Agglutination-labeled complement C1q binding reaction detection technical method based on fusion agglutination reaction and labeled complement C1q binding reaction and application

Abstract

The invention discloses an agglutination-labeled complement C1q binding reaction (A-LCFA) detection technical method based on fusion agglutination reaction and labeled complement C1q binding reaction and application of the A-LCFA detection technical method. According to the method, high simplicity and convenience of the agglutination reaction are combined with high specificity of the complement C1q binding reaction and high sensitivity of a labeling technology; the efficient detection of the target antibody or antigen is realized. The method comprises the following specific steps: reacting a granular antigen or antibody with a sample to be detected to form an agglutination complex, adding a complement C1q binding antigen-antibody complex, and carrying out signal detection by marking a complement C1q antibody. The method is suitable for the field of immunological antibody or antigen detection, and has the characteristics of simplicity and convenience in operation, high specificity and high sensitivity. The embodiment shows successful application of the Brucella antibody detection kit in Brucella antibody detection, and the Brucella antibody detection kit comprises a plate agglutination mode and a column agglutination mode. The technology provides a new solution for immunological detection, and has remarkable practical value and popularization potential.

Description

Technical Field

[0001] This invention relates to the field of immunological antibody or antigen detection techniques and applications, specifically to a detection technique and application based on a fusion agglutination reaction and a labeled complement C1q binding reaction, namely, an "agglutination-labeled complement C1q binding reaction". Background Technology

[0002] There are five types of immunological antigen-antibody reactions: agglutination reactions, precipitation reactions, complement fixation reactions, neutralization reactions, and labeling techniques. Agglutination reactions are characterized by their simplicity, rapid result presentation, easy observation, high quantitative accuracy, low cost, and good stability, but their sensitivity and specificity are relatively poor. Complement fixation reactions are characterized by high specificity, but traditional methods have low sensitivity, making them difficult to commercialize and standardize. Labeling techniques greatly improve detection sensitivity, but specificity remains a challenge.

[0003] The principle of agglutination is that particulate antigens or antibodies with a certain degree of suspension stability react with their corresponding antibodies or antigens, causing the reacting substances to gradually form a network of aggregates. This greatly accelerates their settling speed in the liquid, and the reaction results can be easily obtained by visual inspection or instruments. Agglutination reactions can be divided into direct agglutination reactions and indirect agglutination reactions. Direct agglutination reaction: This is the agglutination phenomenon that occurs when particulate antigens (such as bacteria, red blood cells, etc.) directly bind to their corresponding antibodies. Common techniques include the plate method and the test tube method. The plate method produces visible agglutination within minutes, is simple and rapid, and is often used for identifying bacterial species and determining the ABO blood type of human red blood cells. The test tube method is a classic quantitative test method that can use known antigens to detect the presence and content of specific antibodies in the tested serum. It is often used to assist in clinical diagnosis or for epidemiological surveys. Indirect agglutination reaction: Soluble antigens (or antibodies) are first adsorbed onto the surface of a particulate carrier of a certain size that is not related to immunity, and then react with the corresponding antibody (or antigen). Under suitable conditions, aggregation can occur, a reaction known as indirect agglutination. Microspheres are commonly used particulate carriers; activated carbon particles or aluminum silicate particles are also used, as well as synthetic or natural polymer materials such as polystyrene latex microspheres. Because the carrier particles increase the reaction area of ​​the soluble antigen (antibody), when the antigen (antibody) on the particle binds to a trace amount of antibody (antigen), a visible reaction can occur, making it more sensitive than direct agglutination.

[0004] The principle of complement fixation is as follows: when antigen and antibody highly correspond, a highly specific reaction occurs, altering the antibody conformation and exposing binding sites for C1 in complement components. This allows the largest fragment, C1q, in the C1 molecule to bind to the antigen-antibody reactant, while simultaneously releasing smaller fragments C1r and C1s, initiating the classical complement pathway that activates other complement components. In traditional complement fixation assays, erythrocytes (antigen) and hemolysin (antibody) are used as the display system for the complement fixation reaction. The principle is that if the antibody or antigen to be detected undergoes a highly specific binding reaction, C1q in complement C1 will bind to the antibody in the antigen-antibody conjugate, releasing C1r and C1s, activating the complement system reaction, and consuming the amount of active complement. This results in insufficient active complement to attack the erythrocytes and hemolysin added to the display system, reducing or even preventing hemolysis of the erythrocytes. The lower the degree of hemolysis, the stronger the antigen-antibody reaction; conversely, the higher the degree of hemolysis, the weaker the antigen-antibody reaction. A key advantage of traditional complement fixation reactions is their exceptionally high specificity and accuracy. However, the requirement for fresh sheep red blood cells makes it difficult to produce commercially viable, standardized products, significantly hindering the widespread application of this immunological technique. Furthermore, the use of hemolysis to visualize results results in relatively low sensitivity.

