CN115524485A

CN115524485A - Protein chip kit for quantitative quality control of sample adding accuracy and application

Info

Publication number: CN115524485A
Application number: CN202211226557.8A
Authority: CN
Inventors: 郑俊梅; 吉琛; 丁俊杰; 潘维华; 施启尧
Original assignee: Jiangsu Sanlian Bioengineering Co ltd
Current assignee: Jiangsu Sanlian Bioengineering Co ltd
Priority date: 2022-10-09
Filing date: 2022-10-09
Publication date: 2022-12-27
Anticipated expiration: 2042-10-09
Also published as: CN115524485B

Abstract

The application relates to a protein chip kit for quantitative quality control of sample addition accuracy, which comprises a protein chip coated with a ruthenium capture antibody, an antibody solution containing ruthenium and a labeled antibody solution with a label, wherein the quantitative quality control is carried out on the sample addition accuracy through an anti-ruthenium antibody system of an anti-ruthenium antibody-ruthenium-label. The present application provides a chemiluminescence method for detecting sample application accuracy in an intensive, high-throughput, quantitative manner, with a high degree of sensitivity achieved using chemiluminescence techniques. The method realizes high-speed, simple and convenient detection by utilizing a full-automatic chip reader, and is more suitable for clinical application than other methods requiring a large amount of manual operation.

Description

Protein chip kit for quantitative quality control of sample adding accuracy and application

Technical Field

The application belongs to the technical field of biology, and particularly relates to a protein chip kit for quantitative quality control of sample adding accuracy and application.

Background

Protein chips, also known as protein arrays or protein microarrays, are a method for detecting interactions in vitro. The basic principle is that protein molecules are used as ligands (probe proteins) and are orderly fixed on the surface of a solid phase carrier (a titration plate, a filter membrane, a glass sheet and the like) to form a microarray; specifically binding with the substance to be detected in the sample, washing to remove the unbound components, then binding with peroxidase-labeled protein (or other molecules), washing to remove the unbound components, reacting with a luminescent substrate, catalyzing and emitting light, and capturing a light signal by CCD to obtain a detection result.

The traditional protein chip quality control process for the sampling accuracy comprises the following steps: after the detection result is obtained, the result is preliminarily analyzed, the picture is checked when the result is abnormal, the sample adding accuracy is confirmed by analyzing the position of the liquid level line, and the sample adding accuracy is analyzed and processed by combining part of index signals. In addition, at present, no simple and effective sample adding accuracy quality control method exists.

Disclosure of Invention

Based on this, this application provides a protein chip kit and application for application of sample application degree of accuracy ration quality control is with solving traditional quality control flow inefficiency, complex operation, need to judge the scheduling problem by experienced personnel.

One of the purposes of the application is to provide a protein chip kit for sample adding accuracy quantitative quality control and application, wherein the kit is simple to apply and can efficiently and quickly identify the insufficient sample adding condition.

In order to solve the technical problem, the technical scheme of the application is as follows: providing a protein chip kit for sample application accuracy quantitative quality control:

the kit comprises a protein chip coated with ruthenium capture antibody, ruthenium-containing antibody solution and labeled antibody solution with a label.

The ruthenium capture antibody and the labeled antibody both bind to ruthenium in the ruthenium-containing antibody solution, and the ruthenium capture antibody and the labeled antibody bind to different sites of ruthenium in the ruthenium-containing antibody solution.

In one embodiment, the ruthenium capture antibodies are spotted to form a matrix of (2-6) × (1-6) on the protein chip.

In one embodiment, the protein chip is subjected to alkali solution soaking and silane solution soaking treatment before being coated with the ruthenium capture antibody.

In one embodiment, the alkali liquor is a solution with a pH value of more than 10, and the solution is an ethanol solution with silane mass percent of 0.05-1%.

In one embodiment, the label comprises one or both of a chemiluminescent catalyst and a luminescent label.

In one embodiment, the label comprises one or both of an alkaline phosphatase and a peroxidase chemiluminescent substance.

In one embodiment, the kit further comprises a solution containing a substance capable of catalyzing the emission of light by the chemiluminescent catalyst.

