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How to develop rapid test based on immuno-chromatography?

Posted on: September 11, 2020, by Jason

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What is immunochromatography?
The principle of immunochromatography
The commonly assay method in immunochromatography
The commonly colored particles used in immunochromatography
What is colloidal gold?
The 3 types assay method based on colloidal gold immunochromatography
The raw materials used in immunochromatography development
The equipment used in immunochromatography development
The manufacturing procedure in immunochromatography development
How to use colloidal gold immunochromatography for food safety detection?
Conclusion

What is immunochromatography?

Immunochromatography (immu-nochromatography assay, ICA) is a new type of immunoassay method that appeared in the early 1980s. It is a simple and fast immunoassay established on the basis of immunofiltration (IFA).

The principle of immunochromatography

The principle of immunochromatography is to fix a specific antibody on a certain zone of the nitrocellulose membrane. When the dry nitrocellulose end is immersed in the sample (analyte), due to capillary action, the sample will moving forward along the membrane, when moving to the area where the antibody is fixed, the corresponding antigen in the sample specifically binds to the antibody. If stained with immunogold or immunoenzyme, the area can show a color band to achieve specific immunodiagnosis.

The commonly assay method in immunochromatography

Radioimmunoassay (RIA)

RIA technology uses antigens or antibodies labeled with radioactive isotopes (such as 125I, zhi32P, 3H, etc.) to measure the radioactivity of γ-rays or β-rays with a γdao-ray detector or liquid scintillation counter to determine antibodies Or antigen volume technology. It includes radioimmunoassay characterized by labeled antigen and immunoradiometric assay (IRMA) characterized by labeled antibody. The former mainly uses the liquid phase competitive binding method, which measures both macromolecular antigens and small molecule antigens; the latter uses solid-phase method to measure macromolecular antigens.

RIA occupies a large proportion in the early established pesticide immunoassay methods, and established the radiation of dieldrin, aldrin, 2,4-D and 2,4,5-T, parathion and paraquat. Immunoassay. Although this method is very sensitive (RIA is usually 10-9g, 10-12g, or even 10-15g) and has a wide range of applications, expensive counters are required to perform RIA, and there are also problems such as radiation and pollution. The application and development of the detection field are subject to certain restrictions, and are gradually replaced by other immunoassay methods.

Enzyme immunoassay (enzymeimmunoassay, EIA)

EIA is an immunoassay technology developed after RIA. The detection principle is similar to that of radioimmunoassay, but the label used is an enzyme, which organically combines the specific immune reaction of antigens and antibodies with the high-efficiency catalysis of enzymes. The activity of the enzyme bound to the solid phase is measured to determine the Measure the amount of substance. The enzymes used as markers include horseradish peroxidase (HRP) and alkaline phosphatase (AKP), glucose oxidase (GO), urease (urease) and so on.

The solid support for the enzyme labeling reaction includes polystyrene plastic tubes and membranes. At present, most of the 96-well microtiter plates (MTP) are used as solid support. This kind of plate has a large detection capacity and a large number of samples, and only a simple microplate reader can obtain accurate detection data. Some scholars also use magnetic beads as solid-phase materials for EIA research. The principle is to wrap polymer materials (polystyrene, polyvinyl chloride, etc.) on the outside of small metal particles (Fe2O3, Fe3O4), and then chemically bond with amino groups. (-NH2), carboxyl (-COOH), hydroxyl (-OH) and other active groups are coupled with antibodies or antigens to make immune beads. The advantages of this method are that the microbeads have large specific surface area, strong adsorption capacity, and can be suspended in the liquid phase to quickly and uniformly capture the analyte in the sample. After applying a magnetic field, the microbeads can be separated from the sample solution quickly, thereby reducing detection. Time, improve detection sensitivity.

Because enzyme-labeled reagents are easy to prepare, stable, and inexpensive, and the sensitivity of enzyme immunoassay is close to that of radioimmune technology, EIA technology has developed rapidly in recent years, and a variety of EIA methods have been developed. Among them, enzyme-linked immunosorbent assay (enzymelinked immunosorbent assay, ELISA) is currently the most widely used enzyme immunoassay technique in pesticide residue detection.

Fluorescence immunoassay (fluorescence immunoassay, FIA)

The basic principle of FIA detection is to organically combine the high specificity of antigens and antibodies with the sensitive measurability of fluorescence, and use fluorescent substances as tracers to label antibodies, antigens or hapten molecules to prepare high-quality specific fluorescent reagents. When the fluorescent substance in the antigen-antibody conjugate is irradiated by ultraviolet light or blue light, it can absorb light energy and enter an excited state. When it returns to the ground state from the excited state, it can emit the absorbed light energy in the form of electromagnetic radiation, resulting in fluorescence. Plotting pesticide concentration-fluorescence intensity curve can qualitatively and quantitatively detect pesticide residues in samples.