[0005] The principle of labeling technology is to label antibody / antigen molecules with tracers such as horseradish peroxidase, latex microspheres, and fluorescein. When the antigen and antibody bind, the qualitative and quantitative detection of the target substance is achieved through the visualization of the label (e.g., enzyme substrate color reaction, latex microsphere color display, fluorescence signal intensity observation under a fluorescence microscope). The outstanding advantage of current labeling-based immunological detection technologies is their extremely high sensitivity. They are the preferred method for detecting trace amounts of target antibodies or antigens. However, because the immunological reaction method they employ is a direct antigen-antibody reaction, non-specific reactions with incomplete antigen-antibody correspondence can occur, and this non-specificity increases with increasing sensitivity.

[0006] Mao Kairong et al. disclosed a Brucellosis CF-ELISA antibody detection kit (published on April 26, 2017), which is an assay technique that integrates complement C1q binding reaction and enzyme-linked immunosorbent assay (ELISA). It uses enzyme-labeled complement C1q monoclonal antibodies to link the complement C1q binding reaction with the enzyme colorimetric reaction.

[0007] However, there is still a lack of a detection technique that combines the simplicity of agglutination tests with the high specificity of complement fixation tests and the high sensitivity of labeling tests.

[0008] In contrast, this invention is a technical method that integrates agglutination reaction and complement C1q binding reaction. The particulate conjugates generated by the antigen-antibody agglutination reaction allow the reaction conjugates to be rapidly separated from non-agglutinated substances in solution through methods such as agglutination or filtration. Furthermore, the conjugates are labeled with complement C1q antibodies, simultaneously demonstrating the binding of complement C1q to the antigen-antibody specific reactants. Summary of the Invention

[0009] The purpose of this invention is to provide a detection technique and application of "aggregation-labeled complement C1q binding reaction" based on fusion aggregation reaction and labeled complement C1q binding reaction, so as to solve the problems mentioned in the background art.

[0010] To achieve the above objectives, the present invention provides the following technical solution: a technical method for fusing agglutination reaction and labeled complement C1q reaction. It mainly utilizes the specific agglutination reaction of agglutinating antigen-antibody pairs, followed by further binding of complement C1q and labeled complement C1q antibody, forming a fusion of two traditional reaction types. This results in a complex containing four substances: antigen, antibody, complement C1q, and labeled complement C1q antibody. By identifying whether agglutination occurs and correlating it with the identified complementary C1q binding reaction, the presence of the corresponding target substance (specific antibody or antigen) in the tested sample can be determined.

[0011] A detection technique based on the "agglutination-labeled complement C1q binding reaction" includes the following steps:

[0012] S1, Particulate antigens or antibodies are agglutinated with the corresponding antibodies or antigens in the sample to be tested to form agglutination complex;

[0013] S2, add complement C1q to the agglutination complex to allow it to bind to the antigen-antibody complex;

[0014] S3, add a labeled complement C1q antibody to form a complex containing antigen, antibody, complement C1q and labeled complement C1q antibody;

[0015] S4. By detecting the labeling signal in the complex, the presence of the target antibody or antigen in the sample to be tested is determined.

[0016] Preferably, the particulate antigen or antibody is a natural particulate antigen or antibody in a direct agglutination reaction, or a soluble antigen or antibody adsorbed onto a carrier particle in an indirect agglutination reaction.

[0017] Preferably, the carrier particles are selected from substances that have the particle characteristics required for agglomeration reactions, such as microspheres, aldehyde-modified red blood cells, tanned red blood cells, activated carbon particles, aluminum silicate particles, or particles made of polymer materials.

[0018] Preferably, the labeling agent for the complement C1q antibody is selected from substances with labeling and display properties, such as enzymes, fluoresceins, latex microspheres, or radioisotopes.

[0019] Preferably, the complement C1q is derived from the serum to be tested or from exogenous serum containing complement C1q.

[0020] An "agglutination-labeled complement C1q binding reaction" detection technique, based on fusion agglutination and labeled complement C1q binding reactions, is applied in the field of immunological antibody or antigen detection. If the antigen-antibody specific agglutination reaction binds to complement C1q, complement C1q antibody, and a label, a complex containing antibody, antigen, complement C1q, complement C1q antibody, and label is formed, displaying a detectable signal with both agglutination particle and labeling display characteristics, thus yielding a positive result. If there is no antigen-antibody agglutination reaction, or if the agglutination reaction fails to bind complement C1q, no detectable signal with both agglutination particle and labeling display characteristics is displayed, resulting in a negative result.

