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Direct immunohistochemistry assay

A technology of tissue and tissue sectioning, applied in the direction of immunoglobulin, chemical instruments and methods, biochemical equipment and methods, etc., can solve the problems of time-consuming and concealment of IHC technology

Active Publication Date: 2017-02-15
贵州美鑫达医疗科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Therefore, current IHC techniques are too time-consuming to be used as intraoperative tools
Moreover, current IHC techniques may harbor artefacts caused by the signal amplification techniques used, for example, non-specific binding of secondary antibodies or the presence of endogenous biotin

Method used

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Examples

Experimental program
Comparison scheme
Effect test

preparation example Construction

[0061] Preparation of tissue samples

[0062] Methods of preparing tissue blocks from these particulate samples have been used successfully in previous IHC studies of various prognostic factors and / or are well known to those skilled in the art.

[0063] Briefly, any whole organ or tissue can be cut into relatively small pieces and incubated in various fixatives (eg, formalin, alcohol, etc.) for varying periods of time until the tissue is "fixed." A sample can be almost any intact tissue surgically removed from the body. Samples can be cut into reasonably small piece(s) consistent with instruments routinely used in histopathology laboratories. The size of the cut pieces usually ranges from a few millimeters to a few centimeters.

[0064] In some embodiments, frozen sections can be prepared by rehydrating 50 mg of frozen "crushed" tissue in phosphate-buffered saline (PBS) at room temperature in a small plastic capsule; Centrifuge to pellet pellet; resuspend pellet in viscou...

Embodiment 1

[0303] Example 1. Protocol for Direct IHC on Frozen Tissue Slides

[0304] This example demonstrates a protocol for performing direct IHC on tissue sections obtained from frozen tissue samples.

[0305] Tissue sections were prepared as described in the Exemplary Methods below. Freshly dissected tissue blocks ( Plus, VWR).

[0306] Tissue sections were immunostained as described in the Exemplary Methods below. Tissue sections were fixed for 1 to 2 minutes with a fixative containing 75% methanol, 5% glacial acetic acid, and 20% 37% formaldehyde. Slides with tissue sections are then blocked with blocking buffer, eg, 5% skim milk or 2% BSA, for 2 minutes. After blocking, slides were washed for 10 seconds in 10 mM phosphate buffered saline (PBS) at neutral pH; slides were washed 3 times. The polymerized-HRP / antibody conjugate diluted with 1% BSA was applied to the slide to cover the entire area of ​​the tissue section and incubated at room temperature for 3 to 5 minutes. Aft...

Embodiment 2

[0307] Example 2. Dewaxing and rehydration of tissue slides

[0308] This example demonstrates a protocol for performing direct IHC on tissue sections following deparaffinization, rehydration, and epitope retrieval of the tissue sections.

[0309] Immerse slides with tissue sections in xylene for 5 minutes. The xylene immersion was repeated two more times with clean xylene for 5 minutes each. The slides were then immersed in the following series of ethanol solutions: 100% ethanol for 3 minutes; 100% ethanol for 3 minutes; 95% ethanol for 3 minutes; and 75% ethanol for 3 minutes. Slides were then washed in tap water by immersing slides in clean tap water for 3 minutes; repeated two more times.

[0310] Following rehydration of the tissue sections, protein epitopes were exposed using trypsin. First, a 0.1% trypsin solution in 0.1% calcium chloride was prepared using distilled water. The pH of the trypsin solution was adjusted to 7.2 using 1M sodium hydroxide. The humidifi...

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Abstract

The application is to antibodies which have been labeled with polyenzyme (multiple enzymes), specifically polyperoxidase, for use in direct immunohistochemical assays of tissues. The antibodies used diagnostically may also be antibodies which are used therapeutically.

Description

[0001] Cross References to Related Applications [0002] This application claims priority to US Provisional Serial No. 61 / 990,111, filed May 8, 2014, entitled RAPID IMMUNOHISTOCHEMISTRY ASSAY, which is hereby incorporated by reference in its entirety for all purposes. technical field [0003] The present invention relates to compositions, methods and kits for direct immunohistochemical staining of tissues and uses thereof. Background technique [0004] During surgery, tissue biopsy samples can often be removed from the patient and sent from the operating room to a pathology laboratory for analysis—for example, by frozen tissue section diagnosis. The methodology for frozen tissue section diagnosis may consist of freezing the tissue in a pathology laboratory, sectioning the frozen tissue and performing standard hematoxylin and eosin (H / E) staining. H / E staining is a common technique used to aid medical pathologists in diagnosing histopathology. However, H / E staining has a nu...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/535
CPCC07K16/18C07K16/2803C07K16/289C07K16/3053C07K2317/21G01N33/535A61K39/00G01N2474/20G01N33/581
Inventor S·Q·赵J·J·王J·V·吴Z·张
Owner 贵州美鑫达医疗科技有限公司
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