In one embodiment, the substance capable of emitting light catalyzed by the chemiluminescent catalyst comprises 1 to 3 mass percent of luminol solution.

In one embodiment, the Tris content is 1-3% by mass and the hydrogen peroxide content is 1-3% by mass.

In one embodiment, the luminescent label comprises one or more of adamantane, luminol, isoluminol acridinium ester.

The present application also provides a quantitative quality control method comprising the protein chip kit according to any one of claims 1 to 9.

According to the application, quantitative quality control is carried out on the sample adding accuracy through a ruthenium antibody-ruthenium-marker resisting ruthenium antibody system, the essence control immunology method is a double-antibody sandwich method, optical signals of the essence control immunology method are collected through a CCD camera, intelligent analysis is carried out to obtain optical signals, the ruthenium concentration is different according to different sample adding conditions, the obtained optical signals are different, and therefore quantitative quality control sample adding accuracy is achieved. The present application provides a chemiluminescence method for detecting sample application accuracy in an intensive, high-throughput, quantitative manner, with a high degree of sensitivity achieved using chemiluminescence techniques. The method realizes high-speed, simple and convenient detection by utilizing a full-automatic chip reader, and is more suitable for clinical application than other methods requiring a large amount of manual operation.

Drawings

FIG. 1 shows the relationship between the ruthenium working concentration and the detection signal value.

Detailed Description

The present application will be described in further detail with reference to embodiments and examples. It should be understood that these embodiments and examples are presented only for the purpose of illustrating the present application and are not intended to limit the scope of the present application, which is provided for the purpose of making the present disclosure more thorough and complete. It should also be understood that the present application can be embodied in many different forms and is not limited to the embodiments and examples described herein, and that various changes or modifications may be effected therein by one of ordinary skill in the art without departing from the spirit and scope of the application, and equivalents may be obtained thereby while remaining within the scope of the application. Furthermore, in the following description, numerous specific details are set forth in order to provide a more thorough understanding of the present application, and it is understood that the present application may be practiced without one or more of these details.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.

Term(s) for

Unless otherwise stated or contradicted, terms or phrases used herein have the following meanings:

in the present application, "further", "still further", "specifically" and the like are used for descriptive purposes to indicate differences in content, but should not be construed as limiting the scope of the present application.

In the present application, "optionally", "optional" and "optional" refer to the presence or absence, i.e., to any one of two juxtapositions "present" or "absent". If multiple optional parts appear in one technical scheme, if no special description exists, and no contradiction or mutual constraint relation exists, each optional part is independent.

In this application, the terms "first," second, "" third, "" fourth, "and the like in the terms of first aspect," "second aspect," "third aspect," "fourth aspect," and the like, are used for descriptive purposes only and are not to be construed as indicating or implying a relative importance or quantity, nor is it to be construed as implicitly indicating the importance or quantity of the technical feature indicated. Also, "first," "second," "third," "fourth," etc. are used for non-exhaustive enumeration of description purposes only and should not be construed as a closed limitation to the number.

In the present application, the technical features described in the open manner include a closed technical solution including the listed features, and also include an open technical solution including the listed features.

In the present application, where a range of values (i.e., a numerical range) is recited, unless otherwise stated, alternative distributions of values within the range are considered to be continuous and include both the numerical endpoints of the range (i.e., the minimum and maximum values) and each of the numerical values between the two numerical endpoints. Unless otherwise specified, when a numerical range refers to integers only within the numerical range, both endpoints of the numerical range, and each integer between the two endpoints are included, and herein, it is equivalent to reciting each integer directly, such as t being an integer selected from 1 to 10, meaning t being any integer selected from the group of integers consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10. Further, when multiple range-describing features or characteristics are provided, the ranges may be combined. In other words, unless otherwise indicated, the ranges disclosed herein are to be understood to include any and all subranges subsumed therein.