Fluorescein suitable for labeling of antibodies, antigens or hapten molecules must meet the requirements:

  1. It should have a chemical group that can form a stable covalent bond with the protein molecule, or be easily transformed into this type of reaction without destroying its fluorescent structure;
  2. After labeling, the chemical structure and properties of fluorescein and antibody or antigen will not change;
  3. The fluorescence efficiency is high, and the requirement for binding with protein is small;
  4. The process of binding fluorescein to protein is simple and fast, and free fluorescein and its degradation products are easily removed;
  5. The conjugate has stable performance under general storage conditions and can be stored for a long time.

Chemiluminescence immunoassay (luminescentimmunoassay, LCIA)

LCIA can be divided into chemiluminescent immunoassay (chemiluminescent immunoassay, CLCIA) and bio-luminescent immunoassay (bio-luminescent immunoassay, BLCIA).

In 1976, Shroeder first established a homogeneous chemiluminescence immunoassay technology using the biotin (B)-avidin (A) system. Afterwards, Halman and Velan extended it to heterogeneous systems, which has now been infiltrated into biological research. Various fields. The principle is to indicate the combination of antigen and antibody by luminescence. When the luminescent label is combined with the corresponding antibody or antigen, the substrate reacts with the enzyme, or redox reaction with the luminescent agent, or the fluorescent substance (such as rubrene, etc.) Excite and release light energy. Finally, the luminous intensity was measured with a photometer for quantitative analysis. Commonly used luminescent markers are horseradish peroxidase (HRP), luminol (luminol), isoluminol (isoluminol), lophine (lophine), lucigen (lucigen), double (2, 4, 6) -Trichlorobenzene) oxalate, biphenyltriol and 6[N-(4-diaminobutyl)-N-ethyl]-amino-2,3-dihydrophenazine-1,4-dione (ABEI) etc. When the antibody (or antigen) labeled with the above-mentioned luminescent marker is combined with the corresponding antigen (or antibody) in a certain pH buffer solution, it will emit light under the action of a cooperating factor (such as H2O2, etc.), and its luminous intensity will be the same as that of the test substance. It is proportional to the concentration, so it can be used for quantitative analysis.

Luminescence immunoassay has the advantages of strong specificity, high sensitivity (detection limit of 10-15mol/L), rapid (1~3h), and easy availability of luminescent materials. However, its luminescence process and intensity are often affected by the chemical structure of the luminescent substance itself, the pH of the medium, the co-luminescent substance and metal ion impurities.

Gold immunochromatography assay (goldimmuno-chromatography assay, GICA)

The principle of GICA detection is to coat the ligands (antibodies or antigens) on a microporous membrane such as nitrocellulose membranes in a linear form. The colloidal gold label is also fixed with ligands or other substances in a dry state on the absorbent material. Capillary action causes the sample solution to swim on the chromatographic strip. When it swims to the colloidal gold marker, if the sample contains the receptor to be tested, the first step is a highly specific immune response to form an immune complex When the substance continues to swim to the linear coating area, the second step of highly specific immune response occurs, and the formed immune complex is trapped in the coated linear area, and the red band is shown by the labeled colloidal gold (detection Band), while the free label crosses the detection band and is automatically separated from the bound label. Qualitative or quantitative determination can be achieved by detecting the presence or absence of color or the shade of the color on the belt2.

Colloidal Gold Test Kit Detection

GICA method has the advantages of fast (5-20min), cheap, clear results, no complicated operation skills and special equipment, and easy to carry. However, compared with other immunoassay methods, the detection sensitivity of this method is slightly lower, and it is mainly suitable for on-site rapid qualitative or semi-quantitative determination. At present, this method has been used in many research fields such as medicine and biology, and has been widely used especially in developed countries.

Combination technology of immunoassay and instrumental analysis technology

The use of a single IA technique to analyze pesticide residues has less information, and the selectivity of physical and chemical analysis methods is relatively poor. Kramer et al. used immunoassays and liquid chromatography (LC) in combination, thereby simplifying the analysis method and improving the detection efficiency. The combination of LC-IA combines the high separation ability of LC with the high sensitivity and high specificity of IA. This analysis method is especially suitable for multi-component residue analysis and microanalysis. The combination of immunoassay and gas chromatography or mass spectrometry (GC or MS) can reduce cross-reactions in the analysis of pesticides or metabolites with similar structures to reduce false positives.

