CN110862438B - A kind of compound and its preparation method and application - Google Patents

Abstract

The present invention relates to a complex and its preparation method and application. The present invention provides a complex for reducing the interference of anti-avidin antibodies, the complex comprises biotin and avidin, and the avidin is saturated bind the biotin. The invention also provides the preparation method and its application in immunodetection. Due to the existence of anti-streptavidin antibodies in some human beings, in the process of in vitro immunodiagnosis, the antibodies, as endogenous interfering substances, will affect the detection results, and the complex provided by the present invention can be combined with the antibodies, thereby preventing the antibodies from affecting the detection results. Detection causes interference, reduces corresponding errors or errors, and significantly improves the accuracy of detection results.

Description

Compound and preparation method and application thereof

Technical Field

The invention relates to the technical field of immunodetection, and particularly relates to a compound and a preparation method and application thereof.

Background

The Biotin-Avidin System (BAS) introduced enzyme-linked immune, reflex immune and fluorescence immune detection technology from the end of 70 s, improved the stability and specificity of these methods, greatly improved the sensitivity of the method. A new way is opened up for accurately and rapidly detecting trace antigens and antibodies, and the method is widely applied to qualitative and quantitative detection of the antigens and the antibodies at present.

Biotin (biotin) was found in the early 60's of the 20 th century and is widely present in animals and plants, especially in yolk and liver. Biotin consists of an imidazolone ring and a thiophene ring, and the imidazolone ring is mainly bound to avidin, while carboxyl groups at the side chain ends of the thiophene ring can bind to antibodies and other macromolecular substances.

Avidin (avidin), also known as avidin, was originally a basic glycoprotein extracted from egg white. Streptavidin (SA) secreted by Streptomyces avidinii is a type of avidin commonly used at present and has a molecular weight of 65 kD. The streptavidin molecule consists of 4 identical peptide chains, each of which can bind a biotin and is free of any sugar groups, and 4 biotin molecules, each of which has an affinity constant (K) of 10¹⁵mol/L, at least 1 ten thousand times higher than the affinity between antigen and antibody. The dissociation constant of the compound formed by combining the streptavidin and the biotin is very small, and the compound is irreversible, and the combination of the streptavidin and the biotin is not influenced by acid, alkali, a denaturant, proteolytic enzyme and an organic solvent, so that the combined product of the streptavidin and the biotin has higher stability.

If a human body is infected with streptomyces, an antibody against streptomyces is produced in the body, and the target protein of the antibody is streptavidin, so that the anti-streptavidin antibody is present in part of human serum. In the in vitro diagnosis and detection process, the antibody can be combined with streptavidin coupled on magnetic beads in a detection kit, so that the overall detection result is influenced. If the interference antibody is combined with more SA-magnetic beads, the combination of the SA-magnetic beads and the biotin antibody is reduced due to the steric hindrance effect, so that the magnetic beads of the solid phase combination cannot be connected with the target antigen in the human serum, and the final detection result is false negative. The interference substances are not only combined with the SA-magnetic beads, but also can react with the enzyme-labeled antibody to form a (SA-magnetic beads-interference substances-enzyme-labeled antibody) sandwich structure, and the final detection result is false positive. Therefore, the detection of the target antigen in the human body is seriously influenced by the existence of the anti-streptavidin antibody in the human serum, and an effective solution is not provided in the prior art. Solutions are proposed to eliminate the interference of this substance.

Disclosure of Invention

The invention aims to provide a compound and a preparation method and application thereof.

To this end, in a first aspect of the invention, there is provided a complex for reducing interference of an anti-avidin antibody, said complex comprising biotin and avidin, said avidin being saturated with said biotin.

Further, the avidin is streptavidin. In a specific embodiment, each avidin binds 4 of said biotin in said complex.

In a second aspect of the present invention, a method for preparing the complex is provided, which comprises reacting the avidin with the biotin in a buffer solution to prepare the complex; wherein the molar ratio of the avidin to the biotin is 1: 4-6.

