CN115524485B

CN115524485B - Protein chip kit for quantitative quality control of sample addition accuracy and application

Info

Publication number: CN115524485B
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: 2023-12-05
Anticipated expiration: 2042-10-09
Also published as: CN115524485A

Abstract

The application relates to a protein chip kit for quantitative quality control of sample adding accuracy, which comprises a protein chip coated with a ruthenium capture antibody, a ruthenium-containing antibody solution and a labeled antibody solution with a marker, wherein the quantitative quality control of the sample adding accuracy is carried out through an anti-ruthenium antibody-ruthenium-labeled anti-ruthenium antibody system, an essentially controlled immunological method is a double antibody sandwich method, optical signals of the double antibody sandwich method are acquired through a CCD camera and are intelligently analyzed to obtain optical signals, and the obtained optical signals are different according to different sample adding conditions, so that the quantitative quality control sample adding accuracy is realized. The application provides a chemiluminescence method for detecting sample loading accuracy in an intensive, high-flux and quantitative mode, and high sensitivity is obtained by utilizing a chemiluminescence technology. The full-automatic chip reader is used for realizing high-speed and simple detection, 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 addition 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 loading accuracy and application thereof.

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 taken as ligands (probe proteins) and 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 an object to be detected in a sample, cleaning to remove unbound components, then binding with peroxidase-labeled proteins (or other molecules), cleaning to remove unbound components, reacting with a luminescent substrate, catalyzing luminescence, capturing optical signals by a CCD, and obtaining a detection result.

The traditional quality control flow of the protein chip to the sample adding accuracy is as follows: after the detection result is obtained, the picture is checked when the result is abnormal, the sample loading accuracy is confirmed by analyzing the position of the liquid level line, and the sample loading accuracy of the processing liquid is analyzed by combining part of index signals. Moreover, at present, no simple and effective sample loading accuracy quality control method exists.

Disclosure of Invention

Based on the method, the application provides the protein chip kit for quantitative quality control of sample loading accuracy and application thereof to solve the problems of low efficiency, complex operation, need to be judged by experienced staff and the like of the traditional quality control flow.

The application aims to provide a protein chip kit for quantitative quality control of sample loading accuracy and application thereof, wherein the kit is simple in application and can efficiently and quickly identify the condition of insufficient sample loading.

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

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

The ruthenium capture antibody and the labeled antibody are both capable of binding to ruthenium in the ruthenium-containing antibody solution, and the ruthenium capture antibody and the labeled antibody are different from the binding site of ruthenium in the ruthenium-containing antibody solution.

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

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

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

In one embodiment, the kit further comprises a solution comprising a substance capable of catalyzing luminescence by the chemiluminescent catalyst.

In one embodiment, the substance capable of being catalyzed by the chemiluminescent catalyst to emit light comprises a luminol solution with a mass percent of 1% -3%.

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

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

The application also provides a quantitative quality control mode, which comprises the protein chip kit as claimed in any one of claims 1 to 9.

According to the application, quantitative quality control is carried out on the sample adding accuracy through an anti-ruthenium antibody system of an anti-ruthenium antibody-ruthenium-marker, the essentially controlled immunological method is a double-antibody sandwich method, optical signals are acquired through a CCD camera and are intelligently analyzed to obtain the optical signals, the ruthenium concentration is different according to different sample adding conditions, and the obtained optical signals are different, so that quantitative quality control sample adding accuracy is realized. The application provides a chemiluminescence method for detecting sample loading accuracy in an intensive, high-flux and quantitative mode, and high sensitivity is obtained by utilizing a chemiluminescence technology. The full-automatic chip reader is used for realizing high-speed and simple detection, and is more suitable for clinical application than other methods requiring a large amount of manual operation.

Drawings

FIG. 1 shows the correspondence 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 provided solely for the purpose of illustrating the application and are not intended to limit the scope of the application in order that the present disclosure may be more thorough and complete. It will also be appreciated that the present application may be embodied in many different forms and is not limited to the embodiments and examples described herein, but may be modified or altered by persons skilled in the art without departing from the spirit of the application, and equivalents thereof are also intended to fall 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 application, it being understood that the 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.

