CN117825721A - Buffer composition for removing specific interference and application thereof - Google Patents

Abstract

The application provides a buffer composition for removing specific interference and application thereof. The buffer composition includes 0.1 mg/mL-3.5 mg/mL bromelain, 0.1 mg/mL-3.5 mg/mL cross-reactive carbohydrate determinants of bromelain, 0.1 mg/mL-3.5 mg/mL ascorbate oxidase, and a base buffer. The buffer composition comprises bromelain, cross-reactive carbohydrate determinants of ascopyrin and ascorbate oxidase with specific concentrations, and can be used as sample diluent to be combined with CCD-sIgE produced by a CCD-induced organism in a sample to be tested, so that the false positive result caused by CCD is reduced, and the specificity and accuracy of sIgE detection are improved.

Description

Buffer composition for removing specific interference and application thereof

Technical Field

The application relates to the technical field of allergen detection, in particular to a buffer composition for removing specific interference and application thereof.

Background

Allergic diseases are one of the most common diseases in the twentieth century, timely detection of allergens and avoidance of exposure to allergens are the primary principles of allergy protection, and accurate allergen detection is of great importance for diagnosis and treatment of allergic diseases.

Immunoglobulin E (IgE) is a major effector molecule of allergic diseases, and the content of IgE antibodies in serum of most allergic disease patients is significantly higher than that of healthy people, so detecting the content of allergen-specific IgE in serum of patients is an important method for allergen diagnosis.

Currently, common methods for detecting allergen-Specific IgE (sIgE) levels in patient serum include allergen chip assays. Allergen chip detection is based on a mature high-throughput detection technology, allergen molecules are immobilized on a carrier, and sIgE in human serum is detected through detection of antigen-antibody reaction. The allergen chip can detect allergen molecule sIgE anaphylactic reaction spectrum at one time, and the characteristics of convenience, rapidness, high flux, high sensitivity, high specificity and the like enable the allergen chip to develop rapidly, so the allergen chip becomes an important auxiliary tool for diagnosis and treatment of allergic diseases.

Plant and insect allergens contain glycoproteins with cross-reactive carbohydrate determinants (CCDs-resction carbohudrate determinants). CCDs are found in almost all allergens from plants and insects, but do not cause clinical symptoms. Studies have shown that approximately 20% -37% of people allergic to grass and venom have anti-CCD IgE antibodies (CCD-sIgE). CCD-sIgE is an antibody induced by CCD to produce by the body that is not related to clinical allergic symptoms, and is capable of cross-reacting with multiple allergens, resulting in false positives in the detection of serum-specific IgE. CCDs are glycosyl-modifying moieties common to many cell surface molecules of plant and insect species, and mammals recognize that CCDs produce humoral immune responses to foreign antigens. Therefore, CCD-sIgE is considered to be a significant cause of false positives in clinical detection of sIgE, inconsistent with clinical serum allergen test results. Thus, for patients with CCD-sIgE in the blood, serum-based allergy diagnosis is prone to false positive results.

Thus, there remains a need for improvements in conventional allergen detection methods.

Disclosure of Invention

Based on this, one or more embodiments of the present application provide a buffer composition capable of reducing the binding of anti-CCD IgE antibodies to an allergen as a sample dilution for allergen detection, improving the accuracy of allergen detection. The technical proposal comprises:

according to a first aspect of embodiments of the present application, there is provided a buffer composition comprising 0.1 mg to 3.5 mg/mL bromelain, 0.1 mg to 3.5 mg/mL cross-reactive carbohydrate determinants of bromelain, 0.1 mg to 3.5 mg/mL ascorbate oxidase and a base buffer.

In one embodiment, the buffer composition further comprises papain in an amount of 0.1 mg/mL-3.5 mg/mL based on the total volume of the buffer composition.

In one embodiment, the buffer composition satisfies one or more of the following conditions (1) - (3):

(1) The concentration of the bromelain is 0.8 mg/mL-3.5 mg/mL;

(2) The concentration of the bromelain cross-reactive carbohydrate determinant is 0.6 mg/mL-3.5 mg/mL; and

(3) The concentration of the ascorbate oxidase is 0.6 mg/mL-3.5 mg/mL.

