Immunochromatographic Assay Signal Amplification via Indicator Size Control
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Solution Overview
Problem
Current immunochromatographic assays face challenges in achieving high sensitivity for detecting analytes due to low signal detection sensitivity, requiring improved methods for signal amplification without mechanical control or artificial steps.
Innovation Solution
The method involves binding a primary conjugate body with a first antibody and a first indicator to an analyte, followed by binding to an immobilized second antibody, and then binding a secondary conjugate body with a third antibody and a second indicator, where the primary conjugate body is positioned closer to the immobilized second antibody, and the second indicator is larger than the first, allowing controlled flow and enhanced signal amplification.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If a conventional immunochromatographic assay is used, then the assay can be completed in a short time with simple operation, but the detection sensitivity is low and cannot detect analytes requiring high sensitivity
Solution Approach 1:
The assay is divided into two distinct sites on the membrane: a first site for capturing the analyte-primary conjugate body complex, and a second site for binding the secondary conjugate body to amplify the signal. This segmentation allows each site to perform its specific function optimally, achieving high sensitivity without complicating the overall assay procedure
Solution Approach 2:
The primary conjugate body is pre-formed and disposed nearer to the immobilized second antibody before the actual detection. This preliminary arrangement ensures that when the sample is applied, the amplification components are already in position to enhance the signal, eliminating the need for separate mechanical control or step-by-step manual operations
2Measurement precision
If signal amplification is attempted through multiple binding steps, then detection sensitivity improves, but the assay requires separate mechanical control or artificial step-by-step reactions
Solution Approach 1:
The assay system is designed to perform signal amplification automatically through the inherent flow dynamics of the capillary phenomenon. The secondary conjugate body self-binds to the primary conjugate body at the second site without requiring external mechanical intervention or manual step-by-step manipulation, maintaining ease of operation while achieving high sensitivity
Solution Approach 2:
The capture and amplification functions are merged into a single continuous flow process on the membrane. Both the first and second antibody sites operate simultaneously in one assay run, combining multiple functions into a unified system that does not require separate mechanical control steps
3Measurement precision
If indicator particles of the same size are used, then the assay structure is simple, but the flow rate control and signal amplification are insufficient
Solution Approach 1:
Different sizes of indicator particles are used in different locations: larger particles (40-60 nm) are used in the primary conjugate body at the first site for strong initial signal, while smaller particles (10-20 nm) are used in the secondary conjugate body at the second site for optimized flow rate control and amplification. This local differentiation of particle properties enables both signal amplification and proper flow dynamics
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
This approach significantly increases signal amplification, enabling the development of immunochromatographic kits with enhanced sensitivity, as demonstrated by experiments using troponin I and myoglobin, where the sensitivity is improved by controlling the size and flow rate of indicator particles.
Implementation Method 1
binding a primary conjugate body, which has a first antibody binding specifically to a first epitope of an analyte
Implementation Method 2
binding the analyte bound to the primary conjugate body to an immobilized second antibody binding specifically to a second epitope of the analyte
Implementation Method 3
binding a secondary conjugate body, which has a third antibody binding specifically to the connector of the primary conjugate body
Implementation Method 4
the fluid flows due to a capillary phenomenon
Data Source
Figure 1~2(C)
Figure 3(A)~3(B)
Figure 4(A)~4(B)
AI summary
The present invention relates to a method for amplifying a signal in an immunochro- matographic assay for high- sensitivity detection of an analyte and an immunochromatographic kit using the method, which amplifies a signal by controlling a flow rate by discrimination between the size of a first indicator and the size of a second indicator. According to an aspect of the present invention, a method for amplifying a signal in an im¬ munochromatographic assay includes: binding a primary conjugate body, which has a first antibody binding specifically to a first epitope of an analyte, a connector, and a first indicator to which the first antibody and the connector are bound, to the analyte; binding the analyte bound to the primary conjugate body to an immobilized second antibody binding specifically to a second epitope of the analyte; and binding a secondary conjugate body, which has a third antibody binding specifically to the connector of the primary conjugate body and a second indicator to which the third antibody is bound, to the connector of the primary conjugate body, wherein the primary conjugate body is disposed nearer to the immobilized second antibody than the secondary conjugate body, and the particle of the second indicator is larger than the particle of the first indicator, so that the secondary conjugate body reaches the immobilized second antibody later than the primary conjugate body. According to another aspect of the present invention, an immunochromatographic kit includes: a sample pad to which a liquid sample containing an analyte is applied; a conjugate pad including a primary conjugate body having a first antibody binding specifically to a first epitope of an analyte, a connector, and a first indicator to which the first antibody and the connector are bound, and a secondary conjugate body having a third antibody binding specifically to the connector of the primary conjugate body and a second indicator to which the third antibody is bound, wherein the primary conjugate body is disposed nearer to an immobilized second antibody than the secondary conjugate body and the second indicator is larger than the first indicator so that the secondary conjugate body reaches the immobilized second antibody later than the primary conjugate body; a membrane including a detection site immobilizing thereto the second antibody binding specifically to a second epitope of the analyte to which the primary conjugate body is bound, and a control site for error detection; and an absorbing pad absorbing the liquid sample by a capillary phenomenon. Thus, the present invention can perform signal amplification without separate mechanical control or artificial step-by-step reaction.