Immunochromatography Assay (ICA):
- It is also called as lateral flow dipstick immunoassay or simply strip tests, commonly called as lateral flow test.
- Immunochromatography is union of chromatography and immunoassay.
- Immunochromatography is a simple device proposed to determine the presence or absence of target analyte.
Principle:
- It is based on the same principle as that of ELISA sandwich technique, differing on that the immunological reaction is carried out on the chromatographic paper by capillary action.
- For ICA, two varieties of specific antibodies are used against the antigen. One of the antibodies is immobilized on the chromatographic paper, and the other is labelled with colloidal gold and infiltrated into sample pad.
- An immunographic unit is accomplished by affixing the sample pad at the end of the membrane.
- When the liquid sample is dropped on the sample pad, the antigen in the sample forms an immunocomplex (Ag-Ab complex) with the antibody labelled with colloidal gold.
- Ag-Ab complex migrates along with the liquid sample, and comes in contact with the antibody immobilized on the membrane, followed by the formation of an immunocomplex with the immobilized antibody. As more and more Ag-Ab complexes are halted at the test line, it yields a colored red purple line.
- Appearance of red purple line on the membrane is the suggestive of the presence of antigen of interest in the sample. Since the liquid of the sample travels through the membrane very fast, it makes it possible to determine the presence or absence of antigen within 15 mins.
Basic components of a typical immunochromatographic strip:
- Sample application pad
- Conjugate pad
- Nitrocellulose membrane
- Adsorbent pad
Sample application pad:
- It is composed of cellulose and/or glass fibers.
- The commencement of assay takes place when sample is applied to this pad.
- This pad functions to migrate the sample to other components.
- It should be able to transport sample in a smooth, steady, and homogenous way.
- The pretreatment may consist of segregation of sample components, elimination of interferences, pH adjustment etc.
Conjugate pad:
- Labelled antibodies, usually nano colloid gold particle is dispensed in this pad.
- As soon as the material of the conjugate pad comes in contact with the moving liquid sample, it should release labelled conjugate.
- Labeled conjugate should stay stable throughout the life span of the lateral flow strip.
- Any alterations in distributing, drying or release of conjugate can alter the results of assay significantly.
- Sensitivity of the assay might be largely affected by the poor preparation of labeled conjugate.
- Conjugate pads are synthesized by using materials such as glass fiber, cellulose, polyesters etc.
Nitrocellulose membrane:
- It plays crucial role in the sensitivity of ICA.
- Test and control lines are marked over this piece of membrane.
- An ideal membrane should aid and provide good binding to capture probes (antibodies etc.).
- A good membrane should have lesser non-specific adsorption in the regions of test and control lines for better result.
- For the better sensitivity of the assay, proper dispensing of bio-reagents, drying and blocking plays a major role.
Adsorbent pad:
- It functions as a sink at the end of the strip.
- It also aids in regulating flow rate of liquid over the membrane and prevents back flow of the sample.
All these components are attached over a backing card. Materials for backing card are greatly flexible as they provide platform for preparing assembly of all the components and have no concern with ICA except this. Thus, backing card aids making the handling of strip easier.
Steps involved in ICA
step I: Placing of sample
- For the initiation of the test, a sample is placed on the sample pad at one end of the strip.
- The sample may be used alone as in case with urine or serum or blood tests, or along with buffer which is particular to the test.
step II: Solubilization of molecules
- As soon as the addition of the sample takes place, the detector molecules are solubilized.
- After solubilization, the detector molecules mix up and bind to the analyte in the sample (if analyte present)
step III: Capillary action
- The fluid mixture is drawn up the sample pad and into the membrane by the capillary action.
- The mixture of sample/detector molecule continues to move up the membrane until it reaches the analyte capture molecule.
- A second antibody or antigen immobilized as a thin strip in the nitrocellulose will then capture the Ag-Ab complex if it is positive for the target analyte.
- The control line should always show visible line, or else the test is considered invalid.
- If the test is positive, a colored (generally pink or purple) line develops along with the control line.
step IV: Absorption of excess buffer
- Excess buffer along with any reagents not halted at the test of control line will then travel to the absorbent wicking pad.
Benefits of Immunochromatographic assay:
- Simple and easy to perform
- Rapid test, thus gives quicker results
- Durability over a wide range of climates
- Economical
- Single use
- Commercially accessible
- Small sample volume required
Limitations:
- Mostly qualitative results only
- Less sensitive or less accurate in comparison to other tests, i.e. can detect more than one or two analytes simultaneously.
Applications of immunochromatographic assay:
- Largely used in clinical diagnosis as a screening tests for variety of clinical markers.
- Many markers of clinical values for liver disease, sexually transmitted diseases, cardiac markers as well as markers for men’s and women’s reproductive health can be rapidly detected by immunochromatographic assay
- Detection of human chorionic gonadotropin (HCG), i.e. test of pregnancy.
- Diagnosis of various bacterial and viral infections. Example: rapid immunochromatographic test for syphilis, dengue etc
- Detection of parasitic infections. Eg. Rapid immunochromatographic test for malaria, filariasis etcUsed in early detection of cancer biomarker
References:
- https://www.creative-diagnostics.com/Immunochromatography-guide.htm
- https://www.slideshare.net/amerali6/immunochromatographic-assays
- https://en.wikipedia.org/wiki/Lateral_flow_test