CN116381245A - Reagent combination, kit and application of reagent combination for detecting CEA by photo-excitation chemiluminescence - Google Patents

Reagent combination, kit and application of reagent combination for detecting CEA by photo-excitation chemiluminescence Download PDF

Info

Publication number
CN116381245A
CN116381245A CN202211742330.9A CN202211742330A CN116381245A CN 116381245 A CN116381245 A CN 116381245A CN 202211742330 A CN202211742330 A CN 202211742330A CN 116381245 A CN116381245 A CN 116381245A
Authority
CN
China
Prior art keywords
antibody
reagent
sample
cea
antibodies
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
CN202211742330.9A
Other languages
Chinese (zh)
Inventor
刘庆亚
张黎明
黄正铭
李临
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Kemei Boyang Diagnostic Technology Shanghai Co ltd
Original Assignee
Kemei Boyang Diagnostic Technology Shanghai Co ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Kemei Boyang Diagnostic Technology Shanghai Co ltd filed Critical Kemei Boyang Diagnostic Technology Shanghai Co ltd
Publication of CN116381245A publication Critical patent/CN116381245A/en
Pending legal-status Critical Current

Links

Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
    • G01N21/76—Chemiluminescence; Bioluminescence
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/531—Production of immunochemical test materials
    • G01N33/532—Production of labelled immunochemicals
    • G01N33/533—Production of labelled immunochemicals with fluorescent label
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Landscapes

  • Health & Medical Sciences (AREA)
  • Immunology (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Chemical & Material Sciences (AREA)
  • Hematology (AREA)
  • Biomedical Technology (AREA)
  • Urology & Nephrology (AREA)
  • Molecular Biology (AREA)
  • Physics & Mathematics (AREA)
  • General Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Pathology (AREA)
  • Cell Biology (AREA)
  • Biotechnology (AREA)
  • Food Science & Technology (AREA)
  • Medicinal Chemistry (AREA)
  • Microbiology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Plasma & Fusion (AREA)
  • Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)

Abstract

The invention relates to the field of immunodetection, in particular to a photo-excitation chemiluminescence detection reagent and a preparation method thereof. The invention screens the antibody combination with strong affinity to CEA, prepares the reagent by coating luminous particles and biotin marks, is assisted with universal liquid of a photo-activated chemiluminescence analysis system, quantitatively detects CEA in human serum by adopting a double-antibody sandwich immunofluorescence chemiluminescence method under homogeneous phase condition, and can effectively detect CEA components in samples with low content. In order to solve the HOOK problem, free CEA antibody is added into the reagent, and the use concentration of the CEA antibody after coating and labeling is adjusted, so that the linear range of the reagent is widened under the condition of ensuring the sensitivity.

