CN116380880A

CN116380880A - Light-activated chemical luminescence detection reagent of PG I and application thereof

Info

Publication number: CN116380880A
Application number: CN202211741973.1A
Authority: CN
Inventors: 巢佳佳; 张黎明; 黄正铭; 李临
Original assignee: Kemei Boyang Diagnostic Technology Shanghai Co ltd
Current assignee: Kemei Boyang Diagnostic Technology Shanghai Co ltd
Priority date: 2021-12-31
Filing date: 2022-12-30
Publication date: 2023-07-04

Abstract

The invention relates to the field of photo-excitation chemiluminescence detection, in particular to a photo-excitation chemiluminescence detection reagent of PG I and application thereof. The invention provides a detection reagent for detecting PG I by photo-excitation chemiluminescence, which comprises the following components: r1 and R2 agents; wherein the R1 reagent comprises a receptor capable of reacting with singlet oxygen to generate a detectable signal and a counterpart combined with the receptor, and the counterpart can specifically react with an object to be detected; the R2 reagent comprises another corresponding object which specifically reacts with the object to be detected; the R1 reagent also includes free antibodies. Experimental results show that the addition of free antibodies to the detection reagents can improve the immunoassay linearity.

Description

Light-activated chemical luminescence detection reagent of PG I and application thereof

The present application claims priority from the chinese patent office, application number 202111680567.4, light activated chemiluminescent detection reagent of the invention "PG i and its use" filed on month 12 and 31 of 2021, the entire contents of which are incorporated herein by reference.

Technical Field

The invention relates to the field of photo-excitation chemiluminescence detection, in particular to a photo-excitation chemiluminescence detection reagent of PG I and application thereof.

Background

Pepsinogen (PG) is a precursor of pepsin, and includes two types, pepsinogen I (PGI) and Pepsinogen ii (PG ii). PGI is secreted mainly by the main cells and mucus cells of the stomach, mostly into the stomach cavity, and in small amounts into the blood by some means. PGI is produced mainly by the main cells and neck cells of the mucosa of the pyloric glands of the antrum and Bruner glands of the proximal segment of the duodenum, and small amounts can enter the blood by some means. When the stomach environment changes, such as the gastric mucosa changes pathologically, the secretion amount of PGI can be influenced, so that the content of PGI in blood is influenced, and conversely, the content of PGI in blood can reflect the secretion function of the gastric mucosa. Numerous studies have shown that during the development of gastric lesions: the infection of helicobacter pylori (Hp) is developed into chronic gastritis, then atrophic gastritis and finally gastric cancer, and the PGI and PG II are accompanied with the change, and the content of the PGI in serum is increased and then decreased along with the development of gastric diseases; the PGI content is maintained at a higher level after the PGI content is increased, so that the PGI content can indicate stomach diseases of different degrees. In short, the index of PGI is related to gastritis, hp infection, gastric ulcer, gastrorrhagia, gastric cancer, etc., and they have been attracting attention as an in vitro detection of gastric diseases. The large-scale screening is carried out on the crowd, and the establishment of the sensitive and convenient PGI and PG II content detection method can be beneficial to the auxiliary diagnosis of stomach diseases and the early auxiliary diagnosis of stomach cancer.

Currently, kits for quantitative detection of PGI include Radioimmunoassay (RIA), enzyme-linked immunoassay (EIA), time-resolved fluorescence assay (TRFIA), and the like. The release-free method has potential radioactive pollution, needs special instruments, is operated by professional staff, and is not beneficial to popularization and use; although the ELISA and the time-resolved fluorescence methods are relatively high in use proportion, the operation is long in time consumption and high in price, and the reagent is a liquid-phase homogeneous system and is limited in storage condition and shelf life, so that the reagent is not beneficial to project development of primary hospitals.

The PGI existing reagent for photo-excitation chemiluminescence detection has the problems of poor high-end linearity and low linear R value, and is easy to cause inaccurate high-end measurement value. Therefore, the light-activated chemiluminescent detection reagent for PG I and the application thereof are of great practical significance.

Disclosure of Invention

In view of this, the invention provides a photo-activated chemiluminescent detection reagent of PG I and a preparation method thereof. Experimental results show that the addition of free antibodies to the detection reagents can improve the immunoassay linearity.

In order to achieve the above object, the present invention provides the following technical solutions:

in a first aspect, the invention provides the use of free antibodies in a detection reagent to improve the linearity of an immunoassay.

