CN115097123A - Alkaline phosphatase marker, preparation method, application and method for marking antibody by alkaline phosphatase marker - Google Patents

Alkaline phosphatase marker, preparation method, application and method for marking antibody by alkaline phosphatase marker Download PDF

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CN115097123A
CN115097123A CN202210696869.9A CN202210696869A CN115097123A CN 115097123 A CN115097123 A CN 115097123A CN 202210696869 A CN202210696869 A CN 202210696869A CN 115097123 A CN115097123 A CN 115097123A
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alkaline phosphatase
biotin
labeled
marker
antibody
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石彩娟
蒋明君
林�源
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Sichuan Xinjian Kangcheng Biological Co ltd
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Sichuan Xinjian Kangcheng Biological Co ltd
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/531—Production of immunochemical test materials
    • G01N33/532—Production of labelled immunochemicals
    • G01N33/535—Production of labelled immunochemicals with enzyme label or co-enzymes, co-factors, enzyme inhibitors or enzyme substrates
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G01N33/581—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with enzyme label (including co-enzymes, co-factors, enzyme inhibitors or substrates)

Abstract

The invention discloses an alkaline phosphatase marker, a preparation method, application and a method for marking an antibody by using the alkaline phosphatase marker, wherein the alkaline phosphatase marker is alkaline phosphatase marked by biotin; the alkaline phosphatase marker and the marking method can effectively improve the marking amount of the alkaline phosphatase and further improve the sensitivity of a detection project.

