Principles of radioimmunoassay



Radioimmunoassay is a labelled immunoassay technique created by Yalow and Berson in 1960. Due to the detection sensitivity of the labeled radionuclide, the sensitivity of this method is as high as ng or even pg level. The accuracy of the assay was good, with recoveries in ng amounts close to 100%. This method is especially suitable for the quantitative determination of trace proteins, hormones and peptides.

1. Basic Principles

The basic principle of radioimmunoassay is the competitive binding reaction of labeled antigen (Ag*) and unlabeled antigen (Ag) to specific antibody (Ab).

In this reaction system, the amounts of labeled antigen and antibody as reagents are fixed. The amount of antibody is generally taken to be able to bind 40% to 50% of the labeled antigen, while the non-labeled antigen in the test specimen varies. Depending on the amount of antigen in the specimen, different reaction results are obtained.

Assuming that the test sample does not contain antigen, the reaction conditions are:
In the presence of antigens in the specimen, for example:

When the labeled antigen, the unlabeled antigen and the specific antibody coexist in a reaction system, the labeled antigen and the unlabeled antigen have the same binding force to the specific antibody, so they compete with each other for binding the specific antibody. Since the amount of labeled antigen and specific antibody is fixed, the amount of labeled antigen-antibody complex formed varies with the amount of unlabeled antigen. The amount of non-labeled antigen increases, and more antibodies are bound accordingly, thereby inhibiting the binding of labeled antigen to antibody, reducing the labeled antigen-antibody complex correspondingly, and correspondingly increasing the free labeled antigen, that is, the radioactive intensity in the antigen-antibody complex is similar to that of the antibody. The concentration of antigen in the tested specimen is inversely proportional (Figure 1).

If the antigen-antibody complex is separated from the free labeled antigen and its radioactivity is measured separately, the ratio (B/F) of the bound labeled antigen (B) to the free labeled antigen (F) can be calculated, or the binding rate can be calculated. [B/(B+F)], which is a function of the amount of resistance in the specimen. A dose-response curve can be drawn by reacting with a series of different doses of standard antigen and calculating the corresponding B/F (Figure 2). The tested sample is measured under the same conditions, and the B/F value is calculated, and the antigen content in the sample can be found on the dose-response curve.

(1) Markers

There are two types of nuclides used for labeling: γ-rays and β-rays. The former is mainly 131I, 125I, 57Cr and 60Co; the latter has 14C, 3H and 32P. The selection of radionuclides first considers specific activity. For example, the theoretical value of the specific activity of 125I is 64.38×104GBq/g (1.74×104Ci/g), and the maximum specific activity of 14C with a longer half-life is 166.5GBq/g (4.5Ci/g). Compared with the two, 1mol125I or 14C binds to the antigen, and the sensitivity of 125I is about 3900 times greater than that of 14C. And because 125I has a suitable half-life, low-energy gamma rays are easy to label, so 125I is currently a commonly used RIA marker.

(2) Marking method

The methods of labeling 125I can be divided into two categories, namely direct labeling and indirect labeling.

The direct labeling method is to directly bind 125I to the tyrosine residues in the protein side chain. The advantage of this method is that it is easy to operate, and it is a single-step binding reaction between 125I and protein. It can make more 125I bind to the protein, so the marker has a high specific radioactivity. But this method can only be used to label tyrosine-containing compounds. In addition, if tyrosine-containing residues have protein specificity and biological activity, the activity is easily damaged by labeling.

The indirect labeling method (also known as the linking method) is to label the carrier with 125I, and then combine with the protein after purification. Due to the complicated operation, the specific radioactivity of the labeled protein is significantly lower than that of the direct method. However, this method can label peptides and certain proteins lacking tyrosine. For example, when direct labeling causes changes in protein tyrosine structure and damages its immunity and biological activity, indirect methods can also be used. Its labeling reaction is relatively mild, which can avoid the loss of biological activity caused by the direct addition of protein to 125I solution. The most commonly used direct labeling of chloramine T is described below.

