Dual Antibody Detection via Denaturation and Blocking

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Solution Overview

Problem

Reliable detection of CD3 and CD16 proteins in cancer samples is hindered by undesirable cross-reactivity of secondary antibodies when using primary antibodies from the same species, leading to inconsistent and inaccurate results.

Innovation Solution

An automated immunohistochemistry procedure is developed for dual detection of CD3 and CD16 using primary antibodies from the same host species, involving specific buffer conditions, denaturation, and blocking steps to minimize cross-reactivity, allowing for consistent and precise staining across various assay parameters.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Ease of manufacture

If primary antibodies from the same host species are used for CD3 and CD16 detection, then assay simplicity and cost are improved, but cross-reactivity of secondary antibodies occurs leading to poor detection accuracy

Engineering Contradiction:

Improveassay simplicityVSAvoiddetection accuracy

Core Design Contradiction:

Ease of manufactureVSMeasurement precision

Solution Approach 1:

The patent divides the detection system into separate detection channels for CD3 and CD16 antibodies, using different secondary antibody conjugates (e.g., fluorophore-conjugated vs. enzyme-conjugated) to eliminate cross-reactivity while maintaining the use of same-species primary antibodies

Inventive Principle:

Principle #1Segmentation

Solution Approach 2:

The patent introduces blocking antibodies as intermediaries that bind to remaining primary antibody sites after denaturation, preventing cross-reactivity of secondary antibodies while allowing the detection system to function

Inventive Principle:

Principle #24Intermediary (Mediator)

2Device complexity

If conventional IHC procedures are used without denaturation and blocking steps, then procedure simplicity is maintained, but cross-reactivity and background staining increase

Engineering Contradiction:

Improveprocedure simplicityVSAvoidcross-reactivity

Core Design Contradiction:

Device complexityVSObject-affected harmful factors

Solution Approach 1:

The patent performs denaturation and blocking steps before the actual antibody detection, pre-treating the tissue section to eliminate cross-reactivity pathways and reduce background staining before primary and secondary antibodies are applied

Inventive Principle:

Principle #10Preliminary action

Solution Approach 2:

The patent changes physical parameters (temperature, time, buffer composition) during denaturation and blocking steps to optimize the elimination of cross-reactivity while maintaining tissue integrity and staining quality

Inventive Principle:

Principle #35Parameter changes

Applied Scientific Principles

This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.

Function Achieved in This Case

The method provides accurate and specific detection of CD3 and CD16 proteins in formalin-fixed, paraffin-embedded tissues, enabling reliable scoring and prediction of tumor response to anti-EGFR targeted therapies like GA201.

Implementation Method 1

contacting the sample with a CD3 antibody... contacting the sample with a CD16 antibody

Methodology Applied

Scientific EffectAntibody binding:

Implementation Method 2

The sample is denatured by incubating the sample at about 80° C.-100° C. for at least 5 minutes

Methodology Applied

Scientific EffectDenaturation:

Implementation Method 3

The sample is contacted sample with a blocking antibody, such as a chicken anti-rabbit antibody

Methodology Applied

Scientific EffectBlocking:

Data Source

PatentUS10145852B2Multiples multiplex immunohistochemical assay using primary antibodies of the same host species

Publication Date: 2018.12.04 VENTANA MEDICAL SYSTEMS INC

AI summary

Disclosed herein are methods and systems for detecting CD3 and CD16 in the same sample of a colorectal, head & neck, or lung tumor using immunohistochemical methods using primary antibodies from the same host species and secondary antibodies immunoreactive with antibodies of the host species of the primary antibodies. Methods to denature and block the first primary antibody contacted with the sample are provided.