Terminology

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

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

In the present application, "optional" means optional or not, that is, means any one selected from two parallel schemes of "with" or "without". If multiple "alternatives" occur in a technical solution, if no particular description exists and there is no contradiction or mutual constraint, then each "alternative" is independent.

In the present application, the terms "first", "second", "third", "fourth", etc. are used for descriptive purposes only and are not to be construed as indicating or implying a relative importance or quantity, nor as implying an importance or quantity of a technical feature being indicated. Moreover, the terms "first," "second," "third," "fourth," and the like are used for non-exhaustive list description purposes only, and are not to be construed as limiting the number of closed forms.

In the application, the technical characteristics described in an open mode comprise a closed technical scheme composed of the listed characteristics and also comprise an open technical scheme comprising the listed characteristics.

In the present application, a numerical range (i.e., a numerical range) is referred to, and optional numerical distributions are considered to be continuous within the numerical range and include two numerical endpoints (i.e., a minimum value and a maximum value) of the numerical range and each numerical value between the two numerical endpoints unless otherwise specified. Where a numerical range merely refers to integers within the numerical range, including both end integers of the numerical range, and each integer between the two ends, unless otherwise indicated, each integer is recited herein as directly, such as where t is an integer selected from 1-10, and where t is any integer selected from the group of integers consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10. Further, when a plurality of range description features or characteristics are provided, these 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 disclosure are incorporated by reference in this disclosure as if each were individually incorporated by reference. Unless otherwise indicated to the contrary by the intent and/or technical scheme of the present application, all references to which this application pertains are incorporated by reference in their entirety for all purposes. When reference is made to a cited document in the present application, the definitions of the relevant technical features, terms, nouns, phrases, etc. in the cited document are also incorporated. In the case of the cited documents, examples and preferred modes of the cited relevant technical features are also incorporated into the present application by reference, but are not limited to being able to implement the present application. It should be understood that when a reference is made to the description of the application in conflict with the description, the application is modified in light of or adaptive to the description of the application.

In the present application, "about" or "approximately" means within an acceptable error range of a particular value as determined by one of ordinary skill in the art, which depends in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, according to practice in the art, "about" may mean within 3 or more than 3 standard deviations. 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, in particular with respect to biological systems or processes, the term may mean within one order of magnitude of the value, preferably within a factor of 5, and more preferably within a factor of 2. In the application, the technical characteristics described in an open mode comprise a closed technical scheme composed of the listed characteristics and also comprise an open technical scheme comprising the listed characteristics.

Protein chip: the research object of protein chip technology is protein, its principle is that it carries on special chemical treatment to the solid phase carrier, then fixes the known protein molecule product (such as enzyme, antigen, antibody, receptor, ligand, cytokine, etc.), according to the characteristics of these biological molecules, captures the protein to be tested (existing in serum, plasma, lymph, interstitial fluid, urine, exudates, cell dissolving fluid, secretion, etc.), then carries on washing, purification, then combines with the marked protein, catalyzes and shines, gets the optical signal; the method provides powerful technical support for obtaining important life information (such as unknown protein components, sequences, in vivo expression level biological functions, mutual regulation and control relation with other molecules, drug screening, drug target selection and the like).

Capture antibody: an antibody which is immobilized on a solid phase carrier and can specifically bind to an analyte.

Detection of antibodies: an alkaline phosphatase or peroxidase is labeled, and an antibody specifically binding to the analyte that has bound to the capture antibody is labeled.