In a second aspect, the present invention provides a detection reagent for photoexcitation chemiluminescent detection, comprising: r1 and R2 agents;

the R1 reagent comprises a receptor capable of reacting with singlet oxygen to generate a detectable signal and a counterpart combined with the receptor, wherein the counterpart can specifically react with an object to be detected;

the R2 reagent comprises another corresponding object which specifically reacts with the object to be detected;

the R1 reagent also includes free antibodies.

In some embodiments of the invention, the R1 reagent comprises a luminescent microsphere coated antibody that specifically reacts with the test agent; and/or

The R2 reagent comprises a biotin-labeled another antibody which specifically reacts with the to-be-detected object;

the antibody is identical or different from the epitope recognized by the other antibody.

In some embodiments of the invention, the free antibody comprises:

(I) The affinity of the free antibody is lower than the affinity of the other antibody; and/or

(II) the specificity of the free antibody is lower than the specificity of the other antibody; and/or

(III) the free antibody is present in the reagent in an amount greater than the amount of the other antibody.

In some embodiments of the invention, the free antibodies include, but are not limited to, one or more of coated antibodies, labeled antibodies, or other antibodies associated with the test item.

In some embodiments of the invention, the free antibodies comprise one or more of polyclonal antibodies, monoclonal antibodies, synthetic antibodies, or synthetic antibody fragments.

In some embodiments of the invention, the free antibody is added in an amount of 37.5% of the concentration of the other antibody in the R2 reagent.

In a third aspect, the invention provides the use of the detection reagent in the preparation of a photo-activated chemiluminescent detection kit, detection system or detection device.

In a fourth aspect, the invention provides a photo-activated chemiluminescent detection kit comprising the detection reagent and acceptable adjuvants, adjuvants or carriers.

In a fifth aspect, the present invention provides a method for improving the linearity of light-activated chemiluminescence detection, wherein an analyte is mixed with the detection reagent and detected.

In a sixth aspect, the invention also provides a photoexcited chemiluminescent detection system comprising said detection reagent or said detection kit, and an acceptable module.

In a seventh aspect, the invention also provides a photoexcited chemiluminescent detection device comprising the detection reagent, the detection kit or the detection system, and acceptable components.

In some embodiments of the invention, the test substance includes, but is not limited to, a body fluid.

In some embodiments of the invention, the test substance includes, but is not limited to, one or more of whole blood, serum, plasma, urine.

In some embodiments of the invention, the test substance includes, but is not limited to pepsinogen, preferably, the test substance includes, but is not limited to PGI.

In some embodiments of the invention, the high-end linearity involved in the application, the method, has an R value of not less than 0.998.

The invention provides a detection reagent for detecting PG I by photo-excitation chemical luminescence, which comprises the following components: r1 and R2 agents; wherein the R1 reagent comprises a receptor capable of reacting with singlet oxygen to generate a detectable signal and a counterpart combined with the receptor, and the counterpart can specifically react with an object to be detected; the R2 reagent includes another counterpart that specifically reacts with the receptor; the R1 reagent also includes free antibodies. Experimental results show that the addition of free antibodies to the detection reagents can improve the immunoassay linearity.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below.

FIG. 1 shows a scatter plot with a smooth line corresponding to the test results of example 2, indicating that the addition of free antibody improves high-end linearity.

Detailed Description

The invention discloses a photo-excitation chemiluminescence detection reagent of PG I and a preparation method thereof, and a person skilled in the art can properly improve the process parameters by referring to the content of the photo-excitation chemiluminescence detection reagent. It is expressly noted that all such similar substitutions and modifications will be apparent to those skilled in the art, and are deemed to be included in the present invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those skilled in the relevant art that variations and modifications can be made in the methods and applications described herein, and in the practice and application of the techniques of this invention, without departing from the spirit or scope of the invention.

Where a range of values is provided, it is understood that each intervening value, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.

Unless defined otherwise, all terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, the preferred methods and materials are now described.

Terminology

The term "analyte" as used herein refers to a mixture that may contain an analyte, including but not limited to a protein, hormone, antibody or antigen. Typical analytes that may be used in the methods disclosed herein include body fluids such as blood, blood derivatives, serum, plasma, urine, cerebrospinal fluid, saliva, synovial fluid, and emphysema effusion, among others. The analyte may be a solution obtained by diluting a sample possibly containing the analyte with a diluent or a buffer solution as needed before use. For example, in order to avoid the HOOK effect, the analyte may be diluted with a sample diluent before the on-machine detection and then detected on the detecting instrument, and in this case, the diluted solution possibly containing the analyte is collectively referred to as the analyte.