In one embodiment, the buffer composition further comprises 0.05 v to 5 v/v% blocker, 0.01 v to 5 v/v% surfactant, 0.01 v to 1 v/v% bacteriostat, and 0.001 mg to 0.5 mg/mL anticoagulant, based on the total volume of the buffer composition.

In one embodiment, the blocking agent comprises one or more of casein, bovine serum albumin, rabbit serum, goat serum, horse serum, mark33 blocking agent, HIER-R-014 blocking agent, HIER-C-003 blocking agent, murine IgG, HIER-E-010-D blocking agent, and HBR-PLUS blocking agent.

In one embodiment, the surfactant comprises one or more of triton X-100, tween-20, ethylphenyl polyethylene glycol, surfactant S9, and octylphenyl-polyethylene glycol.

In one embodiment, the bacteriostatic agent comprises one or more of PC-150 bacteriostatic agent, PC-300 bacteriostatic agent, PC-950 bacteriostatic agent, BND-10 bacteriostatic agent, BND-99 bacteriostatic agent, PC-8020 bacteriostatic agent, BIT-10 bacteriostatic agent and GML-2 bacteriostatic agent.

In one embodiment, the anticoagulant includes one or more of heparin salt, edetate, citrate, and oxalate.

In one embodiment, the buffer composition satisfies one or more of the following conditions (1) - (2):

(1) The basic buffer solution comprises one or more of 3-morpholinopropanesulfonic acid buffer solution, 3-morpholinopropanesulfonic acid sodium salt, tris buffer solution, carbonate buffer solution, phosphate buffer solution and tris buffer solution;

(2) The concentration of the solute in the basic buffer solution is 10 g/L-20 g/L.

In one embodiment, the buffer composition satisfies one or more of the following conditions (1) - (4):

(1) The concentration of the blocking agent is 0.05 v/v% -1 v/v%;

(2) The concentration of the surfactant is 0.1 v/v% -5 v/v%;

(3) The concentration of the bacteriostat is 0.5 v/v% -1 v/v%;

(4) The concentration of the anticoagulant is 0.01 mg/mL-0.5 mg/mL.

Use of the above-described buffer composition in allergen detection.

Compared with the prior art, the method has the following beneficial effects:

the buffer composition comprises bromelain, cross-reactive carbohydrate determinants of ascalase and ascorbate oxidase with specific concentrations, and can be used as sample diluent to be combined with anti-CCD IgE produced by a CCD-induced organism in a sample to be tested, but not combined with sIgE, so that false positive results caused by CCD are reduced, and specificity and accuracy of sIgE detection are improved.

Drawings

In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the drawings that are needed in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present application, and other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.

FIG. 1 is a graph of the dot matrix light signal of the allergen detection chip of example 1 of the present application;

FIG. 2 is a graph showing the optical signal of the array of the allergen detection chip of comparative example 1.

Detailed Description

The detailed description of the embodiments of the present application will be presented in order to make the above objects, features and advantages of the present application more obvious and understandable. In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present application. This application is, however, susceptible of embodiment in many other forms than those described herein and similar modifications can be made by those skilled in the art without departing from the spirit of the application, and therefore the application is not to be limited to the specific embodiments disclosed below.

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. The terminology used herein in the description of the application is for the purpose of describing particular embodiments only and is not intended to be limiting of the application. Unless specifically indicated otherwise, the various raw materials, reagents, instruments, equipment, and the like used in the present application are commercially available or may be prepared by existing methods.

The terms "plurality", "plural", "multiple", and the like are used herein, and refer to a number of 2 or more, unless otherwise specified. For example, "one or more" means one kind or two or more kinds.

In this application, "optional," "optional," and "optional" refer to the presence or absence of, that is, either one of the two parallel schemes is selected from "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 technical features described in an open manner include a closed technical scheme composed of the listed features, and also include an open technical scheme including the listed features.

In this application, reference is made to a numerical interval (i.e., a numerical range), where the optional numerical distribution is considered continuous, and includes 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 indicated. 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.

In this application,% (w/w) and wt% both represent weight percent, v/v% means volume percent, and w/v% means mass volume percent.

