CN1098201A - One-hole Assay of Multiple Immune Markers of Hepatitis Virus - Google Patents

Abstract

The most commonly used hepatitis virus immune markers are HBsAg, anti-HBs, HBeAg, anti-HBe, anti-HAV, and anti-HCV. A hepatitis virus immune marker in serum. In the present invention, the wells of the reaction plate are divided into 2-5 small wells according to the actual needs, which are respectively coated with different antigens and antibodies. React in medium for 1 hour, and use TBM substrate system to develop color. Through one operation, more than two immune markers of hepatitis virus can be measured in the same well at the same time, which is 1-4 times more effective than the conventional operation method.

Description

The invention belongs to the enzyme immune method of hepatitis virus immune marker.

The immune markers of hepatitis virus mainly include anti-HAV (anti-Hepatitis A virus, hepatitis A virus antibody), HBsAg (Hepatitis B surface Antigen, hepatitis B virus surface antigen), HBeAg (Hepatitis B e Antigen, hepatitis B virus e antigen), anti-HBc (anti-HBc Hepatitis B core, hepatitis B virus surface antibody), anti-HBe (anti-Hepatitis B e, hepatitis B virus e antibody), anti-HCV (anti-Hepatitis C virus, hepatitis C virus antibody). These viral immune markers are often checked using ELISA. However, this enzyme immunoassay method needs to use a multi-hole enzyme-labeled reaction plate, and only one immune marker can be checked in each well, which is very inconvenient to use.

The purpose of the present invention is to provide a fast and simple assay method that can simultaneously display two or more hepatitis virus immune markers through one operation in one well of an enzyme-labeled reaction plate.

Another object of the present invention is to provide an enzyme-labeled reaction plate corresponding to the above-mentioned hepatitis virus immune marker assay method.

The usual enzyme immunosorbent assay method to determine the immune markers of hepatitis mainly uses the sandwich method and the competition method. The application of the hepatitis B virus surface antigen (HBsAg), hepatitis B virus surface antibody (anti-HBs) and hepatitis B virus e antigen (HBeAg) is Sandwich method, when there are immune markers, it develops color; otherwise, it does not develop color. Hepatitis B virus e antibody (anti-HBe) and hepatitis B virus core antibody (anti-HBc) adopt a competition method, and no color will be developed when the antibody to be tested is positive, otherwise color will be developed. In this enzyme immunoassay, the antigen or antibody must first be coated (that is, adsorbed) in the well of the enzyme-labeled reaction plate. When the corresponding antibody or antigen exists in the serum to be tested, it will bind to the reaction plate through the reaction between the antigen and the antibody. superior. Then the horseradish peroxidase (Horseradish peroxidase abbreviated as HRP)-labeled antigen or antibody added at the same time binds to the enzyme-labeled reaction plate through the antibody or antigen in the serum to be tested (sandwich method), or is stimulated by the antibody in the serum. The competition cannot be bound to the plate (competition method), and after adding the enzyme substrate solution, the enzyme catalyzes the substrate to develop color, so as to determine whether there is an antigen or antibody in the serum to be tested. Because the antigen will react with the corresponding antibody, enzyme-labeled HBsAg and enzyme-labeled anti-HBs cannot be in the same solution. Therefore, only one immune marker can be measured in the same well on the reaction plate, and various enzyme markers need to be stored separately. However, research by the inventors has confirmed that if each hole of the microtiter plate is divided into 2-5 small holes, and each small hole is coated with different antigens or antibodies, then they will not interfere with each other. Various hepatitis virus immune markers in the serum will combine with the antigens or antibodies coated in the small wells respectively. Also, the enzyme labels in the same solution: anti-HBs-HRP, anti-HBe-HRP and anti-HBc-HRP will not react with each other. This enzyme-labeled mixture is added into the large well, and can be combined with the antigen and antibody in the small well respectively. Therefore, by adding one blood sample and one enzyme conjugate, multiple immune markers of hepatitis virus can be displayed simultaneously in one large well.