Description

Reagent combination, kit and application of reagent combination for detecting CEA by photo-excitation chemiluminescence
The present application claims priority from the chinese patent office, application number 202111680240.7, entitled "photo-activated chemiluminescent detection reagent and method of making same," filed on day 31, 12, 2021, the entire contents of which are incorporated herein by reference.
Technical Field
The invention relates to the field of immunodetection, in particular to a reagent combination, a kit and application of the reagent combination for detecting CEA by light excitation chemiluminescence.
Background
Carcinoembryonic antigen (carcinoembryonic antigen, CEA) is a tumor-associated antigen extracted from colon cancer and embryonic tissue, is an acidic glycoprotein with human embryonic antigen properties, exists on the surface of cancer cells differentiated from endodermal cells, and is a structural protein of cell membranes. Formed in the cytoplasm, secreted out of the cell through the cell membrane and then into the surrounding body fluids. Therefore, the sample can be detected from various body fluids and excretions such as serum, cerebrospinal fluid, milk, gastric juice, hydrothorax and ascites, urine, feces, and the like. Serum CEA is a relatively broad-spectrum tumor marker. Clinically, the method can be used for auxiliary diagnosis of common tumors such as colon cancer, rectal cancer, lung cancer, breast cancer, esophagus cancer, pancreatic cancer, gastric cancer, metastatic liver cancer and the like. Other malignant tumors such as medullary thyroid carcinoma, cholangiocarcinoma, urinary malignant tumors and the like have positive rates with different degrees.
According to physical examination data of healthy people, the CEA content in serum is generally below 5ng/mL, so that on the basis of guaranteeing the linear range of the reagent, the sensitivity of the reagent is very necessary to be increased, otherwise, the problem of more false positive samples can occur in clinical examination, and clinical diagnosis is interfered. Therefore, there is a need to develop a carcinoembryonic antigen detection kit with high sensitivity, good repeatability, strong anti-HOOK capability, and convenient and quick operation, which can be used on a full-automatic analyzer.
Disclosure of Invention
In view of the above, the invention provides a carcinoembryonic antigen detection kit which has high sensitivity, good repeatability, strong HOOK resistance and convenient and quick operation and can be used on a full-automatic analyzer.
In order to achieve the above object, the present invention provides the following technical solutions:
in a first aspect, the present invention provides a reagent combination for photoexcitation chemiluminescent detection of CEA, comprising:
(1) A first reagent comprising luminescent particles and a first antibody; and
(2) A second reagent comprising a label and a second antibody;
the first antibody or the second antibody specifically reacts with an object to be detected respectively;
the first antibody or the second antibody further comprises a free antibody;
the addition modes of the free antibody comprise:
(I) Adding to the first reagent; and/or
(II) adding to the second reagent; and/or
(III) simultaneously adding to the first reagent and the second reagent.
In some embodiments of the invention, the free antibody is capable of competing with the first antibody and/or the second antibody for binding to the test substance.
In some embodiments of the invention, the concentration of luminescent microspheres and first antibodies in the first reagent, the concentration of labels and second antibodies in the second reagent, and the concentration ratio of free antibodies comprises 50:1:1.
preferably, the concentrations of the first reagent, the second reagent and the free antibody are 100ug/mL, 2ug/mL and 2ug/mL, respectively.
In some embodiments of the invention, the free antibody is added to the R1 reagent, not to the R2 reagent and/or the R3 reagent.
In some embodiments of the invention, the free antibodies comprise one or more of polyclonal antibodies, monoclonal antibodies, synthetic antibody fragments.
In some embodiments of the invention, the test substance comprises a body fluid; the body fluid includes one or more of blood, serum, plasma, urine.
In a second aspect, the invention also provides application of the reagent combination in preparing a detection kit, a detection system or a detection device for detecting CEA by using light-activated chemiluminescence.
In a third aspect, the invention also provides a detection kit comprising said combination of reagents.
In a fourth aspect, the invention also provides a detection system comprising said reagent combination or said detection kit, and an acceptable module.
In a fifth aspect, the invention also provides a test device comprising the reagent combination, the test kit, the test system, and acceptable components.
In a sixth aspect, the present invention also provides a method for improving the anti-HOOK ability of a light-activated chemiluminescent detection CEA antibody, wherein the detection reagent is mixed with an analyte for detection.