All documents mentioned in this application are incorporated by reference into this application as if each were individually incorporated by reference. Citations referred to herein are incorporated by reference in their entirety for all purposes unless otherwise conflicting with respect to the purpose and/or technical disclosure of the present application. Where a citation document is referred to in this application, the definition of the relevant feature, term, noun, phrase, etc. in the citation document is also incorporated by reference. In the case of citations in the present application, examples and preferred embodiments of the cited features of the related art are also incorporated by reference into the present application, but are not intended to limit the applicability of the present application. It should be understood that where the citation conflicts with the description herein, the application will control or be adapted in accordance with the description herein.

In this application, "about" or "approximately" means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, "about" can mean within 3 or more than 3 standard deviations, according to practice in the art. Alternatively, "about" may mean a range of up to 20%, preferably up to 10%, more preferably up to 5%, and still more preferably up to 1% of a given value. Alternatively, particularly with respect to biological systems or processes, the term may mean within one order of magnitude, preferably within 5-fold, and more preferably within 2-fold of the value. In the present application, the technical features described in the open manner include a closed technical solution including the listed features, and also include an open technical solution including the listed features.

Protein chip: the research object of the protein chip technology is protein, the principle is that a solid phase carrier is subjected to special chemical treatment, known protein molecule products are fixed on the protein chip (such as enzyme, antigen, antibody, receptor, ligand, cytokine and the like), according to the characteristics of biomolecules, proteins to be detected (existing in serum, plasma, lymph, interstitial fluid, urine, exudate, cytolysis fluid, secretion and the like) which can be specifically combined with the biomolecules are captured, and the proteins are washed, purified, combined with labeled protein and subjected to catalytic luminescence to obtain optical signals; it provides powerful technical support for obtaining important life information (such as unknown protein components and sequences, in vivo expression level biological functions, mutual regulation and control relation with other molecules, drug screening, selection of drug target positions and the like).

Capture antibody: and the antibody is fixed on the solid phase carrier and can be specifically combined with the object to be detected.

Detecting an antibody: an antibody labeled with alkaline phosphatase or peroxidase, which specifically binds to the analyte to which the capture antibody has bound.

A CCD camera: the CCD is a charge coupled device, which is a detecting element that uses charge to represent the signal size and transmits the signal in a coupling mode, has a series of advantages of self-scanning, wide sensing spectrum range, small distortion, small volume, light weight, low system noise, low power consumption, long service life, high reliability and the like, and can be made into an assembly with very high integration level.

The application provides a protein chip kit for quantitative quality control of sample adding accuracy: the kit comprises a protein chip coated with ruthenium capture antibody, ruthenium-containing antibody solution and labeled antibody solution with a label. The ruthenium capture antibody and the labeled antibody can be combined with ruthenium in the ruthenium-containing antibody solution, and the ruthenium capture antibody and the labeled antibody have different combination sites with ruthenium. The binding format is a double antibody sandwich method in which the capture antibody is immobilized on a solid support, the sample is added, and the protein of interest is bound and immobilized on a plate. A conjugated detection antibody is then added to bind to another epitope on the target protein. The substrate is added and a signal is generated that is proportional to the amount of analyte present in the sample. The double antibody sandwich method has a high degree of specificity because two antibodies are required to bind to the protein of interest. In determining the concentration of an analyte in a biological sample, a sandwich method is often used, which has the highest specificity and sensitivity.

In one specific example, the protein chip is subjected to a treatment of soaking in an alkaline solution and a silane solution before being coated with the ruthenium capture antibody. Optionally, the carrier of the protein chip is a black glass slide, and the black glass slide is subjected to soaking treatment and then drying treatment before spotting, for example, the treatment method is that the soaked black glass slide is placed into an oven after being purged with nitrogen and is baked for 0.5 to 1 hour at the temperature of 80 to 120 ℃. For example, the temperature is 80 deg.C, 90 deg.C, 100 deg.C, 110 deg.C, 120 deg.C, and the time is 0.5h, 0.6h, 0.7h, 0.8h, 0.9h, and 1.0h.

Optionally, the lye is a solution having a pH of greater than 10. For example, sodium hydroxide and potassium hydroxide solution, and the soaking time is 15 to 25 hours. For example, the soaking time is 15h, 16h, 17h, 18h, 19h, 20h, 21h, 22h, 23h, 24h and 25h.