The commonly colored particles used in immunochromatography

Colloidal gold nanoparticle
Latex particle
Carbon particle
Magnetic beads
Fluorescent dyes
Quantum dots
Upconversion luminescent

What is colloidal gold?

colloidal-gold-particle
colloidal-gold-particle

Colloidal gold, also known as gold nanoparticles, is a stable, uniform, and single-dispersed suspension of gold particles suspended in a liquid after the gold salt is reduced to gold. The gold  particles are composed of a gold atom and a double ion layer surrounding it.

The color of the solution depends on the color of the dispersed phase material, the dispersion of the dispersed phase material, and the type of incident light, whether it is scattered light or transmitted light. The smaller the particles and the higher the dispersion, the shorter the wavelength of the scattered light. For the hydrosol of the same substance, the particle size is different, and the color is also different.

For example, the colloidal gold particles size between 5-20 nm, the absorption wavelength of 520 nm, are red wine color; the size between 20-40 nm mainly absorbs the green light with the wavelength of 530 nm, and the solution is dark red; the 60 nm colloidal gold particle mainly absorbs the wavelength 600 nm orange light, the solution is blue-violet. The colloidal gold particles generally used in immunohistochemistry are in the range of 5-60 nm, and the solution appears red.

The 3 types assay method based on colloidal gold immunochromatography

The most popular colored particle is gold nanoparticle. Colloidal gold technology that combines antigen-antibody immune response and colloidal gold labeling technology for qualitative and quantitative detection of antigen and antibody content. Due to its advantages such as rapidness, simplicity, low cost and good stability, colloidal gold detection has been widely used in the field of clinical detection.

At present, the commonly used detection methods of colloidal gold detection platforms include double-antibody sandwich method, competition method and indirect method. There are big differencies between assay method and analytes.

Double-Antibody Sandwich Method

The double-antibody sandwich method is the most commonly used detection method for colloidal gold detection platforms, which is mainly used to detect relatively large biomolecules and particulate antigens. The double-antibody sandwich method requires preparation of the paired antibody of the antigen to be tested. One antibody is labeled with colloidal gold and fixed on the conjugate pad, and the other antibody is fixed on the detection line (T line) of the NC membrane. In addition, it is necessary to prepare a secondary antibody that specifically binds to the gold-labeled antibody (ie, colloidal gold-labeled antibody) and fix it on the control line (C line) of the NC membrane.

lateral-flow-immunoassay
lateral-flow-immunoassay

When the sample have been added to the strip, it will move to the sample pad, conjugate pad, T line, C line and absorbent pad (wicking pad) in sequence through capillary flow principle. The reaction of each stage is shown in the following table:

 Sample move T conjugate padSample move to T lineSample move to C lineResult
Anaylte contain antigenGold-labeled antibody captured antigen, become a complexThe antibody of fixed on T line will capture the complex and color band will be visibleThe secondary antibody will capture the gold-labeled antibody and color band visibleTwo line visible
Anaylte without antigenNo reactionNo color changeSame aboveOnly C line visible

Therefore, when two red lines are displayed on strip, it means that the substance to be tested in the sample is positive; when there is only C line on strip, it means that the substance to be tested in the sample is negative. The high concentration of the substance will has a stronger color band of T-line.

Competition Method

Small molecule antigens are difficult to prepare paired antibodies (molecular weight too small, it is difficult to find two binding sites for two antibodies to bind at the same time), so small molecule antigens cannot be detected by double antibody sandwich method. For small molecule antigens, usually use the competition method for assay.

In the competition method, the gold-labeled antibody is fixed on the cojugate pad o, and the T line on the NC membrane is fixed with the macromolecular coupled antigen to be tested (small molecule antigens cannot be directly fixed on the NC membrane. Therefore, it is necessary to chemically couple small molecules to BSA and other macromolecular substances and then immobilize them on the NC membrane). The second antibody that can specifically bind to the gold-labeled antibody is immobilized on the C line.

lateral-flow-assay-development
lateral-flow-assay-development

When the sample have been added to the strip, it will move to the sample pad, conjugate pad, T line, C line and absorbent pad (wicking pad) in sequence through capillary flow principle . The reaction of each stage is shown in the following table:

 Sample move T conjugate padSample move to T lineSample move to C lineResult
Anaylte contain antigenGold-labeled antibody captured antigen, become a complexNo antigen can be captured. No color bandThe secondary antibody will capture the gold-labeled antibody and color band visibleOnly C line visible
Anaylte without antigenNo reactionCapture the gold-labeled antibody and color band visibleSame aboveTwo line visible

Therefore, when two red lines are displayed on strip, it means that the substance to be tested in the sample is negative; when there is only C line on strip, it means that the substance to be tested in the sample is positive. The high concentration of the substance will has a stronger color band of T-line.