Further, the pH of the buffer is 7.0-8.0, e.g. 7.0, 7.2, 7.4, 7.5, 7.6, 7.8, 8.0.

Further, the buffer is PB, PBS or PBST, and the concentration of the buffer is 10-100mM, such as 10mM, 20mM, 40mM, 60mM, 80mM, 100 mM.

Further, the temperature of the reaction is 25 to 37 ℃, preferably 37 ℃.

Further, the preparation method also comprises the step of carrying out ultrafiltration after the avidin reacts with the biotin.

Further, the ultrafiltration is carried out by adopting an ultrafiltration tube.

Further, the molecular weight cut-off of the ultrafiltration tube is 10 to 50kD, preferably 10 to 20kD, and more preferably 20 kD.

In a third aspect of the invention, there is provided a use of the complex of the invention in an immunoassay comprising detecting a sample to be tested using avidin, biotin and the complex of the invention.

Further, the avidin is bound with an enzyme, a fluorescent dye, radioactive iodine, a microsphere, or a chromatography pad; the microspheres are preferably magnetic beads.

Further, the biotin is bound with an antibody, and the antibody can specifically bind to a protein to be detected.

Further, the immunoassay is one of enzyme-linked immunoassay, chemiluminescence immunoassay and immunochromatography assay.

Further, the sample for immunoassay is blood, serum, plasma, extracellular fluid, interstitial fluid, lymphatic fluid, cerebrospinal fluid or aqueous humor.

In a fourth aspect, the present invention provides an immunoassay method, wherein the immunoassay method comprises detecting a sample to be detected by using avidin, biotin and the complex of the present invention.

Further, the avidin is bound with an enzyme, a fluorescent dye, radioactive iodine, a microsphere, or a chromatography pad; the microspheres are preferably magnetic beads.

Further, the biotin is bound with an antibody, and the antibody can be specifically bound with an antigen to be detected.

Further, the immunoassay is one of enzyme-linked immunoassay, chemiluminescence immunoassay and immunochromatography assay.

Further, the sample for immunoassay is blood, serum, plasma, extracellular fluid, interstitial fluid, lymphatic fluid, cerebrospinal fluid or aqueous humor.

In a specific embodiment, the immunoassay method is a chemiluminescent immunoassay comprising the steps of:

(1) mixing the magnetic beads coupled with the avidin, the compound, the biotinylated antibody and a sample to be detected, and then sequentially incubating and washing;

(2) adding an enzyme-labeled antibody into the reaction solution obtained in the step (1), uniformly mixing, and then sequentially incubating and washing;

(3) and (3) adding a substrate corresponding to the enzyme into the reaction solution obtained in the step (2), and detecting after incubation.

The enzyme-labeled antibody can be specifically combined with the antigen to be detected and has enzyme activity, after a corresponding substrate is added, the substrate can be enzymatically catalyzed into a colored product or a luminous product, and qualitative or quantitative analysis on the antigen to be detected can be realized by detecting the product.

In specific embodiments, the enzyme is alkaline phosphatase (ALP) or horseradish peroxidase (HRP); accordingly, when the enzyme is alkaline phosphatase, the luminescent substrate corresponding to the enzyme is 32(2 '2 spiroadamantane) 242 methoxy 242 (3' 2 phosphoryloxy) 2 phenyl 21,22 cyclodioxane (AMPPD); when the enzyme is horseradish peroxidase, the luminescent substrate corresponding to the enzyme is luminol or isoluminol.

Further, the concentration of the complex in step (1) is 30 to 150. mu.g/ml, for example, 30. mu.g/ml, 50. mu.g/ml, 70. mu.g/ml, 90. mu.g/ml, 110. mu.g/ml, 130. mu.g/ml, 150. mu.g/ml.

