Colloidal gold is a commonly used labeling technology. It is a new type of immunolabeling technology that uses colloidal gold as a tracer marker and is applied to antigen and antibody. It has its unique advantages. It has been widely used in various bioanalytical studies in recent years. Almost all immunoblotting techniques used in clinical use its markers. At the same time, it may be used in flow, electron microscopy, immunology, molecular biology and even biochips.
In 1971, Faulk and Taytor introduced colloidal gold into immunochemistry. Since then, immunocolloidal gold technology has been widely used in various fields of biomedicine as a new immunological method. At present, the main applications in medical testing are immunochromatography (immunochromatogra-phy) and rapid immunogold filtration assay (DIGFA), which are used to detect HBsAg, HCG and anti-double-stranded DNA antibodies, etc. , fast, accurate and pollution-free.
1. The basic principle of immunocolloidal gold technology
Colloidal gold is made of chloroauric acid (HAuCl4) under the action of reducing agents such as white phosphorus, ascorbic acid, sodium citrate, tannic acid, etc., which can be polymerized into gold particles of a certain size, and become a stable colloidal state due to electrostatic action. A negatively charged hydrophobic glue solution is formed, which becomes a stable colloidal state due to electrostatic action, so it is called colloidal gold. Colloidal gold is negatively charged in a weak base environment and can form a firm bond with the positively charged groups of protein molecules. Since this bond is electrostatic, it does not affect the biological properties of proteins.
In addition to binding to proteins, colloidal gold can also bind to many other biological macromolecules, such as SPA, PHA, ConA, etc. According to some physical properties of colloidal gold, such as high electron density, particle size, shape and color response, coupled with the immunological and biological properties of the conjugate, colloidal gold is widely used in immunology, histology, pathology and cell biology, etc.
Colloidal gold labeling is essentially a coating process in which macromolecules such as proteins are adsorbed to the surface of colloidal gold particles. The adsorption mechanism may be the negative charge on the surface of the colloidal gold particles, which forms a firm bond with the positively charged groups of the protein due to electrostatic adsorption. A variety of colloidal gold particles with different particle sizes, that is, different colors, can be easily prepared from chloroauric acid by the reduction method. The spherical particles have a strong adsorption function for proteins, and can be non-covalently bound to Staphylococcus protein A, immunoglobulins, toxins, glycoproteins, enzymes, antibiotics, hormones, bovine serum albumin polypeptide conjugates, etc. Therefore, it has become a very useful tool in basic research and clinical experiments.
Immunogold labelling technology mainly utilizes the high electron density of gold particles. At the binding site of gold-labeled proteins, dark brown particles can be seen under the microscope. When these labels aggregate at the corresponding ligands, the Red or pink spots are visible to the naked eye and are therefore used in qualitative or semi-quantitative rapid immunodetection methods. This reaction can also be amplified by the deposition of silver particles, called immunogold-silver staining.
2. Commonly used immunocolloidal gold detection technology
(1) Immunocolloidal gold light microscopy staining
Cell suspension smears or tissue sections can be stained with colloidal gold-labeled antibodies, or on the basis of colloidal gold labeling, the silver developing solution can be used to enhance the labeling, so that the reduced silver atoms are deposited on the surface of the labeled gold particles. Can significantly enhance the sensitivity of colloidal gold labeling.
Immunocolloidal gold electron microscopy staining
Colloidal gold-labeled antibodies or anti-antibodies can be combined with negatively stained virus samples or ultrathin tissue sections, and then negatively stained. It can be used for the observation of virus morphology and virus detection.
(2) Dot immunogold percolation method
Using a microporous membrane (such as a membrane) as a carrier, first spot the antigen or antibody on the membrane, add the sample to be tested after blocking, and use colloidal gold-labeled antibody to detect the corresponding antigen or antibody after washing.
(3) Colloidal gold immunochromatography
The specific antigen or antibody is fixed on the membrane in a strip, and the colloidal gold labeling reagent (antibody or monoclonal antibody) is adsorbed on the binding pad. When the sample to be tested is added to the sample pad at one end of the test strip, the capillary action Move forward, dissolve the colloidal gold-labeled reagent on the binding pad and react with each other, and then move to the area of the immobilized antigen or antibody, the conjugate of the object to be tested and the gold-labeled reagent binds specifically to it and is trapped and aggregated On the test strip, the color development can be observed with the naked eye. The method has now been developed into a diagnostic test strip, which is very convenient to use.