In the present invention, the enzyme-labeled reaction plate is designed into the following shape (as shown in Figure 1-7), the general length is 110-140mm, the width is 45-70mm, the thickness is 10-15mm, and 4×10 large holes can be arranged . Its size and number of macropores are practically unlimited. The key is the design of the large holes. The diameter of the large hole (1) is generally 6-12mm, and the hole depth is 10-14mm. Each large hole is divided into 2-5 small holes. The bottom area of each small hole (2) is 5-15mm ² , and the separation layer ( 3) The height is 5-8mm and the thickness is 0.7-1.1mm. Figure 1 is a schematic diagram of the structure of an enzyme-labeled reaction plate. Figure 2 and Figure 3 are schematic diagrams of dividing a large hole into two small holes. Fig. 4 and Fig. 5 are schematic diagrams of a structure in which a large hole is divided into three small holes. Fig. 6 and Fig. 7 are schematic diagrams of a structure in which a large hole is divided into five small holes. The steps for measuring the immune markers of hepatitis virus proposed by the present invention are as follows: 2-5 separate small holes of each large hole are coated with different immune markers of hepatitis virus respectively, that is, different antigens or antibodies are coated respectively, and the coating material The concentration is 0.5-20ug/ml, and the reaction conditions are generally 39°C±3°C for 2-5 hours, or 6°C±2°C for 15-48 hours. During the test, the serum to be tested is added to each large well, generally 0.05-0.2ml, and at the same time, 0.1-0.1ml of the enzyme-labeled mixture corresponding to the antigen or antibody coated in the small well is added to each large well. 0.3ml, react at 39℃±3℃ for 30-60 minutes. The liquid in the pores is then removed. And wash off unbound material with washing solution, plus tetramethylbenzidine (TMB) substrate system. Generally, there is 45-55ul in each small hole, and after about 10 minutes, the negative or positive results of various immune markers in the serum to be tested will be displayed in a large hole.

In the method proposed by the present invention, as long as the coating is arbitrarily selected in the partitioned small wells of the large holes of the enzyme-labeled reaction plate, and the corresponding mixed solution of enzyme markers is added, any combination of hepatitis virus immune markers can be determined. Two or more of the immune markers. The following are examples of various commonly used combinations:

If anti-HBs, anti-HBe, and HBcAg are respectively coated in separate small wells in a large well of the enzyme labeling reaction plate, add the corresponding anti-HBs-HRP, anti-HBe-HRP, and anti-HBc in the enzyme labeling reaction step. -HRP enzyme marker mixture, which can measure the three immune markers of hepatitis B surface antigen (HBsAg), hepatitis B e antigen (HBeAg) and hepatitis B core antibody (anti-HBc).

If HBsAg and HBeAg are respectively coated in the separate wells of a large hole in the enzyme labeling reaction plate, then the corresponding HBsAg-HRP and anti-HBe-HRP enzyme labeling mixture is added in the enzyme labeling reaction step, and B can be determined. Hepatitis surface antibody (anti-HBs) and hepatitis B e antibody (anti-HBe) are two immune signs of hepatitis.

If anti-HBs, HBsAg, anti-HBe, HBeAg, and HBcAg are respectively coated in a large well of the enzyme labeling reaction plate, add the corresponding anti-HBs-HRP, anti-HBe-HRP in the enzyme labeling reaction step And anti-HBc-HRP enzyme marker mixture, can measure HBsAg, HBeAg, anti-HBc, anti-HBs and anti-HBe five immune markers.

If the anti-HBs and HCV antigens are respectively coated in a large well of the enzyme-labeled reaction plate, add the corresponding anti-HBs-HRP and anti-human IgG-HRP (anti-human immunoglobulin) in the enzyme-labeled reaction step. Protein G enzyme marker) or HCV-HRP (hepatitis C virus antigen enzyme marker) enzyme marker mixture, can detect hepatitis B virus surface antigen (HBsAg) and hepatitis C virus antibody (anti-HCV).

If anti-HBs, anti-HBe and HCV antigens are respectively coated in a large well of the enzyme-labeled reaction plate, the corresponding anti-HBs-HRP, anti-HBe-HRP and anti-human IgG-HRP (or HCV-HRP) enzyme marker mixture can detect HBsAg, HBeAg and anti-HCV.