In a seventh aspect, the present invention provides a method for determining a free antibody against HOOK ability, comprising the steps of:
step 1, selecting antibodies to be selected, wherein two antibodies to be selected form a group to form an antibody pair;
step 2, detecting samples without a to-be-detected object and samples with different measured values by adopting the antibody, and respectively obtaining signal values of the samples without the to-be-detected object and signal values of the samples with different measured values;
step 3, selecting antibody pairs with high sensitivity and high discrimination of the samples with different measured values;
the free antibody is selected from the antibody pair described in step 3;
the sensitivity is high as follows: the sample without the object to be detected and the low-value sample can be distinguished, namely the detection signal of the low-value sample/the detection signal of the sample without the object to be detected is high;
the different measured value samples comprise a high value sample and a low value sample;
the differential heights of the different measured value samples are as follows: the ratio of the detection signal of the high-value sample to the detection signal of the low-value sample is high;
the ratio of the detection signal of the high value sample to the low value sample is greater than 2.
In some embodiments of the invention, step 3 further comprises, after: and determining the addition amount of the free antibody according to the requirement of sensitivity and/or HOOK resistance by combining the detection signals.
The invention screens the antibody combination with strong affinity to CEA, prepares the reagent by coating luminous particles and biotin marks, is assisted with universal liquid of a photo-activated chemiluminescence analysis system, quantitatively detects CEA in human serum by adopting a double-antibody sandwich immunofluorescence chemiluminescence method under homogeneous phase condition, and can effectively detect CEA components in samples with low content. Where sensitivity refers to the observability of the reaction of an antibody when detecting a low value sample, which is manifested on the LICA platform as a difference from the background and how much of the signal value is when detecting a low value sample.
In order to solve the HOOK problem, free CEA antibody is added into the reagent, and the use concentration of the CEA antibody after coating and labeling is adjusted, so that the linear range of the reagent is widened under the condition of ensuring the sensitivity.
The LICA technology used by the invention has excellent repeatability, and can be matched with a detection platform specially designed for the LICA system to realize automatic detection to a certain extent.
Detailed Description
The invention discloses a reagent combination for detecting CEA by photo-excitation chemiluminescence, a kit and application thereof, and a person skilled in the art can properly improve process parameters by referring to the content of the reagent combination. 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 terms "primary antibody" and "secondary antibody" as used herein refer to antibodies that specifically bind to an antigen (e.g., tumor marker). For the same antigen (e.g., tumor marker), the corresponding first and second antibodies may be different or the same, and may bind to the antigen at the same time.
In the present invention, the "primary antibody" may also be referred to as "coated antibody", i.e., an antibody coated with luminescent microparticles.
In the present invention, the "second antibody" may also be referred to as "labeled antibody", i.e., an antibody labeled with a label (e.g., biotin). 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. Or may be 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, especially in the field of immunoassay, in order to simplify naming of different components in a commercial kit, each manufacturer usually marks or simply refers to a first reagent, a reagent 1, a reagent R1 or a reagent R1, a second reagent, a reagent 2, a reagent R2 or a reagent R2 … …, and so on for different components in the kit, so that the identification, the assembly and the use of customers are facilitated, and the aim of technical confidentiality is also achieved. 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 present invention, the first reagent or reagent 1 comprises an antibody specifically reacting with the analyte, which is coated with a luminescent microsphere; the second reagent or reagent 2 comprises a biotin-labeled further antibody that specifically reacts with the analyte; the antibody is identical or different from the epitope recognized by another antibody. In order to solve the sensitivity problem, we select the antibody combination with strong affinity to CEA, prepare the reagent by coating luminous particles and biotin mark, and assist the general liquid of the photo-activated chemiluminescence analysis system, and quantitatively detect CEA in human serum by adopting a double-antibody sandwich immunofluorescence chemiluminescence method under homogeneous phase condition, so that CEA components in samples with less content can be effectively detected. Where sensitivity refers to the observability of the reaction of an antibody when detecting a low value sample, which is manifested on the LICA platform as a difference from the background and how much of the signal value is when detecting a low value sample.