The term "free antibody" as used herein includes, but is not limited to, one or more of coated antibodies, labeled antibodies, or other antibodies associated with the test item; preferably, the free antibodies include, but are not limited to, one or more of polyclonal antibodies, monoclonal antibodies, synthetic antibody fragments.

The term "monoclonal antibody" as used herein refers to an immunoglobulin secreted by monoclonal B lymphocytes, which can be prepared by methods well known to those skilled in the art.

The term "polyclonal antibody" as used herein refers to a collection of immunoglobulins derived from more than one B lymphocyte clone, which may be prepared by methods well known to those skilled in the art.

The term "synthetic antibody" as used herein refers to an affinity reagent that is produced entirely in vitro, thus eliminating animals entirely during production. Synthetic antibodies include recombinant antibodies, nucleic acid aptamers, and non-immunoglobulin scaffolds. Because of its in vitro method of manufacture, the antigen recognition site of a synthetic antibody can be designed to any desired target and may be beyond the typical immune repertoire provided by natural antibodies. Synthetic antibodies are being developed for research, diagnostic and therapeutic applications. Synthetic antibodies can be used with traditional monoclonal antibodies or with polyclonal antibodies and offer many inherent advantages over animal-derived antibodies, including relatively low production costs, reagent reproducibility, and increased affinity, specificity, and stability under a range of experimental conditions. Synthetic antibodies have shown utility in a number of applications. Their use in research fields is mainly in life sciences as protein capture reagents and protein inhibitors. In diagnostics, they have been used for applications ranging from infection and cancer screening to mycotoxin detection in cereal samples. Synthetic antibodies are the fastest growing class of therapies.

The term "antigen" as used herein refers to a substance that stimulates the body to produce an immune response and binds to antibodies and sensitized lymphocytes, which are the products of the immune response, in vivo and in vitro, resulting in an immune effect.

The term "epitope" as used herein refers to any protein determinant capable of specific binding to an immunoglobulin or T cell receptor. In some embodiments of the invention, an epitope is a region of an antigen surface that can be assembled specifically by an antibody. Epitope determinants may generally include chemically active surface groupings of molecules such as, but not limited to: amino acids, sugar side chains, phosphoryl and/or sulfonyl groups. In other embodiments of the invention, epitopes may be specifically specific for three-bit structural features as well as specific charge features.

The term "binding" as used herein refers to the direct association between two molecules due to interactions such as covalent, electrostatic, hydrophobic, ionic and/or hydrogen bonding, including but not limited to interactions such as salt and water bridges.

The term "specific binding" as used herein refers to the mutual recognition and selective binding reaction between two substances, and from a steric perspective, corresponds to the conformational correspondence between the corresponding reactants.

The term "biotin" is widely used in animal and plant tissues, and has two cyclic structures, namely an imidazolone ring and a thiophene ring, on the molecule, wherein the imidazolone ring is the main part combined with streptavidin. Activated biotin can be coupled to almost all known biomacromolecules, including proteins, nucleic acids, polysaccharides, lipids, etc., mediated by protein cross-linking agents; and "streptavidin" is a protein secreted by Streptomyces and has a molecular weight of 65kD. The "streptavidin" molecule consists of 4 identical peptide chains, each of which is capable of binding to a biotin. Thus, each antigen or antibody can be conjugated to multiple biotin molecules simultaneously, thereby producing a "tentacle effect" that enhances assay sensitivity. In any case where desired, any agent used in the present invention, including antigen, antibody, receptor or donor, may be conjugated to any member of the biotin-streptavidin specific binding pair member as desired.

Any antibody species may be used as the free antibody in the present invention, and the present invention is not limited thereto. The like numbers of antibody 1, antibody 2, antibody 3, etc. are merely for distinguishing between different groups of antibodies, and antibody 1, antibody 2, antibody 3 may be the same or different. Any antibody known in the art may be used in the present invention.

Basic principle of photoexcitation chemiluminescence:

the basic principles of photo-activated chemiluminescence are well known to those skilled in the art. Conventionally, the photosensitive particles and the luminescent particles are combined in a certain range to generate the transfer of ionic oxygen energy and emit optical signals so as to detect the object to be detected. Wherein the photosensitive particles are internally filled with a photosensitive compound, and the luminescent particles are internally filled with a luminescent compound and a lanthanoid. Under the excitation of red laser (600-700 nm), the photosensitive particles release singlet oxygen ions (4 mu S) with high energy states, and the propagation distance is about 200nm. When the distance between the photosensitive particles and the luminescent particles is close enough, the singlet oxygen ions released by the photosensitive particles can reach the luminescent particles and emit light with high energy level of 520-620 nm through a series of chemical reactions, so that the light is detected by an instrument.