If HBs, anti-HBe and HAV antigens are respectively coated in the separate wells of a large well of the enzyme labeling reaction plate, the corresponding anti-HBs-HRP, anti-HBe-HRP and anti-u- HRP (anti-human u-chain enzyme marker) enzyme-labeled mixture can detect HBsAg, HBeAg and hepatitis A virus antibody (anti-HAV) (IgM).

Compared with the usual measuring method, the present invention improves the work efficiency by 2-4 times and reduces the amount of serum used. According to the usual method, for the determination of the five immune markers of hepatitis B virus, each serum needs to be added 5 times at the same time, and reacted in 5 wells. reaction. In the present invention, five immune markers can be measured in one (or two) wells, and the results of five hepatitis B immune markers can be displayed simultaneously by adding serum and enzyme markers once (or twice). For the mixed detection of other hepatitis virus immune markers, the work efficiency is also significantly improved, and the amount of serum is correspondingly reduced.

Figure 1 is a schematic diagram of the structure of the enzyme-labeled reaction plate (strip)

Figure 2 is a side view of the structure of a large well divided into two small wells on an enzyme-labeled reaction plate (strip).

Figure 3 is a top view of the structure of a large well divided into two small wells on an enzyme-labeled reaction plate (strip).

Figure 4 is a side view of the structure of a large well divided into three small wells on an enzyme-labeled reaction plate (strip).

Figure 5 is a top view of the structure of a large well divided into three small wells on an enzyme-labeled reaction plate (strip).

Figure 6 is a side view of the structure of the large wells divided into 5 small wells on the enzyme-labeled reaction plate (strip).

Figure 7 is a top view of the structure of the large wells divided into 5 small wells on the enzyme-labeled reaction plate (strip).

Example 1

Coat anti-HBe, anti-HBs and HBcAg in 1 well with 3 intervals, add 0.2ml of serum containing HBeAg, HBsAg and anti-HBc positive at the same time, add anti-HBs-HRP, anti-HBe-HRP and anti-HBc-HRP at the same time The mixed solution was 0.1ml, reacted in a 37°C incubator for 1 hour, and after adding TMB substrate solution, all three interval small wells in the wells showed positive results.

Example 2

In 1 well with 2 spaced wells, coat HBsAg and HBeAg at the same time, add 0.2ml of anti-HBs and anti-HBe positive serum (or add known anti-HBe positive serum to anti-HBs positive serum), and add HBsAg- HRP and anti-HBe-HRP mixed solution 0.1ml, react in a 37°C incubator for 1 hour, after adding TMB substrate solution, there are 2 spacers in the well, the well for measuring anti-HBs develops color, while the well for measuring anti-HBe shows no color color, that is, both wells show positive results.

Example 3

Coat anti-HBs, HBsAg, anti-HBe, HBeAg and HBcAg plus HBsAg, HBeAg, anti-HBc positive serum 0.2ml in each well of the enzyme-labeled reaction plate, react at 37°C for 1 hour, add TMB substrate system, the result is as above In each of the coated wells, there are color development, color development, color development, color development and colorless respectively, that is, the test results are positive for HBsAg, HBeAg and anti-HBc, but negative for anti-HBs and anti-HBe. After adding anti-HBs and anti-HBe positive sera to the above-mentioned coated wells, and adding the above-mentioned enzyme marker mixture at the same time, the color development sequence of the substrate system is colorless, colorless, colorless, colorless and color, that is, the detection result Positive for anti-HBs and anti-HBe.

Example 4

Coat anti-HBs and HCV antigens in each well of the enzyme-labeled reaction plate, and add 0.1-0.2 ml of HBsAg and anti-HCV positive serum. Simultaneously add 0.1-0.2ml of anti-HBs-HRP and HCV-HRP mixture, react at 37°C for 1 hour, after adding TMB substrate, the two small wells develop color respectively, that is, the test results are positive for HBsAg and positive for anti-HCV.

Example 5

Coat anti-HBs and HAV antigens in each well of the enzyme-labeled reaction plate, add HBsAg and anti-HAV positive serum 0.1-0.2ml, add anti-HBs-HRP and anti-human u-HRP mixed solution 0.1-0.2ml at the same time, 37 After reacting at ℃ for 1 hour, TMB substrate solution was added, and the two small wells developed color, showing that both HBsAg and anti-HAV were positive.