In a first aspect of the present application, a buffer composition is provided comprising 0.1 mg/mL-3.5 mg/mL bromelain, 0.1 mg/mL-3.5 mg/mL cross-reactive carbohydrate determinant (MUXF) of bromelain, 0.1 mg/mL-3.5 mg/mL ascorbate oxidase, and a base buffer.

It will be appreciated that the concentration of each substance in the buffer composition is the working concentration, and the buffer composition may be a preformed mixture or an as-prepared mixture. That is, the concentrations of the above-mentioned respective substances are based on the relative concentrations at the time of mixing the three substances, but the form of the composition provided in the present application is not limited thereto, and the three substances may be allowed to stand separately, so long as the three substances can be mixed in the same system at the time of use.

In the present application, the concentration of bromelain cross-reactive carbohydrate determinants and the concentration of ascorbyl oxidase are all "0.1 mg/mL to 3.5 mg/mL", i.e. the minimum and maximum values in the range of 0.1 mg/mL to 3.5 mg/mL can be taken, and each value between such minimum and maximum values. Specific examples include, but are not limited to, the following dot values: 0.1 mg/mL, 0.2/mL, 0.3/mL, 0.4/mL, 0.5/mL, 0.6/mL, 0.7/mL, 0.8/mL, 0.9/mL, 1.0/mL, 1.1/mL, 1.2/mL, 1.3/mL, 1.4/mL, 1.5/mL, 1.6/mL, 1.7/mL, 1.8/mL, 1.9/mL, 2.0/mL, 2.1/mL, 2.2/mL, 2.3/mL, 2.4/mL, 2.5/mL, 2.6/mL, 2.7/mL, 2.8/mL, 2.9/mL, 3.0/mL, 3.1/mL, 3.2/mL, 3.3/mL, 3.4/mL or 3.5/mL; or a range of any two of these values, such as 1.5 mg/mL to 2.5 mg/mL.

In some alternative embodiments, the concentration of bromelain is 0.8 mg/mL to 3.5 mg/mL; further alternatively, the concentration of bromelain is 0.8 mg/mL.

In some alternative embodiments, the concentration of the cross-reactive carbohydrate determinants of bromelain is 0.6 mg/mL to 3.5 mg/mL; further alternatively, the concentration of the cross-reactive carbohydrate determinant of bromelain is 0.6 mg/mL.

In some embodiments, the cross-reactive carbohydrate determinant of bromelain is selected from the group consisting of the cross-reactive carbohydrate determinant of bromelain Man2XylFucGlcNAc2, available from the company of biotechnology, no tin-linked biosciences; the commercial model is NAG0000040.

In some alternative embodiments, the concentration of ascorbate oxidase is 0.6 mg/mL to 3.5 mg/mL; further alternatively, the concentration of ascorbate oxidase is 0.6 mg/mL.

In some embodiments, the buffer composition further comprises papain in an amount of 0.1-mg/mL-3.5-mg/mL.

In the application, the concentration of papain is 0.1 mg/mL-3.5 mg/mL, namely, the minimum value and the maximum value of the range of 0.1 mg/mL-3.5 mg/mL can be taken, and each value between the minimum value and the maximum value. Specific examples include, but are not limited to, the following dot values: 0.1 mg/mL, 0.2/mL, 0.3/mL, 0.4/mL, 0.5/mL, 0.6/mL, 0.7/mL, 0.8/mL, 0.9/mL, 1.0/mL, 1.1/mL, 1.2/mL, 1.3/mL, 1.4/mL, 1.5/mL, 1.6/mL, 1.7/mL, 1.8/mL, 1.9/mL, 2.0/mL, 2.1/mL, 2.2/mL, 2.3/mL, 2.4/mL, 2.5/mL, 2.6/mL, 2.7/mL, 2.8/mL, 2.9/mL, 3.0/mL, 3.1/mL, 3.2/mL, 3.3/mL, 3.4/mL or 3.5/mL; or a range of any two of these values, such as 0.8 mg/mL to 2.3 mg/mL.

In some alternative embodiments, the concentration of papain is 0.1 mg/mL to 0.35 mg/mL; further alternatively, the concentration of papain is 0.6 mg/mL.