In order to solve the HOOK problem, free CEA antibody is added into the reagent, and the use concentration of the CEA antibody after coating and labeling is adjusted, so that the linear range of the reagent is widened under the condition of ensuring the sensitivity.
The LICA technology used by the invention has excellent repeatability, and can be matched with a detection platform specially designed for the LICA system to realize automatic detection to a certain extent.
The basic principle of photoexcitation is a homogeneous immune response. It is based on two particles surface coated antigens or antibodies, forming immune complexes in the liquid phase, drawing the two particles closer together. Under the excitation of laser, the transfer of ionic oxygen between particles occurs, and then high-energy red light is generated, and the photon number is converted into the target molecule concentration through a single photon counter and mathematical fitting. When the sample does not contain target molecules, immune complexes cannot be formed between the two particles, the distance between the two particles exceeds the transmission range of the ionic oxygen, the ionic oxygen is rapidly quenched in a liquid phase, and no high-energy-level red light is generated during detection.
In the invention, the expression "the antibody has strong affinity" means that the antibody has high sensitivity (can obviously distinguish sample without detected object and low value sample) and higher differentiation (ratio of high value sample to low value sample) of different measured value samples after the antibody is subjected to platform marking treatment;
in particular, the higher sample 2/sample 1 is, the better, and sample 3/sample 2, sample 4/sample 3, sample 5/sample 4, sample 6/sample 5 should be greater than 2.
The free antibody has strong antigen affinity and good specificity, and can bind to the antibody of the epitope 1 or the epitope 2, namely, can form competition with the antibody 1 or the antibody 2, can be the antibody 1 or the antibody 2, and can find the 3 rd antibody additionally; it is recommended that the free antibody be used either directly as antibody 1 or as antibody 2, or both.
The free antibody may be added to the reagent 1 or the reagent 2, or the same or different free antibodies may be added to both reagents.
Experimental raw material and equipment
TABLE 1
Raw materials Preservation conditions
Luminescent particles Sealing and light-shielding at 2-8 deg.c
Biotin ≤-20℃
CEA antibody Preserving according to the requirements of manufacturers
TABLE 2
Figure BDA0004032177180000081
Figure BDA0004032177180000091
The reagent combination, the reagent kit and the application thereof for detecting CEA by using the photo-excitation chemiluminescence can be obtained from the market.
The invention is further illustrated by the following examples:
example 1 preparation of reagents
1. Luminescent microparticle coating of antibodies
1.1 antibody treatment: the antibody is taken into a dialysis bag, dialyzed in a magnetic stirrer, and diluted to 1mg/mL after the dialysis is completed;
1.2 microparticle treatment: taking the particles into a centrifuge tube, weighing and balancing by a balance, centrifuging for 15min in the centrifuge, discarding the supernatant, adding a cleaning buffer solution, and uniformly mixing by ultrasonic; repeating the steps for one time, and measuring the particle size for later use;
1.3 coating reaction: mixing the particles and the antibody according to the proportion of 10:1, putting the mixture into a multitube adjustable rotary mixer in a constant temperature incubator, and uniformly mixing and reacting for 24 hours to obtain a coating concentrated solution;
2. biotin labelling of antibodies
2.1 antibody treatment: the antibody is taken into a dialysis bag, dialyzed in a magnetic stirrer, and diluted to 1mg/mL after the dialysis is completed;
2.2 biotin labeling: according to the requirement of 5uL of Biotin (DMSO) for each 1mg of antibody, rapidly adding the Biotin (DMSO) into a centrifuge tube, rapidly and uniformly mixing, and reacting for 24 hours at 30rpm by using a multi-tube adjustable rotary mixer at the temperature of 2-8 ℃ to obtain a marked concentrated solution;
3. reagent preparation
Diluting the coating concentrated solution to 50ug/mL to obtain a reagent 1; the labeling concentrated solution was diluted to 2ug/mL to give reagent 2.
Example 2 antibody pair screening
The antibodies with different recognition epitopes are respectively coated with luminous particles and labeled with biotin, and the concentrated solution is prepared by using the company platform for standby. The antibody pairs of each group were paired as follows:
TABLE 3 Table 3
Antibody pairs Microsphere coated antibodies Biotin-labeled antibody
Antibody pair 1 Antibody 1a Antibody 1b
Antibody pair 2 Antibody 1a Antibody 2b