Further, the incubation temperature is 25-37 ℃, and preferably 37 ℃; the incubation time is 1-10min, such as 1min, 3min, 5min, 7min, 10min, preferably 3-10 min; the incubation humidity is 28-32%, preferably 30%.

Further, the pH of the solution used for washing is 6.5 to 8.0, preferably 7.0 to 7.4, and more preferably 7.4.

Further, the washing is performed with a solution selected from the group consisting of: phosphate buffers such as disodium hydrogenphosphate-sodium dihydrogenphosphate buffer, disodium hydrogenphosphate-potassium dihydrogenphosphate buffer, potassium dihydrogenphosphate-sodium hydroxide buffer, disodium hydrogenphosphate-citric acid buffer, Tris-hydrochloric acid buffer, Triethanolamine (TEA) buffer, Tricine buffer, HEPES buffer, PIPES buffer and MOPS buffer.

The compound provided by the invention is biotin-saturated avidin, namely 4 peptide chains on the surface of avidin are saturated and combined with biotin, and other protein binding sites are exposed. Therefore, the avidin in the complex can not be combined with the biotinylated antibody in the immunoassay system any more, but can be combined with the endogenous interferon anti-streptavidin antibody in the sample to be detected, so that the interference of the anti-streptavidin antibody on the detection is reduced. In a preferred embodiment, the avidin in the immunoassay system is coupled to the magnetic beads, and since the complex is in a free state, the avidin coupled to the magnetic beads is more likely to bind to the anti-streptavidin, so that the interference of the anti-streptavidin antibody on the detection can be better eliminated.

Compared with the prior art, the invention has the beneficial effects that:

(1) the invention provides a compound which can be combined with an anti-streptavidin antibody in an immunoassay system, thereby effectively reducing the interference caused by the anti-streptavidin antibody in-vitro diagnosis immunoassay and avoiding false positive and false negative false detection results caused by the interference.

(2) The compound provided by the invention has lower cost, and can realize the anti-interference effect under the condition of not obviously increasing the cost of the immunoassay kit.

(3) The complex provided by the invention has a wide application range, and because the complex can not react with a biotinylated antibody in a detection system and can not influence the original functions of all reagents in the detection system, errors or mistakes caused by corresponding endogenous interferents can be reduced by adding the complex to all immunoassay systems using a biotin-avidin system.

Detailed Description

Exemplary embodiments of the present disclosure will be described in greater detail below, with the understanding that the present disclosure may be embodied in various forms and should not be limited by the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the disclosure to those skilled in the art.

EXAMPLE 1 preparation of biotin-saturated streptavidin (SA-B)

a) 0.6ml SA (10mg/ml, 10mM PB, pH 7.5) was taken

b) 0.1ml of D-biotin (1mg/ml, 10mM PB, pH 7.5) was added to SA, and the reaction was spun at 37 ℃ for 1 hour;

c) ultrafiltering the product obtained in step B) for 4 times (UFC901096, Millipore Amicon Ultra ultrafiltration centrifuge tube [15ml 10KD ])10mM PB, pH 7.5), to obtain biotin-saturated streptavidin (SA-B). The obtained SA-B was used in example 2.

Example 2 detection of carcinoembryonic antigen (CEA)

First, main reagents and materials:

streptavidin magnetic bead (Nanjing Pangu gene), concentration 10mg/ml, 100. mu.l/tube (1 mg/tube)

Coupling antibody: fenpeng CEA-23, Fenpeng CEA-100

Coupling reagent: activators Traut's (Thermo), SM (PEG)4(Thermo)

Biotin: NHS-PEG12-Biotin (thermo)

A sample to be detected: 16 clinical serum samples with the serial numbers of 1,2, 3-15 and 16

Substrate: AMPPD (Shenzhen MeiKaite)

Buffer 10D：20mM 7.2PBS+0.1％BSA+0.05％TW-20+0.05％PC300

Tris buffer: 50mM Tris + 0.9% NaCl + 0.1% Tween20+ 0.05% Pc300, pH 7.4

Step two, step:

(1) biotin antibody preparation (Bio-Ab)

1. Antibody buffer replacement

a. Ultrafiltration tube (0.5mL 50K) +100 μ g antibody + PBS buffer (10mM, pH7.2) to 0.4 mL;

b, 8000g, centrifuging;

c. and (c) repeating the step b 2 times and recovering.