It is understood that glycoproteins containing N-glycans in bromelain, MUXF, ascorbate oxidase and papain are capable of binding to anti-CCD IgE antibodies produced by the CCD-induced organism, but not to sIgE, thereby reducing the false positive results caused by CCD and improving the consistency of sIgE detection with Skin Prick Test (SPT).

Further, the concentration of each component is controlled within a specific range, and the prepared buffer composition is used as a sample diluent for allergen detection, so that the nonspecific binding of an anti-CCD IgE antibody and an allergen can be reduced, the binding of sIgE and the allergen can not be influenced, and the specificity and the accuracy of allergen detection can be improved.

In some embodiments, the base buffer comprises one or more of 3-morpholinopropanesulfonic acid buffer (Mops buffer), 3-morpholinopropanesulfonic acid sodium salt (Mops sodium salt), tris buffer, carbonate buffer, phosphate buffer, and Tris-sodium chloride-tween buffer (TTN buffer).

Buffer is understood to mean a solution containing a certain amount of acid or base, typically a weak acid and its conjugate base or a weak base and its conjugate acid, which changes less in pH when diluted with small amounts of water or small amounts of strong acid, strong base, i.e. a buffer solution.

In some embodiments, the concentration of solute in the base buffer is 10 g/L to 20 g/L. It will be appreciated that the above concentrations are the final working concentrations of the solutes present in the base buffer in the mixture.

In the application, the concentration of the solute in the basic buffer solution is 10 g/L-20 g/L, namely, the minimum value and the maximum value of the range of 10 g/L-20 g/L can be taken, and each value between the minimum value and the maximum value can be taken. Specific examples include, but are not limited to, the following dot values: 10 g/L, 10.5 g/L, 11 g/L, 11.5 g/L, 12 g/L, 12.5 g/L, 13 g/L, 13.5 g/L, 14 g/L, 14.5 g/L, 15 g/L, 15.5 g/L, 16 g/L, 16.5 g/L, 17 g/L, 17.5 g/L, 18 g/L, 18.5 g/L, 19 g/L, 19.5 g/L, or 20 g/L; or a range of any two of these values, such as 10.8 g/L to 18.2 g/L.

Optionally, the concentration of the solute in the base buffer is 15 g/L-20 g/L; further alternatively, the concentration of solute in the base buffer is 18 g/L.

In some embodiments, the buffer composition further comprises 0.05 v to 5 v/v% blocker, 0.01 v to 5 v/v% surfactant, 0.01 v to 1 v/v% bacteriostat, and 0.001 mg to 0.5 mg/mL anticoagulant.

In some embodiments, the blocking agent comprises one or more of casein, bovine serum albumin, rabbit serum, goat serum, horse serum, mark33 blocking agent, HIER-R-014 blocking agent, HIER-C-003 blocking agent, murine IgG, HIER-E-010-D blocking agent, and HBR-PLUS blocking agent.

Specifically, the HIER-R-014 blocker, HIER-C-003 blocker and HIER-E-010-D blocker are all commercially available from the Phpeng BioCo., ltd.

In the application, the concentration of the blocking agent is 0.05 v/v% -5 v/v%, i.e. the minimum value and the maximum value of the range of 0.05 v/v% -5 v/v% can be taken, and each value between the minimum value and the maximum value. Specific examples include, but are not limited to, the following dot values: 0.05 v/v%, 0.06 v/v%, 0.07 v/v%, 0.08 v/v%, 0.09 v/v%, 0.1 v/v%, 0.2 v/v%, 0.3 v/v%, 0.4 v/v%, 0.5 v/v%, 0.6 v/v%, 0.7 v/v%, 0.8 v/v%, 0.9 v/v%, 1.0 v/v%, 1.2 v/v%, 1.4 v/v%, 1.6 v/v%, 1.8 v/v%, 2.0 v/v%, 2.5 v/v%, 3.0 v/v%, 3.5 v/v%, 4.0 v/v%, 4.5 v/v%, or 5.0 v/v%; or any two of these values, such as 1.1 v/v% to 3.8 v/v%.

In some alternative embodiments, the concentration of blocking agent is 1 v/v% -5 v/v%; further alternatively, the concentration of blocking agent is 1.05 v/v%.