Description

Alkaline phosphatase marker, preparation method, application and method for marking antibody by alkaline phosphatase marker
Technical Field
The invention relates to the technical field of enzymatic chemiluminescence detection, and in particular relates to an alkaline phosphatase marker, a preparation method, application and an antibody labeling method thereof.
Background
The chemiluminescence method has the advantages of high sensitivity, wide linearity and the like, but the sensitivity is still insufficient for some projects, the patent proposes a special labeling method which can effectively improve the sensitivity of the reagent, and the technology is suitable for almost all projects.
Enzymatic chemiluminescence is the predominant technique in vitro diagnostic reagents, and among them, alkaline phosphatase (ALP) represented by beckmann and Acridinium Ester (AE) represented by yaperu are the most widely used markers.
The existing alkaline phosphatase (ALP) enzymatic chemiluminescence method is characterized in that magnetic particles are usually connected with a fixed antibody, a detection antibody (Ab) is connected with ALP through a common cross-linking agent, the two antibodies recognize different antigen sites of an object to be detected to form a double-antibody sandwich structure, excessive components except a compound can be eluted through the solid phase action of the magnetic particles, finally, the ALP on the compound can act on a substrate (AMPPD) to promote the light emission of the substrate, and the concentration level of a light-emitting signal is reflected by the concentration level of the object to be detected.
Therefore, the method of bridging using a cross-linking agent in labeling ALP is that the amino group of the detection antibody reacts with 2IT, the amino group of ALP reacts with succinimide-4- (N-maleimide) cyclohexane-1-1 hydroxy acid ester (SMCC), the group of SMCC is cross-linked with 2IT, and finally, the amount of ALP in the complex is limited by the number of amino groups of Ab. For some items requiring particularly high sensitivity, such as hypersensitive muscle calcium, competitive method items, etc., the ALP labeling rate is relatively low and the sensitivity is insufficient.
Disclosure of Invention
The technical problem to be solved by the invention is as follows: the invention provides an alkaline phosphatase marker, a preparation method, application and a method for labeling an antibody, which solve the problems that the existing antibody labeling alkaline phosphatase enzymatic technology limits the ALP in a labeled product to a certain extent by the number of amino groups of Ab and cannot meet the requirement for individual projects with particularly high requirement sensitivity.
The invention is realized by the following technical scheme:
an alkaline phosphatase marker which is biotin-labeled alkaline phosphatase; for labeling streptavidin-labeled antibodies by a streptavidin-biotin amplification reaction.
Further optionally, the biotin comprises (+) -biotin-N-hydroxysuccinimide ester.
A preparation method of an alkaline phosphatase marker comprises the step of mixing biotin and alkaline phosphatase for reaction to prepare the biotin-labeled alkaline phosphatase.
Further optionally, the molar ratio of biotin to alkaline phosphatase is 10/1-30/1; more preferably 15/1-25/1.
Further optionally, the molar ratio of biotin to alkaline phosphatase is 20/1.
A method for preparing an alkaline phosphatase label, comprising the following steps:
dissolving biotin in an organic solvent to prepare a biotin solution;
and (3) adding biotin solution into the alkaline phosphatase after dialysis treatment, and standing for reaction at room temperature to prepare the alkaline phosphatase marked by the biotin.
Further alternatively, the molar concentration of biotin in the biotin solution is 6mM to 15mM, more preferably 10 mM.
The application of the alkaline phosphatase marker in preparing an alkaline phosphatase enzymatic chemiluminescence detection reagent or in an alkaline phosphatase enzymatic chemiluminescence detection method.
A method for marking an antibody with an alkaline phosphatase marker comprises the steps of mixing alkaline phosphatase marked by biotin with an antibody marked by streptavidin, and carrying out amplification reaction on the biotin and the streptavidin to realize the marking of the antibody with the alkaline phosphatase; the biotin-labeled alkaline phosphatase is the alkaline phosphatase marker or the biotin-labeled alkaline phosphatase prepared by the preparation method of the alkaline phosphatase marker.
Further optionally, the mass ratio of the biotin-labeled alkaline phosphatase to the streptavidin-labeled antibody is 1:1 to 3: 1.
The streptavidin-labeled antibody can be prepared by the following method:
step 1: subjecting the antibody (Ab) and a reducing agent (2IT) to a first contact reaction to obtain a reduced thiol-group-containing antibody (Ab-SH);
step 2: reacting Streptavidin (SA) with succinimide-4- (N-maleimide) cyclohexane-1-1 hydroxy acid ester (SMCC) to obtain maleimide modified streptavidin (SA-SMCC);
and step 3: and (3) carrying out a second contact reaction on the antibody containing the sulfydryl and the maleimide modified streptavidin to obtain the antibody-streptavidin conjugate (Ab-SA).
The invention has the following advantages and beneficial effects:
in the existing antibody-labeled alkaline phosphatase enzymatic technology, the amount of ALP in a labeled product is determined by the number of amino groups of Ab, and the ALP labeling rate is relatively low and the sensitivity is insufficient for individual projects requiring particularly high sensitivity, such as hypersensitive muscle calcium, competitive method projects and the like. The invention provides alkaline biotin-labeled alkaline phosphatase, which is used for labeling a streptavidin-labeled antibody through streptavidin-biotin amplification reaction, and an Ab-ALP compound with a stable structure is obtained through an affinity amplification system of SA and BHNS.
The existing antibody-labeled alkaline phosphatase enzymatic technology (i.e., the amino group of an antibody reacts with 2IT, the amino group of ALP reacts with SMCC, and the group of AMCC is crosslinked with 2IT) can meet the requirement of sensitivity, because the large luminous flux is large, the detection time of a reagent can be properly prolonged, and the detection efficiency is not influenced. For single-person POCT luminescence, rapid detection is a competitive advantage, so that it is particularly important to further improve the sensitivity of the reagent in order to obtain a measurement result at a higher speed.
Drawings
The accompanying drawings, which are included to provide a further understanding of the embodiments of the invention and are incorporated in and constitute a part of this application, illustrate embodiment(s) of the invention and together with the description serve to explain the principles of the invention. In the drawings:
FIG. 1 is a flowchart of a method for labeling an antibody with an alkaline phosphatase marker according to the present invention.
FIG. 2 is a graph of the clinical identity of the control group and Beckmann of example 3.