Indirect Method

The indirect method is mainly used to detect antibodies. If indirect method, the conjugate pad fixed with protein A labeled with colloidal gold (Protein A can bind to the antibody non-specifically). The T line on the NC membrane is fixed with an antigen that can specifically bind to the antibody to be tested, and the C line on the NC membrane is fixed with an anti-Protein A antibody.

immunochromatographic-test
immunochromatographic-test

When the sample have been added to the strip, it will move to the sample pad, conjugate pad, T line, C line and absorbent pad (wicking pad) in sequence through capillary flow principle . The reaction of each stage is shown in the following table:

 Sample move T conjugate padSample move to T lineSample move to C lineResult
Anaylte contain antibodyGold-labeled Protein A captured antibody, become a complexAntigen capture antibody, color band visibleProtein A captured by anti-proten A antibody and color band visibleTwo line visible
Anaylte without antibodyNo reactionAntigen can’t capture antigen, color no changeThe gold-labeled antibody captured by secondary antibody, color band visibleOnly C line visible

Therefore, when two red lines are displayed on strip, it means that the substance to be tested in the sample is positive; when there is only C line on strip, it means that the substance to be tested in the sample is negative. The high concentration of the substance will has a stronger color band of T-line.

The raw materials used in immunochromatography development

  1. Antigen and antibody
  2. Bovine Serum Albumin
  3. Backing card lateral flow
  4. Nitrocellulose membrane
  5. Sample pad (Wicking Pad)
  6. Conjugate pad
  7. Absorbent pad
  8. Blood separation membrane (option)
  9. Cassette case (option)

The equipment used in immunochromatography development

  1. Robotic dispensing system (Dispensor)
  2. Strip cutter for lateral flow assay strip
  3. Micropipette
  4. Vacuum freeze dryer
  5. Vacuum oven
  6. High speed refrigerated centrifuge
  7. Liquid handling system (option)
  8. Cassette case processor (option)

The manufacturing procedure in immunochromatography development

  1. Colloidal gold prepared
  2. Colloidal gold labeling
  3. Dispensing antigen or antibody to Nitrocellulose membrane
  4. Nitrocellulose membrane stick to backing card
  5. Laminate conjugate pad on backing card
  6. Laminate sample pad on backing card
  7. Laminate absorbent pad on backing card
  8. Stick Logo label on absorbent pad (option)
  9. Cutting the uncut sheet in to strips
  10. Load strips in to foil pouch or bottle
  11. Packing in box

More details, please refer to How To Develop Lateral Flow Assay Rapid Test?

How to use colloidal gold immunochromatography for food safety detection?

Colloidal gold immunochromatography widely used in food testing for many years. For example, how the milk test work on antibiotic residues in raw milk?

The antibiotic drug is small molecule, so, the assay choose competition method. What is the reaction mechanism?

Let’s see the strip structure:

lateral-flow-assay
lateral-flow-assay
gold-nanoparticles
gold-nanoparticles

The colloidal gold labeled antibody lyophilizated in well plates. Seperation from the strip, increase incubation time and enhance detection sensitivity.

Lateral flow reaction mechanism

lateral-flow-technology
lateral-flow-technology
immunochromatography
immunochromatography

In Positive Sample:

  1. Take raw milk sample and add into the micro wells
  2. If the raw milk contain small molecule, such as, beta lactam, tetracycline, chloramphenicol, Sulfonamides, Cephalosporins, Melamine, etc....
  3. The gold-labeled antibody (Au-Ab) in micro wells will capture the small molecules from the milk samples, become a new complex (Au-Ab binded with small molecule).
  4. While the complex move to T line position, there is no position for BSA-antigen (the antigen that fixed on T lin) to conjugate, because the gold-nanoparticle already conjugated with small molecules, so, color band will not change.
  5. The secondary antibody that fixed on C line will capture the gold-labeled antibody and color band visible.
  6. The result will be positive.

In Negative Sample:

  1. The gold-labeled antibody(Au-Ab) can not capture small molecules from raw milk, but will be captured by T line’s antigen, then color band visible.
  2. The C line’s secodary antibody still capture the gold-labeled antibody(Au-Ab), color band visible.

The interpretation

The Colorimetric method for interpretation of milk test. The test line compare with control line to determine positive or negative result. While manufacturing to control the quantity of Au-Ab to achieve colorimetric assay.

Conclusion

Immunochromatography is very popular assay technology, widely used in in vitro diagnostics products, not only used in food testing. Sepcially in lateral flow assay, colloidal gold immunochromatography assay no need costly equipment, eays to use, cheaper cost to help human being live better, for example, home pregnancy test.

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