It is understood that casein, BSA, rabbit serum, goat serum, horse serum, mark33 blocker, HIER-R-014 blocker, HIER-C-003 blocker, mouse IgG, HIER-E-010-D blocker and HBR-PLUS blocker all contain a large amount of protein, and can be combined with non-specifically combined protein in serum, so that the combination of non-specific protein and antigen is reduced, and the specificity of sIgE detection is improved.

In some of these embodiments, the surfactant includes one or more of triton X-100, tween-20, ethylphenyl polyethylene glycol (NP-40), surfactant S9, and octylphenyl-polyethylene glycol (CA-630).

In the present application, the concentration of the surfactant is "0.01 v/v% -5 v/v%", i.e., the minimum value and the maximum value in the range of 0.01 v/v% -5 v/v%, and each value between the minimum value and the maximum value can be taken. Specific examples include, but are not limited to, the following dot values: 0.01 v/v%, 0.02 v/v%, 0.03 v/v%, 0.04 v/v%, 0.05 v/v%, 0.06 v/v%, 0.07 v/v%, 0.08 v/v%, 0.09 v/v%, 0.1 v/v%, 0.2 v/v%, 0.3 v/v%, 0.4 v/v%, 0.5 v/v%, 0.6 v/v%, 0.7 v/v%, 0.8 v/v%, 0.9 v/v%, 1.0 v/v%, 1.2 v/v%, 1.4 v/v%, 1.6 v/v%, 1.8 v/v%, 2.0 v/v%, 2.5 v/v%, 3.5 v/v%, 4.0 v/v%, 4.5 v/v%, or 5.0.37/v%; or any two of these values, such as 1.3 v/v% to 4.2 v/v%.

In some alternative embodiments, the concentration of surfactant is 0.01 v/v% to 1 v/v%; further alternatively, the concentration of surfactant is 0.1 v/v%.

It is understood that surfactants can provide a compound having significantly reduced surface tension or interfacial tension between two liquids, liquid-gas, liquid-solid. The molecular structure of the modified polyurethane has amphiprotic, one end is a hydrophobic group, and the other end is a hydrophilic group.

In some embodiments, the bacteriostatic agent comprises one or more of PC-150 bacteriostatic agent, PC-300 bacteriostatic agent, PC-950 bacteriostatic agent, BND-10 bacteriostatic agent, BND-99 bacteriostatic agent, PC-8020 bacteriostatic agent, BIT-10 bacteriostatic agent, and GML-2 bacteriostatic agent.

Specifically, PC-150 bacteriostatic agent, PC-300 bacteriostatic agent, PC-950 bacteriostatic agent, BND-10 bacteriostatic agent, BND-99 bacteriostatic agent, PC-8020 bacteriostatic agent, BIT-10 bacteriostatic agent and GML-2 bacteriostatic agent are Niu Sha bacteriostatic agent.

In the application, the concentration of the bacteriostatic agent is 0.01 v/v% -1 v/v%, namely, the minimum value and the maximum value in the range of 0.01 v/v% -15 v/v% can be taken, and each value between the minimum value and the maximum value. Specific examples include, but are not limited to, the following dot values: 0.01 v/v%, 0.02 v/v%, 0.03 v/v%, 0.04 v/v%, 0.05 v/v%, 0.06 v/v%, 0.07 v/v%, 0.08 v/v%, 0.09 v/v%, 0.1 v/v%, 0.2 v/v%, 0.3 v/v%, 0.4 v/v%, 0.5 v/v%, 0.6 v/v%, 0.7 v/v%, 0.8 v/v%, 0.9 v/v% or 1 v/v; or any two of these values, such as 0.2 v/v% to 0.8 v/v%.

In some alternative embodiments, the concentration of the bacteriostatic agent is 0.5 v/v% -1 v/v%; further alternatively, the concentration of the bacteriostatic agent is 1 v/v%.

Understandably, the bacteriostatic agent is a substance capable of inhibiting bacterial growth, and the bacteriostatic agent added into the in-vitro diagnostic reagent needs to meet the characteristics of broad-spectrum bacteriostasis, no influence on detection sensitivity, wide application range, economy, environmental protection, low cost and the like.

In some embodiments, the anticoagulant includes one or more of heparin salts, ethylenediamine tetraacetic acid (EDTA) salts, citrate salts, and oxalate salts.

