Immunochromatography Lecture Notes (PDF)

**Immunochromatography** **Immunochromatography** is a testing method for detecting a disease by dropping the sample containing an analyte onto a test strip. This is a speedy and simple technique that produces diagnostic results in 10 to 15 minutes after dropping the sample. Immunochromatography i...

**Immunochromatography** **Immunochromatography** is a testing method for detecting a disease by dropping the sample containing an analyte onto a test strip. This is a speedy and simple technique that produces diagnostic results in 10 to 15 minutes after dropping the sample. Immunochromatography is the preserve of research laboratories as a laboratory technique. Near patient testing, however, utilizes commercially produced 'sticks' which use immunochromatography. The principle is that the sample, e.g. urine, is applied to the stick which is then developed, e.g. by capillary attraction, the analyte of interest binding at a zone where there are antibodies. There are sometimes built-in positive and negative controls. [When the antigen and antibody combine], they develop a **visible color spot or band** which confirms the presence of the compound of interest following Figure. Strips used for ICA contain four main components: **1. Sample Application Pad** It is made of cellulose and/or glass fiber and sample is applied on this pad to start the assay. Its function is to transport the sample to other components. Sample pad should [be capable of transportation of the sample in a smooth, continuous and homogenous manner]. This pretreatment may include separation of sample components, removal of interferences, adjustment of the pH, etc. [analyte sample should be added to the sample application pad to start the test.] **2. Conjugate Pad** It is the place where labeled biorecognition molecules (labeled antibodies, usually nano colloid gold particle) are dispensed. [Material of conjugate pad should immediately release labeled conjugate upon contact with moving liquid sample]. Labeled conjugate [should stay stable over entire life] [span] of the lateral flow strip. Any variations in dispensing, drying or release of conjugate can change the results of assay significantly. Poor preparation of labeled conjugate can adversely affect sensitivity of the assay. Glass fiber, cellulose, polyesters and some other materials are used to make conjugate pad. **3. Substrate (Nitrocellulose) Membrane** [It is highly critical in determining sensitivity of ICA]. Test and control lines are drawn over this piece of membrane. So, an ideal membrane should provide support and good binding to capture probes (antibodies, etc.). Nonspecific adsorption over test and control lines may affect results of assay significantly, thus a good membrane will be characterized by lesser non-specific adsorption in the regions of test and control lines. Proper dispensing of bioreagents, drying and blocking play a role in improving sensitivity of the assay. **4. Adsorbent Pad** It works as sink at the end of the strip. It also helps in maintaining flow rate of the liquid over the membrane and stops back flow of the sample. Adsorbent capacity to hold liquid can play an important role in results of assay. All these components are fixed or mounted over a backing card. Materials for backing card are highly flexible because they have nothing to do with ICA except providing a platform for proper assembling of all the components. Thus, backing card serves as a support and it makes easy to handle the strip. An allergic reaction indicating a positive test result appears as a colored line. Tests results from using immunochromatography are usually judged by a visual check, but using a high-sensitivity reader can find a disease with good accuracy even when the colored line is very thin. Since the intensity of the colored line is proportional to the amount of analyte in the sample, increasing the reader's sensitivity gives good test results from just a small amount of sample which lowers the burden on the person being tested