The term "acceptor" as used herein refers to a substance capable of reacting with singlet oxygen to produce a detectable signal. The donor is induced to activate by energy or an active compound and releases singlet oxygen in a high energy state which is captured by the acceptor in close proximity, thereby transferring energy to activate the acceptor. In some embodiments of the invention, the acceptor is a substance that undergoes a chemical reaction with singlet oxygen to form an unstable metastable intermediate that may decompose while or subsequently emit light. Typical examples of such substances include, but are not limited to: enol ethers, enamines, 9-alkylidene xanthan, 9-alkylidene-N-alkyl acridines, arylvinyl ethers, bisoxyethylene, dimethylthiophene, aromatic imidazoles or gloss concentrates. In other embodiments of the invention, the acceptor is an olefin capable of reacting with singlet oxygen to form a hydroperoxide or dioxetane that can decompose to a ketone or carboxylic acid derivative; stable dioxetanes that can be decomposed by the action of light; acetylenes that can react with singlet oxygen to form diketones; hydrazones or hydrazides of azo compounds or azocarbonyl compounds, such as luminol, may be formed; and aromatic compounds which can form endoperoxides. A specific, non-limiting example of a receptor that can be utilized in accordance with the present disclosure and claimed invention is described in U.S. patent No. US5340716 (which is incorporated herein by reference in its entirety). In other embodiments of the present invention, the acceptor comprises an olefinic compound and a metal chelate that is non-particulated and soluble in an aqueous medium, as described in PCT/US2010/025433 (which is incorporated herein by reference in its entirety).

In the photoexcitation chemiluminescent system, in addition to the donor reagent, other reagents are included according to the requirement of the detection object or detection method, such as: receptor reagents, biotin-coated secondary antibodies, dilutions, and the like. In the field of in vitro diagnosis, in particular in the field of immunoassay, in order to simplify the naming of the different components in the commercial kit, each manufacturer usually marks or simply refers to the components of the different bottles in the kit as reagent 1, reagent R1 or R1, reagent 2, reagent R2 or R2, reagent 3, reagent R3 or R3, … …, and so on, so that the identification, the assembly and the use of customers are facilitated, and the aim of technical confidentiality is also fulfilled. Therefore, the kit products of different in vitro diagnostic manufacturers may contain the reagent 1, the reagent 2, the reagent 3 and the reagent … …, but the corresponding reagent components of different manufacturers are different.

In the invention, the R1 reagent comprises an antibody which is coated by a luminous microsphere and specifically reacts with an object to be detected; the R2 reagent comprises a biotin-labeled other antibody which specifically reacts with the to-be-detected object; the antibody is identical or different from the epitope recognized by another antibody.

In some embodiments of the invention, "FG-PGI" refers to PGI bound to luminescent microspheres; "BIO-PGI" refers to a PGI that binds to biotin.

Experimental raw material and equipment

TABLE 1

Sequence number	Name of the name
		1	Pepsinogen I detection kit (light excitation chemiluminescence method)
2	PGI antibody 1
		3	PGI antibody 2
4	PGI antibody 3

The antibody 1 and the biotin-labeled antibody in the reagent 2 have the same recognition epitope 1, the antibody 2 recognizes epitope 2, the antibody 3 recognizes epitope 3, and the epitopes 1, 2 and 3 are different.

TABLE 2

Name of the name	Device model/number	Manufacturer' s
			LICA 500	BYG-151/A2104	Kemebo positive diagnostic technology (Shanghai) Co., ltd

In the light-activated chemiluminescent detection reagent for PG I and the preparation method thereof, raw materials and reagents used in the detection reagent can be purchased from the market.

The invention is further illustrated by the following examples:

EXAMPLE 1 ELISA method for determination of binding constant of each free antibody

After the pure PGI product is subjected to gradient dilution, ELISA reaction is carried out on the pure PGI product and the antibodies 1, 2 and 3 are tested on an enzyme-labeled instrument, the measured OD450 value and the original concentration value of the antigen and the antibody are brought into a formula to calculate a Kd value, and the result is as follows:

TABLE 3 Table 3

Antibodies to	Antibody 1	Antibody 2	Antibody 3
				Dissociation constant (Kd)	1.0×10 ^-11	5.0×10 ^-10	3.0×10 ^-9

Affinity: antibody 1 > antibody 2 > antibody 3

Example 2

Preparation of reagent 1: FG-PGI of 100 ug/mL;

reagent 2:2ug/mL BIO-PGI;

antibody 1 was added to each of the reagents 1 and 2 at a concentration of 50% of the antibody concentration of the reagent 2, and a group of reagents without free antibody was used as a control group to measure a mixed sample of gradient dilution, and a linear R value was calculated.