In the application, the concentration of the anticoagulant is 0.001 mg/mL-0.5 mg/mL, namely, the minimum value and the maximum value of the range of 0.001 mg/mL-0.5 mg/mL can be taken, and each value between the minimum value and the maximum value. Specific examples include, but are not limited to, the following dot values: 0.001 mg/mL, 0.002 mg/mL, 0.003 mg/mL, 0.004 mg/mL, 0.005 mg/mL, 0.006 mg/mL, 0.007 mg/mL, 0.008 mg/mL, 0.009 mg/mL, 0.01 mg/mL, 0.02 mg/mL, 0.03 mg/mL, 0.04 mg/mL, 0.05 mg/mL, 0.06 mg/mL, 0.07 mg/mL, 0.08 mg/mL, 0.09 mg/mL, 0.1 mg/mL, 0.2 mg/mL, 0.3 mg/mL, 0.4 mg/mL, 0.5 mg/mL; or a range of any two of these values, such as 0.05 mg/mL to 0.3 mg/mL.

In some alternative embodiments, the anticoagulant concentration is 0.01 mg/mL-0.1 mg/mL; further alternatively, the anticoagulant is at a concentration of 0.03 mg/mL.

It is understood that anticoagulants are capable of removing or inhibiting certain coagulation factors from the blood.

The concentration of each of the above substances is a working concentration, and the above buffer composition may be a pre-formed mixture or an on-the-fly mixture.

By using the buffer composition containing the specific concentration and the specific component as a sample diluent, the buffer composition can be combined with nonspecific proteins in a sample to be detected instead of sIgE, so that false positive and false negative of allergen detection caused by CCD can be reduced, and the true positive results of other allergens can not be interfered, so that the detection accuracy can be improved.

Further embodiments of the present application provide for the use of the above-described buffer compositions in allergen detection.

Some embodiments of the present application also provide a method of allergen detection comprising steps S10 to S20.

Step S10: the buffer composition of any of the above embodiments is used to dilute the sample to be tested.

Step S20: and detecting the diluted sample to be detected.

In some embodiments, in step S10, the sample to be tested comprises a serum sample.

In some embodiments, in step S20, the platform for detection comprises a microarray chemiluminescent protein chip platform manufactured by Jiangsu trigeminy bioengineering, inc.; the platform adopts a luminol-horseradish hydrogen peroxidase enzymatic luminescent system, and the traditional sample diluent contains horseradish hydrogen peroxidase, so that the platform is not suitable for the platform.

The present application will be further described with reference to specific examples and comparative examples, which should not be construed as limiting the scope of the present application. The materials used in the following examples were all commercially available, unless otherwise specified, the equipment used, and the processes involved, unless otherwise specified, were all routinely selected by those skilled in the art.

Example 1:

(1) Sample collection: 4 serum samples were collected clinically for allergen screening. In clinical detection, there is non-specific binding interference between sample one and sample two, and there is no non-specific binding interference between sample three and sample four.

(2) Buffer compositions were formulated as sample dilutions: based on the final concentration of the buffer composition, the buffer composition comprises 0.8 mg/mL bromelain, 0.6 mg/mL MUXF, 0.6 mg/mL ascorbate oxidase, 7 g/L Mops, 2 g/L Mops sodium salt, 9 g/L sodium chloride, 1 v/v% goat serum, 0.05 v/v% MARK33, 0.1 v/v% Tween-20, 1 v/v% Niu Sha PC-300 bacteriostatic agent and 0.03 mg/mL heparin sodium.

(3) And (3) detecting: and (3) adding the buffer composition prepared in the step (2) into a detection instrument, and diluting the serum sample to be detected by 3 times through automatic control of the instrument. The diluted sample solution to be tested is spotted on the surface of an allergen chip (33 allergen component solutions are spotted on the surface of the chip in advance) through an instrument, so that the chip reacts with serum and then reacts with a secondary antibody of a target enzyme to form a sandwich product, a luminescent substrate is added to catalyze and emit light, and a camera is used for collecting optical signals to calculate the signal intensity. The detection results of the first sample are shown in table 2, the detection results of the second sample are shown in table 3, the detection results of the third sample are shown in table 4, and the detection results of the fourth sample are shown in table 5.