Antibody pair 3 Antibody 1a Antibody 3b
Antibody pair 4 Antibody 2a Antibody 1b
Antibody pair 5 Antibody 2a Antibody 2b
Antibody pair 6 Antibody 2a Antibody 3b
Antibody pair 7 Antibody 3a Antibody 1b
Antibody pair 8 Antibody 3a Antibody 2b
Antibody pair 9 Antibody 3a Antibody 3b
And (3) preparing reagents by using the antibody pairs of each group, detecting CEA low-value samples, and screening out the antibody pairs with stronger affinity.
Sample 1 is a sample without CEA antigen, the concentrations of samples 2-6 are sequentially increased, and the concentrations of the samples are as follows:
TABLE 4 Table 4
Sample numbering Concentration (ng/mL)
Sample 1 (background) 0
Sample 2 ～0.5
Sample 3 ～1
Sample 4 ～2
Sample 5 ～4
Sample 6 ～8
The test results are shown in the following table:
TABLE 5
Sample numbering Antibody pair 1 Antibody pair 2 Antibody pair 3 Antibody pair 4 Antibody pair 5 Antibody pair 6 Antibody pair 7 Antibody pair 8 Antibody pair 9
Sample 1 (background) 3605 2025 807 562 1150 780 999 1422 855
Sample 2 15332 6048 1866 980 3197 1554 2021 3661 1862
Sample 3 33427 10502 2905 1449 5712 2367 3552 6905 3781
Sample 4 88333 15096 3481 2470 6122 3836 7217 10150 8998
Sample 5 211267 29712 6832 4987 12261 7155 14795 21010 18083
Sample 6 471974 72944 12672 11776 31578 17654 33891 48314 51843
Sample 2/sample 1 4.25 2.99 2.31 1.74 2.78 1.99 2.02 2.57 2.18
Sample 3/sample 2 2.18 1.74 1.56 1.48 1.79 1.52 1.76 1.89 2.03
Sample 4/sample 3 2.64 1.44 1.20 1.70 1.07 1.62 2.03 1.47 2.38
Sample 5/sample 4 2.39 1.97 1.96 2.02 2.00 1.87 2.05 2.07 2.01
Sample 6/sample 5 2.23 2.46 1.85 2.36 2.58 2.47 2.29 2.30 2.87
The results show that the antibody pair 1 detects the highest value of sample 2/sample 1 of the signal, and the signal discrimination of the samples with different measured values is better (sample 2/sample 1, sample 3/sample 2, sample 4/sample 3, sample 5/sample 4, sample 6/sample 5 are all larger than 2), so the affinity strength of the antibody pair 1 is the best.
Example 3 free antibody selection
Two antibodies of antibody pair 1 were added to the corresponding reagents, respectively, at a concentration of 2ug/mL, and a control sample (non-HOOK sample at a concentration of about 20000 ng/mL) and two HOOK samples (HOOK samples at concentrations of about 1# and 2# and 25000 ng/mL) were cross-detected as follows:
TABLE 6
Figure BDA0004032177180000111
Figure BDA0004032177180000121
The results show that the use of antibody 1 or antibody 2 or both antibodies can result in higher signal in 2 cases of HOOK samples than in the control sample, i.e. the use of free antibody can effectively increase the anti-HOOK capacity of the reagent. The method uses a method of adding the antibody 1a to the reagent 1.
EXAMPLE 4 concentration adjustment of the Components
Based on the above results, the concentration of reagent 1 was 50ug/mL, the concentration of reagent 2 was 2ug/mL, and the concentration of free antibody was 2ug/mL.
Reagent 1 concentration, immobilized reagent 2 and free antibody concentration were adjusted:
TABLE 7
Sample numbering 50ug/mL 100ug/mL 150ug/mL
Sample 1 (background) 505 1023 1503
Sample 2 1598 3167 4619
Sample 3 3401 6757 10083
Sample 4 8545 17301 26167
Sample 5 21260 45720 63059
Sample 6 42714 94160 132279
Sample 2/sample 1 3.16 3.10 3.07
Sample 3/sample 2 2.13 2.13 2.18
Sample 4/sample 3 2.51 2.56 2.60
Sample 5/sample 4 2.49 2.64 2.41
Sample 6/sample 5 2.01 2.06 2.10
Control 1008956 1854784 2560038
1# 1307970 2382052 3333229
2# 1376426 2592531 3577759
Reagent 2 concentration was adjusted, immobilized reagent 1 and free antibody concentration:
TABLE 8
Sample numbering 2ug/mL 4ug/mL 6ug/mL
Sample 1 (background) 505 827 546
Sample 2 1598 2578 1775
Sample 3 3401 5461 3847
Sample 4 8545 13416 9478
Sample 5 21260 33845 24006
Sample 6 42714 69222 48771
Sample 2/sample 1 3.16 3.12 3.25
Sample 3/sample 2 2.13 2.12 2.17
Sample 4/sample 3 2.51 2.46 2.46
Sample 5/sample 4 2.49 2.52 2.53
Sample 6/sample 5 2.01 2.05 2.03
Control 1008956 1543950 1131374
1# 1307970 1955093 1406210
2# 1376426 1993079 1447204
Free antibody concentration was adjusted, reagent 1 and reagent 2 concentrations were fixed:
TABLE 9
Figure BDA0004032177180000131
Figure BDA0004032177180000141
From the experimental data, it can be known that the concentration of the reagent 1 is increased, so that the detection signal can be increased; the concentration of the reagent 2 is increased, and in the process of increasing the concentration by 2-4 ug/mL, the detection signal can be increased, and after the concentration exceeds 4ug/mL, the signal value starts to decrease; increasing the concentration of free antibody can reduce the detection signal.
In order to ensure the accuracy of the LICA detection system background signal value, the background detection signal is preferably >1000, and because the upper limit of the LICA detection signal is 600 ten thousand, the detection signal of the control sample is preferably 100-200 ten thousand.
Therefore, when the concentration of the reagent 1 is 100ug/mL, the concentration of the reagent 2 is 2ug/mL, and the concentration of the free antibody is 2ug/mL, the reagent can be brought into a good state.
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 (9)