Chloramine T is the sodium salt of the N-chloro derivative of p-toluenesulfonamide, which gradually decomposes in aqueous solution to form hypochlorous acid, which is an oxidant. In a partial alkaline solution (pH 7.5), chloramine T oxidizes I- of 125I to I+, and I+ replaces the hydrogen of the phenyl ring of protein tyrosine to form diiodotyrosine. The reaction formula is as follows:

The level of radioactive iodine labeling is related to the content of tyrosine in the antigen (protein or polypeptide) molecule and the degree of exposure of tyrosine in the molecule. When the molecule contains more tyrosine and is exposed to the outside, the labeling rate just high.

Labeling method: Add the purified antigen and 125I to the bottom of the small test tube, then quickly flush the freshly prepared chloramine T, mix and shake for tens of seconds to 2 minutes, and then add sodium metabisulfite to stop the reaction. Then add KI solution to dilute. It was then separated on a Dextran G column and collected tube by tube. The radioactivity intensity (number of pulses/min, cpm) was measured by a well-type scintillation counter. The front part was the labeled antigen peak and the back part was the free 125I peak. Add an equal amount of 1% albumin as a stabilizer in the labeled antigen peak test tube, which is the labeled antigen solution.

(3) Identification of markers

1. Content of radioactive free iodine All proteins were precipitated with trichloroacetic acid (bovine serum albumin was added to the identified sample in advance), and the cpm values ​​of the precipitate and supernatant were measured respectively. Generally, free iodine is required to be less than 5% of the total radioactive iodine. After the labeled antigen is stored for a long time, the deiodination of the labeled substance will occur. If the free iodine exceeds 5%, the free iodine should be repurified to remove this part of the free iodine.

2. There is always some loss of antigenic activity during immunological activity labeling, but it should be avoided as much as possible. The inspection method is to add a small amount of labeled antigen to excess antibody, separate B and F after the reaction, measure the radioactivity respectively, and calculate the BT%. This value should be above 80%, and the larger the value, the less antigenic damage.

3. The radioactive ratio-labeled antigen must have sufficient radioactive ratio. Specificity or specific radioactivity refers to the radiation intensity per unit weight of antigen. The specific radioactivity of the labeled antigen is expressed in mCi/mg (or mCi/mmol). The higher the ratio, the more sensitive the assay. The ratio of labeled antigen is calculated based on the availability (or labeling rate) of radioactive iodine:

For example: 5μghGH is labeled with 2mCiNa125I, and the labeling rate is 40%, then

(4) Detection of antiserum

Antiserum containing specific antibodies is the main reagent for radioimmunoassay, and is often obtained by immunizing small animals with antigens to induce the production of polyclonal antibodies. The quality of the antiserum directly affects the sensitivity and specificity of the assay. The main indicators of antiserum quality are affinity constant, cross-reaction rate and titer.

1. Affinity constant Affinity constant (affinity constant) is commonly expressed by K value. It reflects the binding ability of the antibody to the corresponding antigen. The unit of K value is mol/L, which means that when 1 mol of antibody is diluted into several liters of solution, the binding rate to the corresponding antigen reaches 50%. The larger the antiserum K value, the better the sensitivity, precision and accuracy of the radioimmunoassay. The K value of the antiserum reaches 109~1012mol/L to be suitable for radioimmunoassay.

2. Cross-reaction rate Some substances determined by radioimmunoassay have very similar structures, such as T3 and T4 of thyroxine, and estradiol and estriol of estrogen. Antisera directed against an antigen tend to cross-react with its analogs. Therefore, the cross-reaction rate reflects the specificity of the antiserum, and if the cross-reaction rate is too large, the accuracy of the analytical method will be affected. The method for measuring the cross-reaction rate is to use the same method as the corresponding antigen and its analogs of the antiserum, and observe the amount when 50% of the zero standard tube is replaced. Taking T3 antiserum as an example, the replacement zero standard 50% T3 is 1 ng, and its analog T4 needs 200 ng, so the cross-reaction rate is: 1/200=0.5%.

3. Titer The titer refers to the highest dilution that can react with the antigen when the serum is diluted. It reflects the concentration of effective antibodies in the antiserum. In a radioimmunoassay, the titer is the dilution of the antiserum that binds 50% of the labeled antigen in the absence of the test antigen.

2. Measurement method

The determination by radioimmunoassay is divided into three steps, namely, the competitive inhibition reaction of antigen-antibody, the separation of B and F, and the measurement of radioactivity.