Claims (8)

1, a kind of assay method of hepatitis virus immunological marker, its feature is wrapped respectively by different hepatitis virus immunological markers in 2-5 separation aperture of each macropore of enzyme reaction plate, its concentration is 0.5-20ug/ml, reaction conditions is at 39 ± 3 ℃, 2-5 hour, perhaps 6 ± 2 ℃, 15-48 hour, to adding serum 0.05-0.2ml to be checked in each macropore respectively, in each macropore, add simultaneously corresponding to the mixed liquor 0.1-0.3ml that separates the enzyme labeling thing of the hepatitis virus immunological marker of bag quilt in the aperture, reaction is 30-60 minute in 39 ± 3 ℃, remove liquid in the hole then, add tetramethyl benzidine substrate system 45-55ul in every aperture, promptly in a macropore, show the feminine gender or the positive findings of the every immunological marker of serum to be checked after 10 minutes.

2, the assay method of hepatitis virus immunological marker according to claim 1, it is characterized in that, the hepatitis virus of wrapping quilt in the separation aperture in the macropore of enzyme reaction plate respectively is masked as anti-HBs, anti-HBe, HBcAg, then adds corresponding anti-HBs-HRP, anti-HBe-HRP, anti-HBc-HRP enzyme labeling thing mixed liquor to measure three hepatitis immunological markers of the anti-HBc of HBsAg, HBeAg in macropore.

3, the assay method of hepatitis virus immunological marker according to claim 1, it is characterized in that, the hepatitis virus of wrapping quilt in the separation aperture of the macropore of enzyme reaction plate respectively is masked as HBsAg and HBeAg, the mixed liquor that then adds corresponding HBsAg-HRP and anti-HBe-HRP enzyme labeling thing in macropore is to measure two hepatitis immunological markers of anti-HBs and anti-HBe.

4, the assay method of hepatitis virus immunological marker according to claim 1, it is characterized in that, the hepatitis virus of wrapping quilt in the separation aperture of enzyme reaction plate macropore respectively is masked as anti-HBs, HBsAg, anti-HBe, HBeAg and HBcAg, then in macropore, add corresponding anti-HBs-HRP, anti-HBe-HRP and anti-HBc-HRP enzyme labeling thing mixed liquor, to measure HBsAg, HBeAg, anti-HBc, anti-HBs and anti-HBe five-item hepatitis virus immunity sign.

5, the assay method of hepatitis virus immunological marker according to claim 1, it is characterized in that, the hepatitis virus of wrapping quilt in the separation aperture of enzyme reaction plate macropore respectively is masked as anti-HBs and HCV antigen, then in macropore, add corresponding anti-HBs-HRP and anti-human IgG enzyme labeling thing (or HCV-HRP) mixed liquor, to measure HBsAg and anti-HCV.

6, the assay method of hepatitis virus immunological marker according to claim 1, it is characterized in that, the hepatitis virus of wrapping quilt in the separation aperture of the macropore of enzyme reaction plate respectively is masked as anti-HBs, anti-HBe and HCV antigen, then in macropore, add corresponding anti-HBs-HRP, anti-HBe-HRP and anti-human IgG-HRP(or HCV-HRP) enzyme labeling thing mixed liquor, to measure HBsAg, HBeAg and anti-HCV.

7, the assay method of hepatitis virus immunological marker according to claim 1, it is characterized in that, the hepatitis virus of wrapping quilt in the separation aperture of enzyme reaction plate macropore respectively is masked as anti-HBs, anti-HBe and HAV antigen, then in macropore, add corresponding anti-HBs-HRP, anti-HBe-HRP and anti-u-HRP enzyme labeling thing mixed liquor, to measure HBsAg, HBeAg and anti-HAV(IgM).

8, a kind of enzyme reaction plate that is used to measure the hepatitis virus immunological marker, thickness is 10-15mm, and having diameter on it is that 6-12mm, hole depth are the macropore of 10-14mm, it is characterized in that this macropore is separated into 2-5 aperture, and the floorage of aperture is 5-15mm ², the separate layer height is 5-8mm, thickness is 0.7-1.1mm.

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