Method for fixing primary antibodies to a biological sample
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Solution Overview
Problem
Primary antibodies in biomolecular analysis assays can detach from their target proteins under harsh conditions, such as elevated temperatures or high salt concentrations, leading to signal loss and inefficiency in DNA detection processes.
Innovation Solution
Stabilize primary antibody-oligonucleotide conjugates using unlabeled secondary antibodies that cross-link with primary antibodies, maintaining binding during DNA detection procedures without chemical crosslinking agents.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If chemical crosslinking agents (e.g., paraformaldehyde, NHS-esters) are used to fix primary antibodies to the sample, then the stability of antibody binding is improved, but the integrity of the DNA coupled to antibodies is compromised, reducing signal generation efficiency
Solution Approach 1:
The patent introduces an intermediary crosslinking mechanism where the secondary antibody serves as a mediator to crosslink primary antibodies to each other without directly crosslinking the DNA-antibody conjugate. This indirect crosslinking approach stabilizes the antibody complex while preserving the integrity of the coupled DNA sequences, thereby resolving the contradiction between binding stability and DNA integrity.
2Productivity
If DNA detection procedures are performed under harsh conditions (elevated temperatures, organic solvents, high salt concentrations), then the DNA detection efficiency is improved, but the primary antibodies release from the sample, causing signal loss
Solution Approach 1:
The patent applies preliminary crosslinking action by introducing secondary antibodies that crosslink the primary antibodies to each other before the harsh DNA detection conditions are applied. This pre-established crosslinked network provides structural stability to the antibody complex, enabling it to withstand the challenging detection conditions without releasing from the sample, thus maintaining signal integrity while allowing efficient DNA detection.
3Adaptability or versatility
If enzymes (DNA polymerases, ligases) are used for DNA detection, then the detection capability is improved, but the time and temperature elevation required for enzyme activity destabilizes the primary antibody binding
Solution Approach 1:
The secondary antibody acts as a stabilizing intermediary that creates a robust crosslinked structure around the primary antibody-DNA complex. This intermediary framework provides mechanical and thermal stability during the extended enzyme incubation periods, allowing DNA polymerases and ligases to function at elevated temperatures for the required duration without causing the primary antibody to detach from the sample.
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
Enhances the stability of primary antibody binding, ensuring efficient signal generation and detection of multiple targets in biological samples under challenging conditions.
Implementation Method 1
incubating the labeled sample with a secondary antibody that recognizes the primary antibody of the primary antibody-oligonucleotide conjugates, thereby cross-linking the primary antibody and producing a cross-linked sample
Data Source
AI summary
A method for labeling a biological sample is provided. In some embodiments, the method may comprise: labeling the biological sample with a one or more primary antibody-oligonucleotide conjugates to produce a labeled sample, incubating the labeled sample with a secondary antibody that recognizes the primary antibody of the primary antibody-oligonucleotide conjugates, thereby cross-linking the primary antibody and producing a cross-linked sample, performing one or more molecular reactions on the oligonucleotide of the primary antibody-oligonucleotide conjugates to produce a nucleic acid product; and detecting the nucleic acid product. Kits for practicing the method are also provided.