(1) Antigen-antibody reaction

Antigen (standard and test specimen), labeled antigen and antiserum are quantitatively added to a small test tube in sequence, and the reaction is carried out at a certain temperature for a certain period of time, so that the competitive inhibition reaction reaches a balance. Different qualities of antibodies and different amounts of antigen have different requirements for incubation temperature and time. If the antigen content of the tested sample is relatively high and the affinity constant of the antiserum is relatively large, a higher temperature (15-37°C) can be selected for a shorter incubation time; otherwise, a longer time should be performed at a low temperature (4°C). The formation of antigen-antibody complexes is relatively firm.

(2) B, F separation technology

In the RIA reaction, the content of labeled antigen and specific antibody is extremely small, and the formed labeled antigen-antibody complex (B) cannot be precipitated by itself, so it needs to be thoroughly precipitated with a suitable precipitant to complete the interaction with the free labeled antigen. (F) Separation. In addition, for small molecular weight antigens, B and F can also be separated by adsorption.

1. Secondary antibody precipitation method This is the most commonly used precipitation method in RIA. Goat anti-rabbit IgG serum (secondary antibody) is prepared by immunizing another animal (eg, sheep) with IgG from an animal (eg, a rabbit) that produces a specific antibody (primary antibody). Since the first and second antibodies are used in this reaction system, it is called the double antibody method. After the antigen reacts with the specific antibody, the second antibody is added to form a diabody complex composed of the antigen-first antibody-second antibody. However, due to the very low concentration of the primary antibody and its very few complexes, centrifugation cannot be performed. Therefore, a certain amount of serum or IgG of the same species as the primary antibody is added during the separation to form visible IgG with the secondary antibody. The precipitate forms a co-precipitate with the diabody complexes of the above-mentioned antigens. The precipitate containing the bound antigen (B) can be precipitated by centrifugation and separated from the free labeled antigen (F) in the supernatant.

The solid-phase secondary antibody is formed by binding the secondary antibody on the particulate solid-phase carrier. Using solid-phase secondary antibody to separate B and F, the operation is simple and fast.

2. Polyethylene glycol (PEG) precipitation method Recently, various RIA reaction systems have gradually adopted PEG solution instead of secondary antibody as precipitant. The main advantage of the PEG precipitant is that it is easy to prepare and the precipitation is complete. The disadvantage is that the very specific binding rate is higher than with the secondary antibody, and the precipitate is easily reconstituted at temperatures above 30°C.

3. The PR reagent method is a method that combines the diabody with the PEG method. This method maintains the advantages of the two, saves the consumption of the two, and separates them quickly and easily.

4. Activated carbon adsorption method Small molecule free antigens or haptens are adsorbed by activated carbon, and macromolecular complexes remain in solution. For example, a layer of glucan is coated on the surface of activated carbon, so that the surface has a mesh with a certain aperture, and the effect is better. After the antigen reacts with the specific antibody, dextran-activated carbon is added. Place for 5~10min to make the free antigen adsorbed on the activated carbon particles, centrifuge to precipitate the particles, and the supernatant contains the bound labeled antigen. This method is suitable for the determination of steroid hormones, cardiac glycosides and various drugs, because they are relatively non-polar and smaller than antigen-antibody complexes, which are easily adsorbed by activated carbon.

(3) Determination of radioactive intensity

After the separation of B and F, the radioactive intensity can be measured. There are two types of measuring instruments, liquid scintillation counters (beta rays, such as 3H, 32P, 14C, etc.) and crystal scintillation counters (beta rays, such as 125, 131I, 57Cr, etc.).

The counting unit is the number of electrical pulses output by the detector, and the unit is cpm (counts/minute), which can also be expressed in cps (counts/second). If the efficiency of this measurement system is known, the intensity of the radioactive source can also be calculated as dpm (decays per minute) or dps (decays per second).

A standard curve graph is required for each determination, with the different concentrations of the standard antigen as the abscissa and the corresponding radioactive intensities obtained in the assay as the ordinate. The radioactive intensity can be selected as B or F, and the calculated value of B/(B+F), B/F and B/B0 can also be used. The samples should be measured in duplicate, the average value should be taken, and the corresponding tested antigen concentration should be found on the prepared standard curve.