization, Fluorescence/methods , Tyramine/chemistry , Animals , Antigens/analysis , Horseradish Peroxidase , Humans , Immunoenzyme Techniques , Rats , Sensitivity and Specificity , Vimentin/analysisABSTRACT
In a previous publication (Bobrow et al., J. Immunol. Methods (1989) 279-285), we described a novel signal amplification method, catalyzed reporter deposition (CARD), and its application to microplate immunoassays. The method utilizes the analyte-dependent reporter enzyme (ADRE) to catalyze the deposition of additional reporter onto the surface of a solid-phase immunoassay system. In this paper, we describe the utilization of CARD amplification for nonradiometric membrane assays where detection is facilitated by the formation of an insoluble chromogenic product. In the examples described, deposition of reporter is accomplished in two steps: (i) a horseradish peroxidase (HRP) ADRE catalyzes the deposition of either a biotin or fluorescein labeled phenol, and (ii) incubation with either enzyme labeled streptavidin or anti-fluorescein, respectively, results in the deposition of additional enzyme. Using this method, we have improved detection limits from 8- to greater than 200-fold depending on the amplification format and the chromogen used.
Subject(s)
Immunoassay , Animals , Bacterial Proteins , Fluoresceins , Horseradish Peroxidase , Membranes , Mice , Rabbits , Simplexvirus/immunology , StreptavidinABSTRACT
A new 4h rapid enzyme-immunoassay for direct detection of herpes simplex virus (HSV) antigen (Du Pont Herpchek) was evaluated with 743 clinical samples collected at obstetrics and gynecology (OB/GYN), sexually transmitted diseases (STD), and ophthalmology clinics. The sensitivity and specificity of Herpchek was 98.0% and 98.4% respectively compared to virus isolation in cell culture. Confirmatory blocking ELISA tests, clinical history and follow up indicate that the true specificity of the test is 100%.
Subject(s)
Enzyme-Linked Immunosorbent Assay , Herpes Simplex/diagnosis , Simplexvirus/isolation & purification , Adult , Antigens, Viral/analysis , Cells, Cultured , Evaluation Studies as Topic , Eye Infections, Viral/diagnosis , Female , Humans , Male , Middle Aged , Predictive Value of Tests , Reagent Kits, Diagnostic , Sexually Transmitted Diseases/diagnosisABSTRACT
A novel signal amplification method, catalyzed reporter deposition (CARD), and its application to immunoassays is described. The method involves utilizing an analyte-dependent reporter enzyme (ADRE) to catalyze the deposition of additional reporter on the surface in a solid-phase immunoassay. In the examples described, deposition of reporter is facilitated by using a horseradish peroxidase (HRP) ADRE to catalyze the deposition of biotin labeled phenols. The deposited biotins are then reacted with streptavidin-labeled enzyme, thereby resulting in deposition of enzyme. Using the ADRE to catalyze the deposition of additional enzyme results in an amplification of the signal of the ADRE alone and improves the detection limit of the assay. The method is highly sensitive, simple, flexible, and easy to implement.
Subject(s)
Immunoassay/methods , Animals , Antigens, Viral/analysis , Bacterial Proteins , Biotin , Dose-Response Relationship, Immunologic , HIV Antigens/analysis , Horseradish Peroxidase , Immunoglobulin G/analysis , Mice , Phenols , Retroviridae Proteins/immunology , Simplexvirus/immunology , StreptavidinABSTRACT
A method is described for the production of a murine antiserum to human factor VIII, and the quantitation of factor VIII related antigen using this antiserum. C57BL/6J mice were injected intraperitoneally with a mixture of factor VIII and complete Freund's adjuvant. Ascitic fluid developed after repeated innoculations for seven to eight weeks, and was subsequently tapped. Using this antiserum, factor VIII related antigen was quantitated by immunoelectrophoresis and enzyme immunoassay. This method provides a simple and economical way of producing an antiserum to factor VIII, and may also be applicable to other proteins available in submiligram quantities.
