AU2020104302A4

AU2020104302A4 - Target and Hemoglobin Bispecific Immune Complex and Preparation and Detection Method Thereof

Info

Publication number: AU2020104302A4
Application number: AU2020104302A
Authority: AU
Inventors: Weiwei Cui; Dong Li
Original assignee: Jilin University
Current assignee: Jilin University
Priority date: 2020-12-24
Filing date: 2020-12-24
Publication date: 2021-03-18
Anticipated expiration: 2028-12-24

Abstract

The present disclosure provides a target and hemoglobin bispecific immune complex and a preparation and detection method thereof. An anti-target-antigen specific antibody-secondary antibody-anti-hemoglobin specific antibody-hemoglobin structure is used. The complex is added to a specimen to be detected, and an enzyme substrate is added to perform chromogenic reaction to obtain a detection result. In the above way, the immune complex of the present disclosure is bispecific, can be obtained through an antigen-antibody reaction, is simple to prepare and detect, simplifies process, can be stored for a long time, has stable activity and is suitable for large-scale production and application. 6 / FIG. 1 %j~ *i FIG. 2 1 /1

Description

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TARGET AND HEMOGLOBIN BISPECIFIC IMMUNE COMPLEX AND PREPARATION AND DETECTION METHOD THEREOF TECHNICAL FIELD

The present disclosure relates to thefield of immunological experimental technology and immunolabeling technology, and in particular to a target and hemoglobin bispecific immune complex and a preparation and detection method thereof.

BACKGROUND

At present, immunolabeling technology is one of the most widely used experimental techniques in clinical immunoassay and medical immunization experiments all over the world. The majority of the existing immunolabeling technologies use monospecific antibodies and particularly horseradish peroxidase (HRP) for detection; for example, when detecting concentrations of molecules such as endocrine hormones, cytokines, antibodies, and complements of the body, specific antibodies (abbreviation: primary antibodies) must be applied to detect these antigen targets. For positive specimens, after a specific antibody binds to an antigen target to be detected, an antibody against an enzyme-labeled specific antibody (abbreviation: enzyme-labeled secondary antibody) is added to form an immune complex: antigen-monospecific antibody-enzyme-labeled secondary antibody. Enzyme in the enzyme labeled secondary antibody acts on an enzyme substrate, a color change occurs in the reaction mixture, and the change of wavelength is read by using a microplate reader. In the practical work of routine clinical immunoassay, enzyme-labeled primary antibodies are rarely applied, and bispecific antibodies and corresponding reagents thereof are not applied. HRP, which is a commonly used enzyme, has unstable biological activity and is easily degraded naturally. Thus, even under the most stringent storage conditions, such as cryopreservation at 4°C, HRP merely expires in one year, and HRP-based immunoreagents are also susceptible to loss of activity and efficacy during transport and storage.

SUMMARY

The technical problem to be solved by the present disclosure is to provide a target and hemoglobin bispecific immune complex, and a preparation and detection method thereof, with an excellent application effect.

In order to solve the above technical problem, a technical solution adopted by the present disclosure is to provide a target and hemoglobin bispecific immune complex, including an anti target-antigen specific antibody-secondary antibody-anti-hemoglobin specific antibody hemoglobin structure, where the anti-target-antigen specific antibody and the anti-hemoglobin specific antibody are different antibodies derived from the same animal species.

In a preferred embodiment of the present disclosure, the animal may be a murine, a sheep, a chick, a rabbit, a donkey or a horse.

In a preferred embodiment of the present disclosure, the secondary antibody may be an antibody against the anti-target-antigen specific antibody and the anti-hemoglobin specific antibody.

In a preferred embodiment of the present disclosure, two Fab segments of an Ig class secondary antibody may be ligated to Fc segments of the anti-target-antigen specific antibody and the anti-hemoglobin specific antibody, respectively.

Provided is a detection method using a target and hemoglobin bispecific immune complex, including the steps of: step 1, adding a target and a hemoglobin bispecific immune complex to a specimen to be detected, and washing; and step 2, adding an enzyme substrate to perform chromogenic reaction to obtain a detection result.

In a preferred embodiment of the present disclosure, the specimen to be detected may be a normal body fluid, a pathological body fluid or a histopathological section.

In a preferred embodiment of the present disclosure, the body fluid in the normal body fluid and the pathological body fluid may include blood serum, plasma, saliva, urine or tumor tissue fluid.

The present disclosure has the following beneficial effects: in view of the target and hemoglobin bispecific immune complex and the preparation and detection method thereof provided by the present disclosure, the immune complex is bispecific, can be obtained through an antigen-antibody reaction, is simple to prepare and detect, simplifies process, can be stored for a long time, has stable activity and is suitable for large-scale production and application.

