Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Patent
1992-08-31
1994-06-21
Saunders, David
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
424 3, 435 23, 435188, 435267, 435960, 435961, 435962, 436175, 436825, C12N 996, C12Q 137, G01N 134, G01N 3353
Patent
active
053227719
DESCRIPTION:
BRIEF SUMMARY
FIELD OF THE INVENTION
The present invention relates to an improved method for immunohistochemical staining of slides, particularly an automated method, and reagents therefor.
BACKGROUND OF THE INVENTION
Four main methods using peroxidase (HRPO) are now generally recognized for immunostaining. The method are based on the immune reaction of an antigen to be detected in the specimen complexing with an antibody specific for the antigen. The methods differ, primarily in the manner of detecting the antigen-antibody complex. The methods are the direct method, an indirect method using an enzyme-conjugated secondary antibody specific for the species of the primary or first antibody, the peroxidase-anti-peroxidase (PAP) method, and an indirect biotin-avidin method using a biotin-conjugated secondary antibody and a complex of a biotin-conjugated peroxidase and either avidin or strepavidin.
All of the immunohistochemistry methods, as well as other immunochemical methods, are multi-step procedures which consist of a sequence of reagent additions, incubations, and washings. Most of these procedures require highly trained personnel and the results can vary significantly between laboratories. Automated systems have been explored to introduce cost savings, uniformity of slide preparation, and reduction of procedural human errors.
For both automated and manual methods, there are a number of critical points to be considered. Care must be exercised to avoid the loss of specimen from the slide. Thorough washing of the specimen between reagent applications is essential particularly to remove unbound antibody as residues would be amplified. Excess liquid must be removed to avoid unwanted dilution of antibodies, yet specimens must never be allowed to dry out. Enough antibody reagent must be applied to completely cover the slide area where the specimen may occur, but waste has to be kept to an absolute minimum.
In addition, many of the reagents used in immunohistochemical methods as well as immunochemical methods, such as enzyme solutions and peroxidase color development reagents, have limited stability at the working temperature and even at room temperature. This necessitates frequent preparation of the reagents. Furthermore, nonspecific antibody binding, leading to erroneous results, remains a problem.
Methods and reagents that improve results and minimize reagent preparation would facilitate both manual and automated immunohistochemical methods. Many of the improvements could be readily applied to related immunochemical methods such as enzyme-linked immunosorbent assays (ELISA), immunofluorescence assays and in situ hybridization.
DESCRIPTION OF THE PRIOR ART
Cosgrove et al, ACL pp 23-27 (December, 1989) describe immunostaining methods, particularly peroxidase staining methods, and an automated staining apparatus. Brigati and his colleagues [Brigati et al, J. Histotechnology 11:165-183 (1988); Unger et al, J. Histotechnology. 11:253-258 (1988)] describe the Fisher automated work station (which can perform immunohistochemical staining and in situ hybridization methods) and reagents used in the automated methods. The system and reagents are further described in U.S. Pat. Nos. 4,777,020, 4,798,706 and 4,801,431. Each of those devices uses a different method to conserve expensive (antibody-containing) reagents.
Stross et al, J. Clin. Pathol. 42:106-112 (1989) describes an automated tissue staining system which processes the slides following manual application of antibody-containing reagents. Stark et al, J. Immunol. Methods. 107:89-92 (1988) describes a microprocessor-controlled automated staining system.
Each of the above-described references is incorporated herein by reference in its entirety.
SUMMARY OF THE INVENTION
The present invention provides an improved method for staining slides using immunochemical reagents. In a preferred embodiment, the method is used in an automated process. The method comprises the following steps. The assay region of a slide (the region containing the tissue section) is washed with an imp
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Degroff Michael J.
Gizinski Michael J.
Hartman Anthony L.
Miller Phillip C.
Rybski James A.
Saunders David
Ventana Medical Systems, Inc
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