FIG. 1 is a dot matrix optical signal of the allergen detection chip surface after diluting the second sample with the buffer composition of the present embodiment.

Example 2 to example 9:

substantially the same as in example 1, except that the buffer composition was formulated differently, the buffer composition formulations of examples 2 to 9 are shown in Table 1. The test results of samples one to four after diluting the test samples with the buffer compositions prepared in examples 2 to 9 are shown in tables 2, 3, 4 and 5, respectively.

Table 1 buffer composition formulations of examples 1-9

TABLE 2 allergen constituent detection Signal value of sample one

Tri a 19, tri a 28, tri a 34, tri a 37, tri a 40 are the correct positive signal values of the sample, and after interference is removed, more antibodies are correctly bound to the allergen on the chip surface, and the signal values are higher. The signal values of the other components are greatly reduced to interference signals, if not eliminated, the sample cannot be correctly judged to which component generates a correct positive signal.

TABLE 3 allergen component detection Signal values of sample two

Tri a 25, tri a 33, tri a 41 are positive signal values of the sample, and after interference is removed, antibodies which are correctly bound with the allergen on the chip surface are more, and the signal values are higher. The signal values of the other components are greatly reduced to interference signals, if not eliminated, the sample cannot be correctly judged to which component generates a correct positive signal.

TABLE 4 allergen component detection Signal value of sample III

Tri a 2, tri a 4, tri a 5, tri a 12, tri a 14, tri a 20, tri a 21, tri a 25, tri a 26 are the correct positive signal values of the sample, and after interference is removed, more antibodies are correctly bound to the allergen on the chip surface, and the signal values are higher. The signal values of the other components are greatly reduced to interference signals, if not eliminated, the sample cannot be correctly judged to which component generates a correct positive signal.

TABLE 5 allergen constituent detection Signal value of sample four

Tri a 19, tri a 20, tri a 21, tri a 25, tri a 30 are the correct positive signal values for the samples, and after interference is removed, more antibodies are correctly bound to the allergen on the chip surface, and the signal values are higher. The signal values of the other components are greatly reduced to interference signals, if not eliminated, the sample cannot be correctly judged to which component generates a correct positive signal.

Comparative example 1:

substantially identical to example 1, except that no sample dilution was used to dilute the sample to be tested prior to on-board testing.

FIG. 2 is a graph showing the lattice light signal of the allergen detection chip surface without using the sample diluent to treat sample two. In comparison to fig. 2, the binding between non-specific proteins and allergens is significantly reduced in fig. 1 after treatment of sample two with the sample dilution of example 1.

Comparative example 2:

the procedure is substantially as in example 1 except that the sample diluent used in comparative example 2 is a CCD inhibitor (including bromelain, peroxidase and ascorbate oxidase) sold by Shanghai Collision Tech, inc. and is commercially available from Cornikow (tin-free) medical science, inc., under accession number Su Xixie/20210126.

Comparative examples 3 to 6:

substantially the same as in example 1, except that the buffer composition formulations were different, the buffer composition formulations of comparative examples 3 to 6 are shown in Table 6.

Table 6 buffer composition formulations of comparative examples 3-6

The test results of samples one to four after diluting the test samples with the sample dilutions prepared in comparative examples 1 to 6 are shown in tables 7, 8, 9 and 10, respectively.

TABLE 7 allergen constituent detection Signal value of sample one

TABLE 8 allergen component detection Signal values of sample two

TABLE 9 allergen component detection Signal value of sample III

Table 10 allergen constituent detection Signal value of sample four

As can be seen from tables 2 to 3 and 7 to 8, the sample dilutions prepared in examples 1 to 9 of the present application were used to dilute the serum samples to be tested for allergens, and the detection values of the allergen components were significantly reduced as compared with comparative examples 1 and 2. The sample diluent of the application is shown to reduce the combination of anti-CCD IgE antibodies or non-specific binding antibodies and allergens, and allergen detection results can truly reflect the allergic conditions of patients.

It can be seen from tables 4-5 and 9-10 that, for the sample to be tested without CCD interference and non-specific interference, the accuracy of the detection result of the sample to be tested is not affected after the sample diluent is added.