BRIEF DESCRIPTION OF THE DRAWINGS

In order to more clearly illustrate the technical solutions in the embodiment of the present disclosure, the drawings used in the description of the embodiment will be briefly described below. It is obvious that the drawings in the following description are only some embodiments of the present disclosure. For those skilled in the art, other drawings can be obtained according to these drawings without creative efforts, herein:

FIG. 1 is a structural diagram illustrating a preferred embodiment of the target and hemoglobin bispecific immune complex of the present disclosure;

FIG. 2 illustrates a process of a preferred embodiment of a detection method using a target and hemoglobin bispecific immune complex provided by the present disclosure;

reference numerals: 1. hemoglobin, 2. anti-hemoglobin specific antibody, 3. secondary antibody, 4. anti-target-antigen specific antibody, and 5. target antigen to be detected.

DETAILED DESCRIPTION

The technical solutions in the embodiment of the present disclosure will be clearly and completely described below. It is obvious that the described embodiment is only a part of, not all of, the embodiments. All other embodiments obtained by those skilled in the art based on the embodiment of the present disclosure without creative efforts are within the scope of the present disclosure.

Supplementary data

The present disclosure has the following beneficial effects:

1. Bispecificity refers to the specificity of target antigen to be detected and hemoglobin; an immune complex simultaneously binds to an anti-detection target antibody capable of detecting a target antigen and hemoglobin having HRP activity, and has biological activity of a target and hemoglobin bispecific antibody, but monospecificity is found in the prior art;

2. The immune complex is obtained by antigen-antibody reaction, instead of bispecific antibodies prepared by chemical methods and genetic engineering commonly used in the prior art;

3. In the chemical complex, an enzyme molecule is not chemically labeled onto an antibody molecule;

4. The preparation and detection method of the immune complex may be realized in two steps, and the test procedure is simplified. In the conventional enzyme immunoassay, the most commonly used method requires three steps: step 1, adding a primary antibody into a specimen to be detected; step 2, adding an enzyme-labeled secondary antibody; and step 3, adding an enzyme substrate. In theory, enzyme-labeled specific antibodies may be applied, and the test procedure has two steps, but in fact, objectively, the application is rare in medical clinical routine immunoassays. The reason is that there is no commercially available enzyme-labeled specific primary antibody; it is very difficult for a laboratory to prepare this specific primary antibody, and the quality is difficult to guarantee;

5. The immune complex fully and efficiently utilizes the enzymatic activity of hemoglobin, replaces HRP, and is applied in the enzyme-linked immunosorbent assay (ELISA) of soluble substrates and immunohistochemical assay of precipitated substrates. In this way, a problem that the activity of HRP is easily lost is avoided; hemoglobin has extremely stable activity and is readily available infinitely;

6. Secondary antibody-anti-hemoglobin specific antibody in the immune complex can be prepared into a highly standardized general reagent by mass production.

The above description is only an embodiment of the present disclosure and is not intended to limit the patent scope of the present disclosure, and any equivalent structures or equivalent flow variations made by using the content of the specification of the present disclosure, or directly or indirectly applied to other related technical fields, shall be included in the scope of patent protection of the present disclosure.

Claims

What is claimed is:

1. A target and hemoglobin bispecific immune complex, comprising an anti-target-antigen specific antibody-secondary antibody-anti-hemoglobin specific antibody-hemoglobin structure, wherein the anti-target-antigen specific antibody and the anti-hemoglobin specific antibody are different antibodies derived from the same animal species.

2. The target and hemoglobin bispecific immune complex according to claim 1, wherein the animal is a murine, a sheep, a chick, a rabbit, a donkey or a horse.

3. The target and hemoglobin bispecific immune complex according to claim 1, wherein the secondary antibody is an antibody against the anti-target-antigen specific antibody and the anti-hemoglobin specific antibody;

wherein two Fab segments of an Ig class secondary antibody are ligated to Fc segments of the anti-target-antigen specific antibody and the anti-hemoglobin specific antibody, respectively.

4. A detection method using a target and hemoglobin bispecific immune complex, comprising: step 1, adding a target and a hemoglobin bispecific immune complex to a specimen to be detected, and washing; and step 2, adding an enzyme substrate to perform chromogenic reaction to obtain a detection result.

5. The detection method according to claim 4, wherein the specimen to be detected is a normal body fluid, a pathological body fluid or a histopathological section;

wherein the body fluid in the normal body fluid and the pathological body fluid comprises blood serum, plasma, saliva, urine or tumor tissue fluid.