Fig. 3 is a graph of the clinical concordance of the experimental groups of example 3 and beckmann.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail below with reference to examples and accompanying drawings, and the exemplary embodiments and descriptions thereof are only used for explaining the present invention and are not meant to limit the present invention.
In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present invention. However, it will be apparent to one of ordinary skill in the art that: it is not necessary to employ these specific details to practice the present invention. In other instances, well-known structures, circuits, materials, or methods have not been described in detail so as not to obscure the present invention.
Throughout the specification, reference to "one embodiment," "an embodiment," "one example," or "an example" means: the particular features, structures, or characteristics described in connection with the embodiment or example are included in at least one embodiment of the invention. Thus, the appearances of the phrases "one embodiment," "an embodiment," "one example" or "an example" in various places throughout this specification are not necessarily all referring to the same embodiment or example. Furthermore, the particular features, structures, or characteristics may be combined in any suitable combination and/or sub-combination in one or more embodiments or examples. Further, those of ordinary skill in the art will appreciate that the illustrations provided herein are for illustrative purposes and are not necessarily drawn to scale. As used herein, the term "and/or" includes any and all combinations of one or more of the associated listed items.
In the description of the present invention, the terms "front", "rear", "left", "right", "upper", "lower", "vertical", "horizontal", "upper", "lower", "inner", "outer", etc. indicate orientations or positional relationships based on those shown in the drawings, merely for convenience of description and simplification of description, and do not indicate or imply that the device or element referred to must have a particular orientation, be constructed in a particular orientation, and be operated, and therefore, should not be construed as limiting the scope of the invention.
Example 1
This example provides an alkaline phosphatase marker, biotin-labeled alkaline phosphatase; for labeling streptavidin-labeled antibodies by a streptavidin-biotin amplification reaction. The preparation method comprises the following steps:
dissolving (+) biotin-N-succinimidyl ester (BNHS) in an organic solvent dimethyl sulfoxide (DMSO) to prepare a BNHS solution; taking 2 mu L of BNHS solution with the concentration of 10mM for standby;
taking alkaline phosphatase (ALP) according to the molar ratio of BHNS to ALP of 20:1, dialyzing with PBS (dialyzing for 12-24 h, replacing dialysate for one time), adding 2 μ L BNHS solution with concentration of 10mM, standing at room temperature for 30 min;
and performing PBS dialysis treatment (dialysis for 12h-24h, and replacing dialysate once) to obtain biotin-labeled alkaline phosphatase, ALP-BNHS for short.
Example 2
The embodiment provides a method for labeling an antibody with alkaline phosphatase, which comprises the steps of mixing alkaline phosphatase labeled with biotin and an antibody labeled with streptavidin, and carrying out amplification reaction on the biotin and the streptavidin to realize the labeling of the antibody with the alkaline phosphatase, wherein the specific preparation method is as shown in figure 1:
step 1: preparation of biotin-labeled alkaline phosphatase
The method of example 1 was specifically used to prepare ALP-BNHS.
Step 2: preparation of streptavidin-labeled antibody
Subjecting the antibody (Ab) and a reducing agent (2IT) to a first contact reaction to obtain a reduced thiol-group-containing antibody (Ab-SH);
reacting Streptavidin (SA) with succinimide-4- (N-maleimide) cyclohexane-1-1 hydroxy acid ester (SMCC) to obtain maleimide modified streptavidin (SA-SMCC);
and (3) carrying out a second contact reaction on the sulfhydryl-containing antibody and the maleimide-modified streptavidin to obtain the antibody-streptavidin conjugate (Ab-SA).
The specific method comprises the following steps:
(1) prepare 50 mM/L2-IT solution, slowly add 2.4uL to 0.2mg Ab, mix them evenly at room temperature for 30 min.
(2) 20mM/L of SMCC solution is prepared, 10uL of the SMCC solution is slowly dripped into 2mg of SA, and the mixture is evenly mixed at room temperature and reacts for 30 min.
(3) Ab-2IT and SA-SMCC are connected in a mass ratio of 2:1, and are mixed uniformly at room temperature for reaction for 60 min.
And step 3: alkaline phosphatase marker labeled antibody
The alkaline phosphatase labeled with the substance and the antibody labeled with the streptavidin were mixed uniformly at a mass ratio of 2:1, and reacted at room temperature for 30min to obtain an antibody labeled with the alkaline phosphatase (Ab-ALP).
Example 3
Based on the labeling method provided in example 2, antigens were detected, and the specific design experiment was as follows:
test method
1. Source of clinical samples
Beckmann-valued muscle calcium clinical serum was purchased and centrifuged to examine the supernatant.
2. Control group
Immunomagnetic beads: 0.4mg/ml streptavidin magnetic bead, 4.0. mu.g/ml myocalcium monoclonal antibody, 1% bovine serum albumin, 0.05M Tris, 0.9% NaCl, 0.3% PC950, 0.05% tween 20.
Enzyme working solution: : 1mg/0.8mg alkaline phosphatase-labeled myocalcium monoclonal antibody, 1% bovine serum albumin, 0.05M Tris, 0.9% NaCl, pH7.4, 0.3% PC950, 0.05% tween20, 1mM MgCl 2 Buffer, 0.1mM ZnCl 2 And (4) a buffer solution.
Cleaning solution: 0.05M Tris-HCl buffer, 0.05% Tween.
AMPPD substrate.
3. Experimental group
Immunomagnetic beads: the same as the control group.
Enzyme working solution: 2mg/1mg of the alkaline phosphatase-labeled creatine calcium monoclonal antibody prepared in example 2 and 1% bovine serumAlbumin, 0.05M Tris, 0.9% NaCl, pH7.4, 0.3% PC950, 0.05% tween20, 1mM MgCl 2 Buffer, 0.1mM ZnCl 2 And (4) a buffer solution.
Cleaning solution: the same as the control group.
AMPPD substrate was the same as control.
(III) test results
The results of the measurements are shown in tables 1 and 2 and fig. 1 and 2. As shown in Table 1, the sensitivity of the experimental group is much higher than that of the control group by the signal-to-noise ratio; and the light emission value of the experimental group is higher than that of the control group. As shown in Table 2, FIG. 2, and FIG. 3, the clinical values of the experimental groups were closer to the results of the comparison with the manufacturer Beckmann, and the correlation R between the two values 2 Up to 0.97, the control group was found to be relatively poor.
TABLE 1
Figure BDA0003702962980000051
TABLE 2
Figure BDA0003702962980000052
Figure BDA0003702962980000061
The above-mentioned embodiments are intended to illustrate the objects, technical solutions and advantages of the present invention in further detail, and it should be understood that the above-mentioned embodiments are merely exemplary embodiments of the present invention, and are not intended to limit the scope of the present invention, and any modifications, equivalent substitutions, improvements and the like made within the spirit and principle of the present invention should be included in the scope of the present invention.

