Immunochromatography Guide

Immunochromatography assay (ICA), namely lateral flow test, is a simple device intended to detect the presence or absence of the target analyte. The concept of immune-chromatography is a combination of chromatography (separation of components of a sample based on differences in their movement through a sorbent) and immunochemical reactions. The most widespread immunochromatographic system is the test strip (Figure 1).

ICA Format

Lateral flow assay basically combines a number of variants such as formats, biorecognition molecules, labels, detection systems and applications. There are three types of ICAs based on detection format, which are:

1. Sandwich Assay

In this assay format, label (Enzymes or nanoparticles or fluorescence dyes) coated antibody is immobilized at conjugate pad. This is a temporary adsorption which can be flushed away by flow of any buffer solution. A capture antibody against target analyte is immobilized over test line. A secondary antibody against labeled antibody is immobilized at control zone (Figure 2).

To start a test, sample containing the analyte is applied to the sample application pad and it subsequently migrates to the other parts of strip. At conjugate pad, target analyte is captured by the immobilized labeled antibody and results in the formation of analyte-labeled antibody complex. This complex now reaches to nitrocellulose membrane and moves under capillary action. At test line, analyte-labeled antibody complex is captured by another antibody which is primary to the analyte. Analyte becomes sandwiched between labeled and primary antibodies forming labeled antibody-analyte-primary antibody complex. Excess labeled antibody will be captured at the control zone by secondary antibody. Buffer or excess solution goes to absorption pad. Intensity of color at test line corresponds to the amount of target analyte and is measured with an optical strip reader or visually inspected. Appearance of color at control line ensures that a strip is functioning properly.

Figure 2. Schematic diagram of Sandwich ICA.

2. Competitive Assay

Competitive format has two layouts. In the first layout, solution containing target analyte is applied onto the sample application pad and prefixed labeled antibody gets hydrated and starts flowing with moving liquid. Test line contains pre-immobilized antigen (same analyte to be detected) which binds specifically to label conjugate. Control line contains pre-immobilized secondary antibody which has the ability to bind with labeled antibody. When liquid sample reaches at the test line, pre-immobilized antigen will bind to the labeled conjugate in case target analyte in sample solution is absent or present in such a low quantity that some sites of labeled antibody conjugate were vacant. Antigen in the sample solution and the one which is immobilized at test line of strip compete to bind with labeled conjugate (Figure 3.). In another layout, labeled analyte conjugate is dispensed at conjugate pad while a primary antibody to analyte is dispensed at test line. After application of analyte solution, a competition takes place between analyte and labeled analyte to bind with primary antibody at test line.

Such format suits best for low molecular weight compounds which cannot bind two antibodies simultaneously. Absence of color at test line is an indication for the presence of analyte while appearance of color both at test and control lines indicates a negative result.

Figure 3. Schematic diagram of competitive ICA.

3. Multiplex Detection Assay

Multiplex detection format is used for detection of more than one target species and assay is performed over the strip containing test lines equal to number of target species to be analyzed. It is highly desirable to analyze multiple analytes simultaneously under the same set of conditions. Multiplex detection format is very useful in clinical diagnosis where multiple analytes which are inter-dependent in deciding about the stage of a disease are to be detected. Lateral flow strips for this purpose can be built in various ways, for example, by increasing length and test lines on conventional strip, making other structures like parallel threads, stars or T-shapes. Shape of strip for ICA will be dictated by number of target analytes.

Lateral flow immunoassays represent a well-established and very appropriate technology when applied to a wide variety of point-of-care (POC) or field use applications.

The advantages of the lateral flow immunoassay system (LFIA) are well known:

• Relative ease of manufacture – equipment and processes already developed and available. Easily scalable to high-volume production
• Stable – shelf-lives of 12-24 months often without refrigeration
• Ease of use: minimal operator-dependent steps and interpretation
• Can handle small volumes of multiple sample types
• Can be integrated with onboard electronics, reader systems, and information systems
• Relatively low cost and short timeline for development and approval
• Market presence and acceptance – minimal education required for users and regulators

Traditionally designed lateral flow immunoassays, however, have also suffered from performance limitations, most notably sensitivity and reproducibility. Some of these issues are listed below:

• Miniaturization of sample volume requirements below microliter level
• Multiplexing: simultaneous analysis of multiple markers difficult
• Integration with onboard electronics and built-in QC functions challenging
• Sensitivity issues in some systems
• Test-to-test reproducibility challenging – limits applications in quantitative systems

Benefit from its rapid test procedure and naked eyes visible characteristics, lateral flow immunoassays have achieved broad penetration in a variety of markets. Here we summarize some of the applications in figure 4.

Brows All Production Related to Rapid Test Kit

Figure 4. Applications of Immunochromatography assay.

Creative Diagnostics offers extensive experience in the development of rapid, point-of-care, lateral-flow-format diagnostic assays. We can fully develop an immunoassay test (or optimize an existing assay) according to your specifications. Once developed, we will ship the components to you, and all products associated with the project shall become the exclusive property of yours at the conclusion of the project. Full confidentiality is guaranteed.

Learn More about Colloidal Gold Lateral Flow Strips Development Service