Wherein, the silane solution is an ethanol solution with the silane mass percent of 0.05-1%. The soaking time is 20-40 min. For example, 20min, 25min, 30min, 35min, 40min. The silane solution is soaked to act as an adhesion-promoting additive to bind the antibody to the surface of the support, which not only reduces corrosion and peeling of the paint film in a wet environment, but also improves scratch resistance and environmental resistance.

In one specific example, ruthenium capture antibodies are spotted to form a (2-6) × (1-6) matrix on a protein chip. For example, 2 × 1, 2 × 2, 2 × 3, 2 × 4, 2 × 5, 2 × 6, 3 × 1, 3 × 2, 3 × 3, 3 × 4, 3 × 5, 3 × 6, 4 × 1, 4 × 2, 4 × 3, 4 × 4, 4 × 5, 4 × 6, 5 × 1, 5 × 2, 5 × 3, 5 × 4, 5 × 5, 5 × 6, 6 × 1, 6 × 2, 6 × 3, 6 × 4, 6 × 5, 6 × 6. The matrix is formed by automatic machine sample application, and the sample application mode comprises contact sample application (needle point) and non-contact sample application (spray point). The spotting is to spot a DNA sample onto a medium by directly contacting the surface of the medium with a needle. Common types of needles are split needles, solid needles, capillary needles or loop needles.

Further optionally, immersing the spotted black slide in a blocking solution for 1-4 hours, such as 1 hour, 2 hours, 3 hours, 4 hours, wherein the blocking solution is a buffer solution containing blocking protein, and the blocking protein is bovine serum albumin or ovalbumin; the buffer solution is one or more of PBS buffer solution, tris buffer solution, HEPS buffer solution and MOPS buffer solution.

Optionally, the label comprises one or both of a chemiluminescent catalyst and a luminescent label.

In one embodiment, the label comprises one or both of an alkaline phosphatase and a peroxidase chemiluminescent substance. Also included in the kit is a solution containing a substance capable of catalyzing the emission of light by the chemiluminescent catalyst. When in use, the corresponding chemiluminescence is matchedThe substrate can be used for quantitative detection by luminescence, and the chemiluminescence substrate comprises NaOH and H ₂ O ₂ Also included is at least one of adamantane, luminol and derivatives thereof, isoluminol or derivatives thereof, preferably N- (4-aminobutyl) -N-ethyl isoluminol (ABEI).

Optionally, the solution of the substance capable of emitting light catalyzed by the chemiluminescent catalyst comprises 1-3% by mass of luminol, 1-3% by mass of Tris and 1-3% by mass of hydrogen peroxide.

In one embodiment, the luminescent label comprises one or more of adamantane, luminol, isoluminol, and acridinium ester. The luminescent marker refers to a compound which participates in energy transfer in a luminescence reaction and finally releases energy in the form of emitting photons, and the compound can be catalyzed by a catalyst and oxidized by an oxidant to form an excited state intermediate, and when the excited state intermediate returns to a stable ground state, photons (hM) are simultaneously emitted.

The present application also provides a quantitative quality control method, which comprises the white chip kit according to any one of claims 1 to 9.

Example 1

A chip for quantitatively detecting the sampling accuracy is prepared by the following steps:

(1) Pretreating a black glass slide;

(1) and (3) placing the black glass slide into a glass slide pretreatment solution containing NaOH to be soaked for 16h, and then cleaning the black glass slide for 2-8 times by adopting purified water.

(2) The black glass slide is soaked in 1wt% silane solution (25% ethanol as medium) for 20min.

(3) And blowing the soaked black glass slide with nitrogen, putting the black glass slide into an oven, and baking the black glass slide for 0.2h at the temperature of 180 ℃.

(2) Spotting a capture antibody component solution;

adopting a machine to automatically sample, and spotting a 4 multiplied by 1 matrix on a black slide; the matrix is the spot formed by the ruthenium capture antibody solution.

(3) Blocking the capture antibody solution;

and immersing the spotted black slide into a blocking solution (Tris buffer solution containing 3% bovine serum albumin) for 3 hours, then taking out the black slide, and centrifuging to remove residual blocking solution to obtain the chip.