Claims (10)

1. An alkaline phosphatase marker, which is biotin-labeled alkaline phosphatase; for labeling streptavidin-labeled antibodies by a streptavidin-biotin amplification reaction.
2. The alkaline phosphatase marker according to claim 1, wherein the biotin comprises (+) -biotin-N-hydroxysuccinimide ester.
3. A method for producing an alkaline phosphatase-labeled substance, characterized in that biotin is reacted with alkaline phosphatase in a mixed manner to produce the alkaline phosphatase labeled with biotin according to claim 1 or 2.
4. The method for preparing an alkaline phosphatase marker according to claim 3, wherein the molar ratio of biotin to alkaline phosphatase is 10/1-30/1.
5. The method of claim 4, wherein the molar ratio of biotin to alkaline phosphatase is 20/1.
6. The method for preparing an alkaline phosphatase marker according to any one of claims 3 to 5, comprising the steps of:
dissolving biotin in an organic solvent to prepare a biotin solution;
and (3) adding biotin solution into the alkaline phosphatase after dialysis treatment, and standing for reaction at room temperature to prepare the alkaline phosphatase marked by the biotin.
7. The method according to claim 6, wherein the molar concentration of biotin in the biotin solution is 6 mM-15 mM.
8. The application of the alkaline phosphatase marker is characterized in that the alkaline phosphatase marker is used for preparing an alkaline phosphatase enzymatic chemiluminescence detection reagent or used in an alkaline phosphatase enzymatic chemiluminescence detection method.
9. A method for marking an antibody by an alkaline phosphatase marker is characterized in that the alkaline phosphatase marked by biotin and an antibody marked by streptavidin are mixed, and the alkaline phosphatase marking antibody is realized by the amplification reaction of the biotin and the streptavidin; the biotin-labeled alkaline phosphatase is the alkaline phosphatase marker as defined in claim 1 or 2, or the biotin-labeled alkaline phosphatase prepared by the method for preparing the alkaline phosphatase marker as defined in any one of claims 3 to 7.
10. The method according to claim 0, wherein the mass ratio of the biotin-labeled alkaline phosphatase to the streptavidin-labeled antibody is 1:1 to 3: 1.
CN202210696869.9A 2022-06-20 2022-06-20 Alkaline phosphatase marker, preparation method, application and method for marking antibody by alkaline phosphatase marker Pending CN115097123A (en)

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