[0021] Beneficial effects

[0022] This invention provides a detection technique and application based on the "agglutination-labeled complement C1q binding reaction" of fusion agglutination reaction and labeled complement C1q binding reaction, which has the following beneficial effects:

[0023] 1. The detection technology and application of "agglutination-labeled complement C1q binding reaction" based on fusion agglutination reaction and labeled complement C1q binding reaction have outstanding substantive features and significant progress compared with the prior art. The technology and method have the same simplicity as agglutination tests, while also having the high specificity of complement binding tests and the high sensitivity of labeling tests.

[0024] 2. The "agglutination-labeled complement C1q binding reaction" detection technology and its application based on fusion agglutination reaction and labeled complement C1q binding reaction, the invention and its related products are applicable to various fields of immunological detection of antigens and antibodies, such as antibody or antigen detection of infectious diseases, and detection of targets with biological antigenicity, etc., and have significant value in improving the level of immunological detection technology. Attached Figure Description

[0025] Figure 1 The results of Brucella PA-LCFA antibody detection in Example 1 are shown, where + represents a positive serum reaction plate; - represents a negative serum control reaction plate; and 0 represents a dilution control reaction plate.

[0026] Figure 2The results of Brucella CA-LCFA antibody detection in Example 2 are shown, where H+ represents a positive serum reaction tube; H- represents a negative serum control reaction tube; and H represents a dilution control reaction tube. Detailed Implementation

[0027] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0028] Example 1: This invention provides a technical solution: Example 1: Brucella agglutination-red blood cell labeled complement C1q fixation assay (PA-LCFA) antibody detection method.

[0029] Test method:

[0030] Step 1: Preparation of erythrocyte-labeled complement C1q antibody

[0031] Aldehyde-containing erythrocytes: Whole blood from a healthy animal was added to an equal volume of Alder's solution (erythrocyte preservation solution), centrifuged at 2000 rpm for 10 min, and the precipitated erythrocytes were collected and their volume recorded. 25 mL of pH 7.2 PBS was added to each 1 mL of precipitated erythrocytes, mixed well, and then 2 mL of 2.5% glutaraldehyde was slowly added dropwise. After shaking at room temperature for 1 h, the mixture was centrifuged at 2000 rpm for 10 min and washed. The precipitate was collected and reconstituted into a 10% aldehyde-containing erythrocyte suspension with physiological saline containing 0.01% thimerosal.

[0032] Tanninized red blood cells: Prepare a 1% concentration of 10% aldehyde-treated red blood cells by mixing 0.01% tannic acid-containing physiological saline with an equal volume of 0.1 mol / L pH 7.2 PBS. Shake in a 37°C water bath for 15 min, centrifuge at 2000 rpm for 10 min, and wash the precipitate three times with pH 7.2 PBS. Then, prepare a 1% concentration of red blood cells again with 0.1 mol / L pH 6.4 PBS solution and mix thoroughly. This is the tanninized red blood cell preparation.

[0033] Antibody-sensitized red blood cells: Tanned red blood cells and complement C1q antibody (titer 1:1000) were mixed at a ratio of 2:1 and sensitized by shaking in a 37°C water bath for 45 min. The mixture was then centrifuged at 2000 rpm for 10 min, and the precipitate was resuspended in pH 7.2 PBS to prepare a sensitized red blood cell suspension. This is the red blood cell-labeled complement C1q antibody (RL-C1q antibody).

[0034] Step 2: Brucella PA-LCFA antibody detection

[0035] 20 μL of red blood cell agglutination antigen (commercial reagent) on a red blood cell plate, 20 μL of C1q antibody-sensitized red blood cells, and 20 μL of positive serum (or negative serum control) were mixed and reacted for 8 minutes. The results were then observed (see [link to product description]). Figure 1 Brucella PA-LCFA antibody test results).

[0036] Result determination:

[0037] Positive serum reaction results showed that the agglutinated particles were a mixture of dark red and tiger red, and the liquid was clear.

[0038] The results of the negative serum control reaction showed that the liquid was a suspension of mixed red and dark red.

[0039] in conclusion:

[0040] The results of Example 1 demonstrate that the plate agglutination-erythrocyte-labeled complement C1q binding reaction, which integrates agglutination and complement C1q binding reactions, can specifically detect Brucella antibodies. Therefore, it is inferred that it is also applicable to the detection of specific reactions of other immunological antigens and antibodies.