(4) Reagent kit

The chip was packaged together with a secondary antibody solution labeled with HRP enzyme, a MAK-33 antibody solution containing ruthenium, a detection solution A (containing 1% luminol and 2% Tris), and a detection solution B (1% hydrogen peroxide) to make a kit.

(5) Signal acquisition

The instrument automatically sucks quality control sample and MAK-33 antibody solution containing ruthenium in different proportions into a reaction cup to be mixed uniformly, a protein chip is automatically placed into a sample to be detected, the sample is incubated for 40 minutes at 37 ℃, then the chip is taken out by an instrument clamping jaw, the chip is automatically washed by the instrument and then put into a second antibody solution (200 ul, the instrument automatically sucks in advance) marked with HRP enzyme, the chip is taken out by the instrument clamping jaw after the sample is incubated for 40 minutes again, the chip is automatically washed by the instrument and then put into a luminescent substrate solution (formed by mixing 100ul of detection liquid A and 100ul of detection liquid B, the chip is automatically sucked and mixed by the instrument), finally, the protein chip is photographed and imaged, pictures are automatically analyzed by software, and an analysis result is given.

Example 2

A chip for quantitatively detecting the accuracy of sample adding is prepared by the following steps:

(1) Pretreating a black glass slide;

(1) and (3) soaking the black glass slide in a glass slide pretreatment solution containing NaOH for 24h, and then cleaning the black glass slide for 2-8 times by adopting purified water.

(2) The black glass slide is soaked in a silane solution (the medium is 25% ethanol) with the mass concentration of 0.05wt% for 60min.

(3) And blowing the soaked black glass slide with nitrogen, putting the black glass slide into an oven, and baking the black glass slide for 0.4h at the temperature of 140 ℃.

(2) Spotting a specific component solution;

(3) Blocking the capture antibody solution;

and (3) immersing the spotted black slide into a blocking solution (PBS buffer solution containing 6% ovalbumin) for 4 hours, taking out the black slide, and centrifuging to remove residual blocking solution to obtain the chip.

(4) Reagent kit

The chip was packaged together with HRP-enzyme-labeled secondary antibody solution, MAK-33 antibody solution containing ruthenium, detection solution A (containing 1% luminol and 2% Tris), and detection solution B (1% hydrogen peroxide) to prepare a kit.

(5) Signal acquisition

The instrument automatically sucks quality control sample and ruthenium-containing MAK-33 antibody solution with different proportions into a reaction cup to be mixed uniformly, a protein chip is automatically placed into a sample to be tested to be incubated for 40 minutes at 37 ℃, then the chip is taken out by an instrument clamping jaw, the chip is automatically washed by the instrument and then put into a second antibody solution (200 ul, the instrument is automatically sucked in advance), which is marked with HRP enzyme, the chip is taken out again by the instrument clamping jaw after the chip is incubated for 40 minutes again, the chip is automatically washed by the instrument and then put into a luminescent substrate solution (formed by mixing 100ul of detection solution A and 100ul of detection solution B, the chip is automatically sucked and mixed by the instrument), finally, the protein chip is photographed and imaged, pictures are automatically analyzed by software, and an analysis result is given.

Example 3

(1) Pretreating a black glass slide;

(1) and (3) soaking the black glass slide in a glass slide pretreatment solution containing NaOH for 20h, and then cleaning the black glass slide for 2-8 times by adopting purified water.

(2) The black glass slide is soaked in a silane solution (the medium is 25% ethanol) with the mass concentration of 0.5wt% for 30min.

(3) And blowing the soaked black glass slide with nitrogen, putting the black glass slide into an oven, and baking the black glass slide for 0.6h at the temperature of 100 ℃.

(2) Spotting a capture antibody component solution;

(3) Blocking the capture antibody solution;

and (3) immersing the spotted black slide into a sealing solution (MOPS buffer solution containing 4% bovine serum albumin) for 1h, then taking out the black slide, and centrifuging to remove residual sealing solution to obtain the chip.