2. Antibody coupling to biotin

Antibody: biotin 10: 1 (molar weight ratio), adding liquid, mixing evenly, and coupling for 1h at room temperature in a dark place.

3. Desalination by ultrafiltration

a.0.5mL 50K ultrafiltration tube + conjugate antibody + PBS buffer (10mM, pH7.2, 0.5% BSA);

b.4 ℃, 8000g, 5min and 3 times of centrifugation; and (5) immediately replacing a new pipe, turning the pipe with the cover opened inwards, and centrifuging at 1000g for 2min for recovery.

4. Preservation of

Adding equal volume of 50% glycerol, mixing, and storing at-20 deg.C.

(2) Enzyme-labeled antibody preparation (Epsilon-Ab)

1. Buffer replacement

a. Antibody: ultrafiltration tube (0.5mL 50K) + 120. mu.g antibody + PBS buffer (10mM, pH7.2) to 0.4 mL;

AP enzyme: ultrafiltration tube (0.5mL 30K) +250 μ g enzyme + PBS buffer (10mM, pH7.2) to 0.4 mL;

c. centrifuging 8000g of the antibody and the enzyme prepared in the step a and the step b;

d. and c, repeating the step c 2 times, immediately replacing a new pipe, turning the pipe with the cover inwards, centrifuging at 1000g for 2min, and recovering.

2. Activation of antibodies with enzymes

a. Antibody 100 mug + activator Traut's 10 mug, total volume controlled at 100ul, mixing, RT dark reaction 2 h;

b. mu.g of enzyme + SM (PEG) 410. mu.g, mixing well, and reacting for 1h at RT in the dark. (siliconized tube).

3. Coupling of activated antibodies with activating enzymes

According to the enzyme: antibody 2: 1 (mass ratio), adding liquid, mixing gently, standing at room temperature in a dark place for 30 min;

4. desalination by ultrafiltration

a.0.5mL 50K ultrafiltration tube + conjugate antibody + PBS buffer (10mM, pH7.2, 0.5% BSA);

b.4 ℃, 8000g, 5min and 3 times of centrifugation; and (5) immediately replacing a new pipe, turning the pipe with the cover opened inwards, and centrifuging at 1000g for 2min for recovery.

5. Preservation of

Adding equal volume of 50% glycerol, mixing, and storing at-20 deg.C.

(3) Diluting preparation

Respectively taking out Buffer solution Buffer 10D, antigen, substrate, Bio-Ab and epsilon-Ab, and balancing at room temperature;

dilution of Bio-Ab: Bio-Ab was diluted to 1.667. mu.g/ml with Buffer 10D;

dilution of ε -Ab: Epsilon-Ab was diluted to 2. mu.g/ml with Buffer 10D;

interfering substance: diluting the interfering substance to an appropriate concentration using serum of carcinoembryonic antigen (CEA);

the prepared Bio-Ab and ε -Ab were used in the following experimental procedures.

(4) Reaction of

Add 10. mu.l streptavidin magnetic beads (4mg/ml), 10. mu.l serum sample, 10. mu.l SA-B (2mg/ml), 120. mu.l Bio-Ab to the white chemiluminescence plate, incubate 3min at 37 ℃ and wash three times with Tris buffer; a further 150. mu.l of ε -Ab (1.34. mu.g/ml) were added. The liquid is impacted and mixed uniformly during the last liquid adding, the liquid is slightly rotated and mixed uniformly on the table top, and the mixture is incubated for 3min at constant temperature and humidity (37 ℃, 30 +/-2%).