[0041] Example 2: Brucella column agglutination-labeled complement C1q fixed assay (CA-LCFA) antibody detection technique.

[0042] Test method:

[0043] Step 1: Preparation of glass microspheres

[0044] Fill 0.5ml centrifuge tubes with glass microbeads of 150-200μm and 100-200μm in diameter to the 0.5ml mark.

[0045] Step 2: Brucella column agglutination-complement C1q labeling binding reaction antibody detection

[0046] Take 20 μL of Brucella rose benzene plate agglutination test antigen (commercial reagent), 20 μL of positive serum (negative serum control, dilution control), and 20 μL of erythrocyte-labeled complement C1q antibody (see step 1 of Example 1). Mix and react for 8 min. Then transfer the mixture to a centrifuge tube filled with glass beads and observe the results (see...). Figure 2 Brucella CA-LCFA antibody test results).

[0047] Result determination:

[0048] Positive serum reaction results showed that the column surface exhibited a mixture of tiger red and dark red aggregated particles, and the liquid column was tiger red.

[0049] The negative serum control reaction results showed that the column surface contained dark red agglutinating particles consistent with the red blood cell-labeled complement C1q antibody, and the liquid column was tiger red.

[0050] Diluent control: The column surface is colorless, and the liquid column is a mixture of tiger red and dark red.

[0051] in conclusion:

[0052] The results of Example 2 demonstrate that the column agglutination-complement C1q labeling binding reaction, which integrates agglutination, complement C1q binding, and column filtration technologies, can specifically detect Brucella antibodies. Therefore, it is inferred that this method is also applicable to the detection of specific reactions between other immunological antigens and antibodies.

[0053] The above embodiments are merely illustrative of the principles and effects of the present invention and are not intended to limit the invention. Any person skilled in the art can modify or alter the above embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or alterations made by those skilled in the art without departing from the spirit and technical concept disclosed in the present invention should still be covered by the claims of the present invention.

Claims

1. A detection technique based on the "aggregation-labeled complement C1q binding reaction" of fusion agglutination reaction and labeled complement C1q binding reaction, characterized in that, Includes the following steps: S1, Particulate antigens or antibodies are agglutinated with the corresponding antibodies or antigens in the sample to be tested to form agglutination complex; S2, add complement C1q to the agglutination complex to allow it to bind to the antigen-antibody complex; S3, add a labeled complement C1q antibody to form a complex containing antigen, antibody, complement C1q and labeled complement C1q antibody; S4. By detecting the labeling signal in the complex, the presence of the target antibody or antigen in the sample to be tested is determined.

2. The detection technique and method for "aggregation-labeled complement C1q binding reaction" based on fusion agglutination reaction and labeled complement C1q binding reaction as described in claim 1, characterized in that: The particulate antigen or antibody is a natural particulate antigen or antibody in a direct agglutination reaction, or a soluble antigen or antibody adsorbed onto a carrier particle in an indirect agglutination reaction.

3. The detection technique and method for "aggregation-labeled complement C1q binding reaction" based on fusion agglutination reaction and labeled complement C1q binding reaction as described in claim 2, characterized in that: The carrier particles are selected from microspheres, aldehyde-modified red blood cells, tanned red blood cells, activated carbon particles, aluminum silicate particles, or particles made of polymer materials, and are substances with the particle characteristics required for the aggregation reaction.

4. The detection technique and method for "aggregation-labeled complement C1q binding reaction" based on fusion agglutination reaction and labeled complement C1q binding reaction as described in claim 1, characterized in that: The labeling agent for the complement C1q antibody is selected from enzymes, fluorescein, latex microspheres, or radioisotopes, and is a substance with labeling and display properties.

5. The detection technique and method for "aggregation-labeled complement C1q binding reaction" based on fusion agglutination reaction and labeled complement C1q binding reaction as described in claim 1, characterized in that: The complement C1q is derived from the serum to be tested or from exogenous serum containing complement C1q.

6. The application of a detection technique according to any one of claims 1-5 in the field of immunological antibody or antigen detection, characterized in that: If, after a specific antigen-antibody agglutination reaction, complement C1q, complement C1q antibody, and a label are bound, a complex containing antibody, antigen, complement C1q, complement C1q antibody, and a label will be formed. This complex will display a detectable signal exhibiting both agglutination particle characteristics and labeling display characteristics, resulting in a positive result. If there is no antigen-antibody agglutination reaction, or if the agglutination reaction fails to bind complement C1q, no detectable signal exhibiting both agglutination particle characteristics and labeling display characteristics will be displayed, resulting in a negative result.

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