Sample adding mode: 20uL sample+25 uL reagent 1+25uL reagent 2;

the detection was performed on a LICA 500 instrument.

The experimental results are shown in the following table:

TABLE 4 Table 4

The results showed that the addition of free antibody improved the linear R value, and the addition effect was better in reagent 1 than in reagent 2 or in both reagent 1 and reagent 2.

As a result of the test, a scatter diagram with a smooth line was made, and it was found that the addition of the free antibody improved the high-end linearity.

Example 3

Preparation of reagent 1: FG-PGI of 100 ug/mL;

reagent 2:2ug/mL BIO-PGI;

antibody 1, antibody 2 and antibody 3 were added to reagent 1 at a concentration of 50% of the antibody concentration of reagent 2, and a group of reagents without free antibody was used as a control group to measure a mixed sample of gradient dilution and calculate a linear R value.

Sample adding mode: 20uL sample+25 uL reagent 1+25uL reagent 2;

preparing a reaction solution according to a sample adding mode, and incubating for 15min at 37 ℃; 175ul of a universal solution for a light-activated chemiluminescent assay system was added to the reaction wells and incubated at 37℃for 10min using

The analyzer takes readings.

The experimental results are shown in the following table:

TABLE 5

The results showed that the addition of antibody 1, antibody 2, and antibody 3 to reagent 1 all improved the linear R values, with the addition of antibody 3 having the highest R value, the best linearity, the least decrease in signal-to-noise ratio, and the least loss of low-end sensitivity.

Example 4

Preparation of reagent 1: FG-PGI of 100 ug/mL;

reagent 2:2ug/mL BIO-PGI;

the concentration of antibody 3 added to reagent 1 was set to 0%, 25%, 37.5%, 50%, 62.5%, 75% of the concentration of antibody 2 in reagent 1, and the gradient diluted mixed samples were measured.

Sample adding mode: 20uL sample+25 uL reagent 1+25uL reagent 2;

The analyzer takes readings. The experimental results are shown in the following table:

TABLE 6

The results showed that the free antibodies at 37.5%, 50%, 62.5%, 75% reagent 2 concentration had similar linear enhancement, but the latter three lost more signal and decreased signal-to-noise ratio, i.e., lower end sensitivity.

Example 5

Preparation of reagent 1: FG-PGI of 100 ug/mL;

reagent 2:2ug/mL BIO-PGI;

experimental group antibody 3 was added to reagent 1 at a concentration of 37.5% reagent 2;

sample adding mode: 20uL sample+25 uL reagent 1+25uL reagent 2;

The analyzer takes readings.

3 high value samples were selected, 10 wells of the samples were repeatedly assayed using reagents with and without free antibody added as described above, and the accuracy of the sample assay was verified.

TABLE 7

The test result shows that the CV of the high-value sample tested by the kit is lower, the consistency of repeated test is better, and the precision is high.

The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.

Claims

1. The application of adding free antibody in the detection reagent for detecting PG I by light excitation chemiluminescence to improve the immunity detection linearity.

2. A detection reagent for detecting pgi by photoexcitation chemiluminescence, comprising: r1 and R2 agents;

the R1 reagent also includes free antibodies.

3. The test reagent of claim 2, wherein the R1 reagent comprises a luminescent microsphere coated antibody that specifically reacts with the analyte; and/or

4. The detection reagent of claim 3, wherein the free antibody comprises:

5. The test agent of claim 2, wherein the free antibody comprises one or more of a coated antibody, a labeled antibody, or other antibodies associated with the test item.

6. The detection reagent of claim 2, wherein the free antibody comprises one or more of a polyclonal antibody, a monoclonal antibody, a synthetic antibody, or a synthetic antibody fragment.

7. The detection reagent according to claim 3, wherein the free antibody is added in an amount of 37.5% of the concentration of the other antibody in the R2 reagent.

8. Use of the detection reagent according to any one of claims 2 to 7 for preparing a kit, a detection system or a detection device for detecting pgi by photo-excitation chemiluminescence.

9. A kit for the detection of pgi by photo-excitation chemiluminescence, comprising a detection reagent according to any of claims 2 to 7, together with acceptable adjuvants, adjuvants or carriers.

10. A method for improving the linearity of a light-activated chemiluminescent assay for detecting PG i, wherein an analyte is mixed with a detection reagent according to any one of claims 2 to 7 for detection.