At present, microplate reader has become a widely used in medical examination of professional medical equipment. And now immune enzyme technology also has very mature, to the body of enzyme and antibody test has played a very important role.
1, principle:
Immune enzyme technology is the specificity of the antigen-antibody reaction with the efficient enzymes catalysis of the organic combination of a kind of method. It with enzyme as a marker, and antibody or antigen coupling, and the corresponding antigen and antibody action through the substrate for the color of antigen and antibody response of qualitative and quantitative, also can be used in the organization antigen or antibody positioning, namely enzyme immunohistochemical techniques.
At present the most immune enzyme technology application is ELISA experiment (ELISA), it is to make antigen and antibody adsorption on solid phase supports, make the subsequent antigen-antibody reaction on the surface were in the carrier, so as to simplify the separate steps, improve the sensitivity, can test the antigen, also can detect antibodies. The experimental method including the indirect method, method and clip competition law.
In order to detect the antigen available clip method. The specific antibodies adsorption in the solid phase carrier (polystyrene small tube, made of small dish or small holes), and then test solution, if the samples have corresponding antigen and antibody is a complex surface in carrier. After washing to mark the specificity of the enzyme antibody, the latter through the antigen to combine with the surface of the carrier also. Wash away excess mark antibody, join the enzyme substrates, in a certain time from the classic enzyme catalysis non-ferrous products and solution in the amount of antigen is proportional to the content, available macroscopic observation or spectrophotometer to measure.
In order to detect antibodies can be used the indirect method. Make antigen adsorption on carrier, and then join the measured serum, such as to have antibody, with antigen in the carrier is formed on the compound. After washing with the enzyme markers of globulin (fight antibody) and the reaction. After washing with the substrate show color, non-ferrous products with the amount of antibody is proportional to the amount.
Commonly used enzymes have horseradish peroxidase (HRP) and alkaline phosphatase (AP), the corresponding substrate were neighbors benzene 2 amine (OPD) and nitrobenzene of phosphate, the former is color reaction is tan, the latter for blue. Can be used visual qualitative, also can YongMei standard analyzer to measure the light density (OD) value to reflect the antigen content.
2, ELISA type:
ELISA can be used for determination of antigen, also can be used for testing the antibodies. In this kind of determination method of three necessary reagents:
(1) solid phase of the antibiotics or antibody, called "immune adsorbent" (immunosorbent);
(2) the antigen enzyme mark or antibody, called "combined" (conjugate);
(3) the enzyme reaction of the substrate. According to the source of the specimen and reagent and detection of the specific condition, can design various different kinds of test methods.
Used for clinical inspection ELISA basically has the following kinds of types:
2.1 double the antigen antibody clip method: double antibody clip detection method is the most commonly used method antigen, operation procedure is as follows: 1) will be specific antibodies and solid phase carrier coupling, form solid phase antibodies. Washing to remove not bound antibodies and impurities. 2) add client specimens, heat preservation reaction. Specimen of antigen and antibody of solid phase combination, form solid phase antigen antibody compound. Washing to remove not combined with other substances. 3) and enzyme standard antibody, heat preservation reaction. Solid phase immune complex on the enzyme standard antigen and antibody binding. Thoroughly washing of the combination of the enzyme standard not antibody. At this time with solid phase supports the enzyme quantity and samples in the amount of antigen client related. 4) and the substrate show color. Solid phase of the enzyme catalysis substrate become non-ferrous products. Through the than color, measured in the amount of antigen know specimens.
2.2 double antigen clip the dama antibody: reaction model and double antibody clip method is similar. Use specific antigen enzyme preparation for bag was and combined, to detect the corresponding antibody. And the indirect method of antibody of different enzymes to mark antigen enzyme standard instead of resistance to antibodies. This method does not need to dilute the client specimens, can be directly used to measure, so the relative sensitivity than the indirect method. Hepatitis b markers of resistant HBs testing are using this law. This law is the key enzyme preparation of standard antigen, should according to the different antigen structure, searching for the right marker methods.
2.3 the indirect method the antibody: detection antibody indirect method is commonly used methods. Its principle for use of the enzyme mark of antibody (against human immunoglobulin antibody) to detect and solid phase antigen antibody of client, is called the indirect method. Proceed as follows: will specific antigen and solid phase carrier coupling, form solid phase antigen. Washing to remove the antigen and not combined with impurities. Add the client to dilute the serum, heat preservation reaction. In the serum of distinctive antibody and solid phase the antigen, form solid phase antigen antibody compound. After washing, solid phase carrier leaves only specific antibodies, in the serum of other ingredients in washing process been washed away. Add enzyme standard of antibodies.
YongMei standard of people in total Ig detection antibody, but generally more YongMei standard antiviral IgG detection IgG antibodies. Solid phase immune complex antibodies in the standard antibody antibody binding with enzymes, which indirectly and mark the enzyme. After washing, solid phase carriers and the quantity of enzyme specimen examination the amount of antibody positive correlation. 4) and the substrate show color measuring 2.4 competition law antibody: when antigens in the material is not easy to remove interfering substances, or is not easy to get enough antigens purified, can use this method detection antibody specificity. Its principle for samples and a certain amount of the antibodies in the enzyme standard antibody competition and solid phase antigen. Specimen of antibodies in the more, combined with the enzyme in solid phase mark the fewer antibodies, so positive reaction is color is light in negative reaction. Such as antigens for of high purity, can be directly bag was solid phase. Such as antigens of interfering substances, direct bag was not easy success, can use capture bag was method, the first packet is and solid phase of the corresponding antigen antibody, and then join the antigen, form solid phase antigen. Washing to remove impurities antigen, and then add specimens and enzyme standard antibody binding reaction to competition. The competition law antibody many models, but the specimen and enzyme standard antibody and solid phase antigen with competition. Another mode for the specimen and antigen to join to compete in solid phase antibody union, after washing add enzyme standard antibodies, and combined with the in solid phase antigen. The detection of HBe general use this law.
Article : From Perlong medical (http://www.pl999.net/)
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