(4) Reagent kit

(5) Signal acquisition

Test example

The SLXP-001B type biochip analyzer produced by Jiangsu triple bioengineering GmbH is used for detecting the internal quality control of the company, and the working process of the SLXP-001B type biochip analyzer is as follows:

the instrument automatically absorbs quality control product samples and a ruthenium-containing MAK-33 solution in different proportions into a reaction cup to be mixed uniformly (the proportion of 180+20ul is a control group, the proportion of 150+20ul, 100+20ul, 200+20ul, 230+20ul, 180+15ul, 180+10ul, 180+25ul and 180+30ul is an experimental group), the protein chip prepared in the embodiment is automatically placed into blood serum to be detected by the instrument, the blood serum is incubated for 40 minutes at 37 ℃, then the chip is taken out by a clamping jaw of the instrument, the protein chip is automatically flushed by the instrument and then put into a second antibody solution (200 ul, the instrument is automatically sucked in advance), the chip is taken out by the clamping jaw of the instrument again after 40 minutes of incubation, the chip is automatically flushed and then put into a luminescent substrate solution (the detection solution A with 100ul of HRP enzyme and the detection solution B are mixed together), and the instrument automatically absorbs and mixes the image, and finally the chip is analyzed by software to obtain an image.

Characterization data and effect data for the products of examples and comparative examples:

mean value	Working concentration of ruthenium	Sample application conditions	Signal value
				Z1	0.013％	20+180	6930
Z2	0.015％	20+150	8544
				Z3	0.021％	20+100	10122
Z4	0.011％	20+200	7003
				Z5	0.010％	15+180	5497
Z6	0.007％	10+180	4638
				Z7	0.015％	25+180	7490
Z8	0.018％	30+180	10590

TABLE 1

The technical features of the above embodiments can be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the above embodiments are not described, but should be considered as the scope of the present specification as long as there is no contradiction between the combinations of the technical features.

The above examples only express several embodiments of the present application, and the description thereof is more specific and detailed, but not construed as limiting the claims. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the concept of the present application, which falls within the scope of protection of the present application. Therefore, the protection scope of the present patent application shall be subject to the appended claims.

Claims

1. A protein chip kit for quantitative quality control of sample adding accuracy is characterized by comprising a protein chip coated with a ruthenium capture antibody, an antibody solution containing ruthenium and a labeled antibody solution with a label;

2. The protein chip kit for quantitative quality control of sample application accuracy according to claim 1, wherein a matrix of (2-6) × (1-6) is formed on the protein chip after the ruthenium capture antibody is spotted.

3. The protein chip kit for quantitative quality control of sample application accuracy according to claim 1, wherein the protein chip is subjected to alkali solution soaking and silane solution soaking before being coated with the ruthenium capture antibody.

4. The protein chip kit for quantitative quality control of sample application accuracy according to claim 3, wherein the alkaline solution is a solution with pH greater than 10, and the silane solution is an ethanol solution with silane mass percent of 0.05-1%.

5. The protein chip kit for quantitative quality control of sample application accuracy according to any one of claims 1 to 4, wherein the label comprises one or both of a chemiluminescent catalyst and a luminescent label.

6. The protein chip kit for quantitative quality control of sample application accuracy according to any one of claims 1 to 4, wherein the label comprises one or two chemiluminescent catalysts of alkaline phosphatase and peroxidase.

7. The protein chip kit for quantitative quality control of sample application accuracy according to any one of claims 1 to 4, wherein the kit further comprises a substance containing a substance capable of catalytic luminescence by the chemiluminescent catalyst.

8. The protein chip kit for sample application accuracy and quantitative quality control according to claims 1 to 4, wherein the substance capable of being catalyzed by the chemiluminescent catalyst to emit light comprises 1 to 3 mass percent of luminol solution;

optionally, the substance capable of being catalyzed by the chemiluminescence catalyst to emit light further comprises 1-3% by mass of Tris and 1-3% by mass of hydrogen peroxide.

9. The protein chip kit for quantitative quality control of sample application accuracy according to claim 7, wherein the luminescent label comprises one or more of adamantane, luminol, isoluminol, acridinium ester and derivatives thereof.

10. Use of the protein chip kit of any one of claims 1 to 9 for quantitative quality control with sample application accuracy.