(5) Washing machine

Tris buffer was washed three times.

(6) Luminescence detection

And (4) adding 200 mu l of balanced AMPPD substrate into each hole of the chemiluminescent plate washed in the step (4), shaking and uniformly mixing in a 96-hole plate, and incubating for 5min at 37 ℃ in an enzyme labeling instrument to detect luminescence.

The detection is taken as an experimental group, a control group is additionally arranged, and the difference between the control group and the experimental group is that SA-B is not added. The detection results are shown in table 1, the table 1 simultaneously lists the detection results of the CEA Roche detection kit, the Roche detection data, the experimental group results and the control group results are respectively subjected to curve fitting, and the correlation coefficient R is calculated²。

TABLE 1

Endogenous interferents are an important source of detection errors in clinical laboratories, and in-vitro immunodiagnosis, the problem that an anti-streptavidin antibody influences a detection result and even causes the detection result to have false positive or false negative exists. In the detection process, no obvious improvement effect is achieved by adding a commercial active/passive blocking agent into the sample, and by applying the technical scheme provided by the invention, endogenous interference is effectively reduced, so that the correlation between the detection result of the serum sample and the Roche detection result is obviously improved.

The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope of the present invention are included in the scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the protection scope of the appended claims.

Claims (13)

1. Use of a complex in an immunoassay for non-diagnostic purposes, said immunoassay comprising the use of avidin, biotin and said complex for detecting a sample to be tested;

the complex consists of biotin and avidin, which binds to biotin with saturation;

the avidin in the compound cannot be combined with a biotinylated antibody in an immunoassay system, but can be combined with an endogenous interferon anti-streptomycin avidin antibody in a sample to be detected, so that the interference of the anti-streptavidin antibody on the detection is reduced.

2. The use of claim 1, wherein the immunoassay is one of an enzyme-linked immunoassay, a chemiluminescent immunoassay, and an immunochromatographic assay.

3. The use of claim 1, wherein the avidin is bound to an enzyme, a fluorescent dye, radioactive iodine, a microsphere, or a chromatographic pad.

4. The use of claim 3, wherein the microspheres are magnetic beads.

5. The use of claim 1, wherein the biotin is conjugated to an antibody that specifically binds to a test protein.

6. The use of claim 1, wherein the test sample is blood, serum, plasma, extracellular fluid, interstitial fluid, lymphatic fluid, cerebrospinal fluid, or aqueous humor.

7. A method of immunodetection for non-diagnostic purposes, comprising the step of detecting a test sample using avidin, biotin and the complex of claim 1.

8. The immunoassay of claim 7, wherein said immunoassay is one of an enzyme-linked immunoassay, a chemiluminescent immunoassay, and an immunochromatographic assay.

9. The immunoassay of claim 7, wherein said avidin is bound to an enzyme, a fluorescent dye, radioactive iodine, microspheres, or a chromatographic pad.

10. The immunoassay method of claim 9, wherein said microspheres are magnetic beads.

11. The immunoassay of claim 7, wherein said biotin is conjugated to an antibody that specifically binds to an antigen to be detected.

12. The immunoassay of claim 7, wherein said sample to be assayed is blood, serum, plasma, extracellular fluid, interstitial fluid, lymphatic fluid, cerebrospinal fluid or aqueous humor.

13. The immunoassay method of any one of claims 7 to 12, comprising the steps of:

(1) mixing the magnetic beads coupled with avidin, the compound as claimed in claim 1, the biotinylated antibody and the sample to be tested, and then sequentially incubating and washing;

(2) adding an enzyme-labeled antibody into the reaction solution obtained in the step (1), uniformly mixing, and then sequentially incubating and washing;

(3) and (3) adding a substrate corresponding to the enzyme into the reaction solution obtained in the step (2), and detecting after incubation.