Principles and precautions of radioimmunoassay

The principle of radioimmunoassay is to use radionuclide to label antigen or antibody, and then combine with the tested antibody or antigen to form an antigen-antibody complex for analysis. It is divided into two methods.

1. Competitive RIA, also known as traditional RIA

Main feature: The antigen is labeled. The principle: unknown antigen Ag + labeled antigen Ag + known quantitative antibody Ab labeled antigen and antibody complex AgAb + unknown antigen and antibody complex Ag Ab + free labeled antigen Ag (removed) (there are many unknown antigens, the complex of labeled antigen and quantitative antibody binding There are few things, showing a negative correlation).

Clinical detection of alpha-fetoprotein (AFP), carcinoembryonic antigen (CEA), 8-microglobulin (pz-MG), ferritin (Fer), human chorionic gonadotropin p subunit (HCG-13), etc. Both are principles of competitive RIA. Competitive RIA method, according to the difference between the order of loading and the number of incubations, the reaction method is divided into three types: the equilibrium method, the non-labeled antigen (analyte and standard), antibody, and labeled antigen are added to the reaction tube in sequence, after mixing Incubate once until the reaction reaches dynamic equilibrium, and then add a separating agent to separate B (complex) and F (free material) such as: AFP, 8-MG, Fer; sequential addition method, first the non-labeled antigen (test substance) And standard), the first incubation with the antibody in the reaction tube, after the reaction reaches dynamic equilibrium. After adding labeled antigen and incubating for a second time, separate B and F, such as: CEA; emergency test method: the reaction method proposed for the clinical urgent need of test results, the reaction is terminated when the RIA reaction reaches dynamic equilibrium; the separation agent Choices are, PEG (polyethylene glycol), secondary antibody, etc.

At present, the method of combining the second antibody with PEG is generally called dual antibody-PEG method.

PEG principle: When the concentration of PEG reaches 7-9, it can precipitate the antigen-antibody complex.

Advantages: economical, simple and universal.

Disadvantages: poor repeatability and high non-specific binding rate.

Second antibody: The first antibody is an antibody that can specifically bind to the tested antigen, and comes from a rabbit or guinea pig. The primary antibody is used as an antigen and acts on sheep and horses. The antibody produced against the primary antibody is called secondary antibody. The second antibody specifically binds to the first antibody under appropriate conditions, generating an immune complex with a large molecular weight (AgAb × Ab), which can naturally precipitate.

Advantages: good separation effect. Low non-specific binding rate.

Disadvantages: increase funding. A second incubation is required, extending the time.

Therefore, a dual antibody-PEG method combining the two has been produced, which combines the advantages of the above two methods.

2. Non-competitive RIA, also known as Immunoradiation Analysis (IRMA)

Main feature: The antibody is labeled.

According to the reaction principle, it is divided into two types:

(1) Unit point IRMA

The principle: the antigen has only one antigenic determinant, and the tested antigen is a small molecule antigen, Ab + AgAg Ab + Ab + ImAd—Ag ImAd—AgAb + Ag Ab (removed) (negative correlation): (ImAd—Ag) solid phase antigen immunity Adsorbent, usually coated with polystyrene plastic beads; dual-site IRMA: antigen has two epitopes, the two subtype antibodies used do not interfere with ImAd—Ab + AgImAd when binding to the same antigen molecule —Ab—Ag + AbImAd-Ab—Ag-Ab + Ab (excess, free): (ImAd—Ab) solid-phase antibody immunosorbent, usually coated with polystyrene plastic beads.

(2) Double-site IRMA, also known as double antibody sandwich method

From the sequence of sample addition measurement, there are three methods: the solid phase antibody binds to the unknown antigen first, and then to the labeled antibody (positive two-step method). Then remove the free labeled antibody and measure the radioactivity count of the solid-phase antibody-antigen-labeled antibody complex; the unknown antigen is first bound to the labeled antibody and then to the in-phase antibody (reverse two-step method). Then remove the free labeled antibody, and measure the radioactivity count of the solid-phase antibody-antigen-labeled antibody complex; the unknown antigen reacts with the solid-phase antibody and the labeled antibody simultaneously (one-step method, also known as simultaneous sample addition method). The free labeled antibody is then removed and the radioactive count of the solid-phase antibody-antigen-labeled antibody complex is measured.
These three methods all produce sandwich-like antibody-antigen-antibody complexes, so they are also called the double antibody sandwich method.

Carbohydrate antigens CA50 and CA125 use the third method (one-step method). The unknown antigen in the serum reacts with the solid-phase antibody and the labeled antibody at the same time to generate a sandwich-like antibody-antigen-antibody complex, which is then removed For free labeled antibody, the radioactivity count of the solid-phase antibody-antigen-labeled antibody complex is measured.

3. Influencing factors of RIA method

pH and ionic strength; reaction temperature, the temperature is generally 37 ℃, there are also 45 ℃, room temperature, 4 ℃ refrigerator; reaction time, operating precautions: wear gloves, protective glasses, etc., remove the kit from the refrigerator, at room temperature Leave it for 30 minutes before use.

(1) Numbering: the first row is the standard tube: according to the concentration of the standard product from low to high A, B, C, D, E, F, G ...; the second row is the tested samples 1, 2, 3, 4 , 5, 6, ....

(2) The sample addition should be accurate, the use of pipettes (two gears, one suction, two discharge) tip (one specimen and one tip to avoid mutual contamination). Add reagent: first look at the bottle label, then shake well, and finally suck it. (The marker is generally red, and the primary antibody is blue).

(3) Incubation time should be guaranteed: incubation according to the time required in the manual can be extended, not shortened.

(4) Adding the separating agent: the mixing and standing time can be extended, not shortened. Ensure that the antigen-antibody complex is fully bound to the second antibody.

(5) Temperature: Pay attention to observe the water temperature of the water bath, the error should not be too large, it is ± 1 ℃, the room temperature is about 21 ℃.

(6) Centrifugal time: It should also be operated according to the requirements in the manual, and the speed is generally 3 500r / min. When placing the reaction tube in the centrifuge, try to keep it upright (because tilting may cause liquid to flow out). When sucking the supernatant, the precipitate is at the bottom of the test tube. The tip of the vacuum pump's tip cannot be inserted directly into the center of the bottom of the test tube (it will suck up the precipitate, and our purpose is to separate and retain the precipitate). The tube wall of the test tube is lowered, and the test tube should be slightly inclined, but it should stop when it is about to be inserted into the edge of the sediment, and rise quickly to remove it. Keep a little less supernatant. Mainly to ensure the integrity of the sediment.

(7) When measuring into the human immune counter, pay attention to the quality of the curve: whether it needs to be modified to eliminate the bad points.

(8) Reporting results: If you encounter abnormal results, you should check again to see if the sediments are all present, how the patient is, and whether the results are consistent with the patient's conditions before they can be reported. In the non-competitive RIA method, when cleaning the solid-phase coated beads, the cleaning frequency should be strictly in accordance with the instructions in the manual, and must not be reduced. Although there are many factors affecting the RIA method, as long as the above matters are noted during operation, the stability of the RIA method is still quite good. The main reason is that it has radioactive pollution. This problem is not easy to solve and has affected its future development. Nowadays, electrochemiluminescence immunoassay, chemiluminescence immunoassay, enzyme-linked immunoassay, and magnetic enzyme immunoassay have been launched one after another, and they have developed rapidly. The RIA method will be eliminated.

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