CCD camera: CCD is a charge coupled device, which is a detecting element for representing the signal size by charge quantity and transmitting signals by coupling mode, and has the advantages of self-scanning, wide sensing spectrum range, small distortion, small volume, light weight, low system noise, small 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 addition accuracy, which comprises the following components: the kit comprises a protein chip coated with a ruthenium capture antibody, a ruthenium-containing antibody solution and a labeled antibody solution with a label. Both the ruthenium capture antibody and the labeled antibody are capable of binding to ruthenium in the ruthenium-containing antibody solution, and the ruthenium capture antibody and the labeled antibody are different from the binding site of ruthenium. The binding mode 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. Conjugated detection antibodies are 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 is highly specific 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 a specific example, the protein chip is subjected to an alkaline solution soak and a silane solution soak treatment prior to coating with the ruthenium capture antibody. Optionally, the carrier of the protein chip is a black glass slide, and the black glass slide is soaked before spotting and then dried, for example, the soaked black glass slide is put into an oven after being purged by nitrogen, and baked for 0.5 to 1 hour at the temperature of 80 to 120 ℃. For example, the temperature is 80 ℃, 90 ℃, 100 ℃, 110 ℃, 120 ℃, and the time is 0.5h, 0.6h, 0.7h, 0.8h, 0.9h, 1.0h.

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

Wherein the silane solution is ethanol solution with the mass percent of silane of 0.05-1 percent. The soaking time is 20-40 min. For example 20min, 25min, 30min, 35min, 40min. The effect of the soaking silane solution is to bind the antibodies to the support surface as an additive to improve adhesion, which not only reduces corrosion and flaking of the paint film in wet environments, but also improves scratch and environmental resistance.

In one specific example, a matrix of (2-6) × (1-6) is formed on the protein chip after spotting the ruthenium capture antibody. 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 for automatic printing of the machine, and the printing modes comprise contact printing (needle point) and non-contact printing (spray point). By spotting is meant spotting the DNA sample onto the medium by direct contact with the surface of the medium with a needle. Common types of needles are split needles, solid needles, capillary or loop needles, etc.

Further alternatively, the spotted black glass slide is immersed in a blocking solution for 1-4 h, for example, 1h, 2h, 3h and 4h, 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.

Alternatively, 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. Also included in the kit are solutions containing substances capable of being catalyzed by chemiluminescent catalysts to emit light. When in use, the fluorescent light can be quantitatively detected by matching with corresponding chemiluminescent substrates, wherein the chemiluminescent substrates comprise NaOH and H ₂ O ₂ Also included are 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 being catalyzed by the chemiluminescent catalyst to emit light 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 above luminescent marker refers to a compound which participates in energy transfer in a luminescence reaction and finally releases energy in the form of emitted photons, and which is capable of forming an excited state intermediate by the catalysis of a catalyst and the oxidation of an oxidizing agent, and emitting photons (hM) at the same time when such excited state intermediate returns to a stable ground state.

The application also provides a quantitative quality control mode, which comprises the white chip kit as claimed in any one of claims 1 to 9.

Example 1

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

(1) Pretreating a black glass slide;

(1) and placing the black glass slide into a slide pretreatment liquid containing NaOH for soaking for 16 hours, and then cleaning the black glass slide with purified water for 2-8 times.

(2) The black slide was immersed in a 1wt% silane solution (25% ethanol medium) for 20min.

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

(2) Spotting the capture antibody component solution;

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

(3) Blocking the capture antibody solution;

the spotted black slide was immersed in blocking solution (Tris buffer containing 3% bovine serum albumin) for 3 hours, after which the black slide was removed and centrifuged to remove residual blocking solution, thus obtaining the chip.

(4) Kit for detecting a substance in a sample

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

(5) Signal acquisition

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

Example 2

A chip for quantitatively detecting sample addition accuracy, which is prepared by the following steps:

(1) Pretreating a black glass slide;

(1) and placing the black glass slide into a slide pretreatment liquid containing NaOH for soaking for 24 hours, and then cleaning the black glass slide with purified water for 2-8 times.

(2) The black slide was immersed in a silane solution (medium 25% ethanol) having a mass concentration of 0.05% for 60min.