In comparative examples 3-5, bromelain, MUXF and ascorbate oxidase were absent in the sample diluent formulation, respectively, and the final allergen component detection results were higher than the actual results, and CCD interference or non-specific interference may still exist.

In comparative example 6, the concentrations of bromelain, MUXF and ascorbate oxidase in the sample diluent formulation were all 0.05 mg/mL, and the prepared sample diluent could not completely prevent the anti-CCD IgE antibody or the non-specific binding antibody from binding with the allergen, and the detection result was still higher than the real result.

The technical features of the above-described embodiments may be arbitrarily combined, and all possible combinations of the technical features in the above-described 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 above examples merely represent a few embodiments of the present application, which are described in more detail and are not to be construed as limiting the scope of the invention. It should be noted that it would be apparent to those skilled in the art that various modifications and improvements could be made without departing from the spirit of the present application, which would be within the scope of the present application. Accordingly, the scope of protection of the present application shall be subject to the appended claims.

Claims (11)

1. A buffer composition comprising 0.1 mg/mL to 3.5 mg/mL bromelain, 0.1 mg/mL to 3.5 mg/mL bromelain cross-reactive carbohydrate determinant, 0.1 mg/mL to 3.5 mg/mL ascorbate oxidase, and a base buffer.

2. The buffer composition of claim 1, further comprising 0.1 mg/mL to 3.5 mg/mL papain, based on the total volume of the buffer composition.

3. The buffer composition according to claim 1, wherein the buffer composition satisfies one or more of the following conditions (1) - (3):

(1) The concentration of the bromelain is 0.8 mg/mL-3.5 mg/mL;

(2) The concentration of the bromelain cross-reactive carbohydrate determinant is 0.6 mg/mL-3.5 mg/mL; and

(3) The concentration of the ascorbate oxidase is 0.6 mg/mL-3.5 mg/mL.

4. A buffer composition according to any one of claims 1 to 3, further comprising 0.05 v/v% to 5 v/v% blocker, 0.01 v/v% to 5 v/v% surfactant, 0.01 v/v% to 1 v/v% bacteriostat and 0.001 mg/mL to 0.5 mg/mL anticoagulant, based on the total volume of the buffer composition.

5. The buffer composition of claim 4, wherein the blocking agent comprises one or more of casein, bovine serum albumin, rabbit serum, goat serum, horse serum, mark33 blocking agent, HIER-R-014 blocking agent, HIER-C-003 blocking agent, murine IgG, HIER-E-010-D blocking agent, and HBR-PLUS blocking agent.

6. The buffer composition of claim 4, wherein the surfactant comprises one or more of triton X-100, tween-20, ethylphenyl polyethylene glycol, surfactant S9, and octylphenyl-polyethylene glycol.

7. The buffer composition of claim 4, wherein the bacteriostat comprises one or more of a PC-150 bacteriostat, a PC-300 bacteriostat, a PC-950 bacteriostat, a BND-10 bacteriostat, a BND-99 bacteriostat, a PC-8020 bacteriostat, a BIT-10 bacteriostat, and a GML-2 bacteriostat.

8. The buffer composition of claim 4, wherein the anticoagulant comprises one or more of heparin salt, ethylenediamine tetraacetate, citrate, and oxalate.

9. The buffer composition according to any one of claims 1 to 3 and 5 to 8, wherein the buffer composition satisfies one or more of the following conditions (1) - (2):

(1) The basic buffer solution comprises one or more of 3-morpholinopropanesulfonic acid buffer solution, 3-morpholinopropanesulfonic acid sodium salt, tris buffer solution, carbonate buffer solution, phosphate buffer solution and tris buffer solution;

(2) The concentration of the solute in the basic buffer solution is 10 g/L-20 g/L.

10. The buffer composition according to any one of claims 5 to 8, wherein the buffer composition satisfies one or more of the following conditions (1) to (4):

(1) The concentration of the blocking agent is 0.05 v/v% -1 v/v%;

(2) The concentration of the surfactant is 0.1 v/v% -5 v/v%;

(3) The concentration of the bacteriostat is 0.5 v/v% -1 v/v%;

(4) The concentration of the anticoagulant is 0.01 mg/mL-0.5 mg/mL.

11. Use of a buffer composition according to any one of claims 1 to 10 for allergen detection.