(3) And (3) placing the soaked black glass slide into an oven after nitrogen purging, and baking for 0.4h at 140 ℃.

(2) Spotting the specific component solution;

(3) Blocking the capture antibody solution;

the spotted black slide was immersed in blocking solution (PBS buffer containing 6% ovalbumin) for 4 hours, after which the black slide was removed and centrifuged to remove residual blocking solution, thereby obtaining the chip.

(4) Kit for detecting a substance in a sample

(5) Signal acquisition

Example 3

(1) Pretreating a black glass slide;

(1) and placing the black glass slide into a slide pretreatment liquid containing NaOH for soaking for 20 hours, and then cleaning the black glass slide with purified water for 2-8 times.

(2) The black slide was immersed in a silane solution (medium 25% ethanol) having a mass concentration of 0.5% for 30min.

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

(2) Spotting the capture antibody component solution;

(3) Blocking the capture antibody solution;

the spotted black slide was immersed in a blocking solution (MOPS buffer containing 4% bovine serum albumin) for 1h, after which the black slide was removed and centrifuged to remove the residual blocking solution, thus preparing the chip.

(4) Kit for detecting a substance in a sample

(5) Signal acquisition

Test case

The SLXP-001B biochip analyzer manufactured by Jiangsu triple bioengineering Co., ltd was used to detect the internal quality control of the company, and the working process of the SLXP-001B biochip analyzer was as follows:

the instrument automatically absorbs and evenly mixes the quality control sample and the MAK-33 solution containing ruthenium in different proportions into a reaction cup (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 instrument automatically places the protein chip prepared by the embodiment of the application into serum to be detected, incubates for 40 minutes at 37 ℃, then the instrument clamping jaw takes out the chip, the chip is automatically washed by the instrument and then is put into a secondary antibody solution (200 ul) marked with HRP enzyme, the instrument automatically absorbs in advance, the chip is again taken out after incubation for 40 minutes, the chip is automatically washed by the instrument and then is put into a luminescent substrate solution (formed by mixing 100ul of detection solution A and 100ul of detection solution B), finally the protein chip is automatically absorbed and mixed by the instrument, the image is formed by shooting is carried out, and the software automatically analyzes the image, and the analysis result is given.

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

mean value of	Ruthenium working concentration	Sample application case	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 may be arbitrarily combined, and all possible combinations of the technical features in the above embodiments are not described for brevity of description, however, as long as there is no contradiction between the combinations of the technical features, they should be considered as the scope of the description.

The foregoing examples illustrate only a few embodiments of the application, which are described in detail and are not to be construed as limiting the scope of the claims. It should be noted that it will be apparent to those skilled in the art that several variations and modifications can be made without departing from the spirit of the application, which are all within the scope of the application. Accordingly, the scope of protection of the present application is to be determined by the appended claims.

Claims

1. A protein chip kit for quantitative quality control of sample addition accuracy, which is characterized by comprising a protein chip coated with a ruthenium capture antibody, a ruthenium-containing MAK-33 antibody solution and a labeled antibody solution with a label;

the marker comprises one or two of a chemiluminescent catalyst and a luminescent marker;

the chemiluminescent catalyst comprises one or two of alkaline phosphatase and peroxidase;

the luminescent marker comprises one or more of adamantane, luminol, isoluminol, acridinium ester and derivatives thereof;

the kit also comprises 1-3% of luminol solution by mass percent, 1-3% of Tris by mass percent and 1-3% of hydrogen peroxide by mass percent;

the ruthenium capture antibody and the labeled antibody are both capable of binding to ruthenium in the ruthenium-containing MAK-33 antibody solution, and the ruthenium capture antibody and the labeled antibody are different from the binding site of ruthenium in the ruthenium-containing MAK-33 antibody solution;

the protein chip is subjected to alkali liquor soaking and silane solution soaking treatment before being coated with the ruthenium capture antibody;

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

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

3. The use of the protein chip kit according to any one of claims 1-2 in quantitative quality control of sample application accuracy.