1. A reagent combination for detecting CEA by photoexcitation chemiluminescence, comprising:
(1) A first reagent comprising luminescent particles and a first antibody; and
(2) A second reagent comprising a label and a second antibody;
the first antibody or the second antibody specifically reacts with an object to be detected respectively;
the first antibody or the second antibody further comprises a free antibody;
the addition modes of the free antibody comprise:
(I) Adding to the first reagent; and/or
(II) adding to the second reagent; and/or
(III) simultaneously adding to the first reagent and the second reagent.
2. The reagent combination of claim 1, wherein the free antibody is capable of competing with the first antibody and/or the second antibody for binding to the test substance.
3. The reagent combination of claim 1, wherein the concentration of luminescent microspheres and first antibodies in the first reagent, the concentration of labels and second antibodies in the second reagent, and the concentration ratio of free antibodies comprises 50:1:1.
4. the combination of reagents according to claim 1, wherein the free antibody is added to the R1 reagent without being added to the R2 reagent and/or the R3 reagent.
5. The combination of reagents of claim 1, wherein the free antibodies comprise one or more of polyclonal antibodies, monoclonal antibodies, synthetic antibody fragments.
6. The combination of reagents according to claim 1, wherein the analyte comprises a body fluid; the body fluid includes one or more of blood, serum, plasma, urine.
7. Use of a combination of reagents according to any one of claims 1 to 6 for the preparation of a detection kit, detection system or detection device for the detection of CEA by photo-activated chemiluminescence.
8. A test kit comprising a combination of reagents according to any one of claims 1 to 6.
9. A method for improving the anti-HOOK ability of a CEA antibody by photoexcitation chemiluminescence, comprising mixing the detection reagent according to any one of claims 1 to 6 with a test substance.
CN202211742330.9A 2021-12-31 2022-12-30 Reagent combination, kit and application of reagent combination for detecting CEA by photo-excitation chemiluminescence Pending CN116381245A (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN202111680240 2021-12-31
CN2021116802407 2021-12-31

Publications (1)

Publication Number Publication Date
CN116381245A true CN116381245A (en) 2023-07-04

Family

ID=86968177

Family Applications (1)

Application Number Title Priority Date Filing Date
CN202211742330.9A Pending CN116381245A (en) 2021-12-31 2022-12-30 Reagent combination, kit and application of reagent combination for detecting CEA by photo-excitation chemiluminescence

Country Status (1)

Country Link
CN (1) CN116381245A (en)

Similar Documents

Publication Publication Date Title
CN109975536B (en) Anti-mullerian hormone latex enhanced turbidimetry detection kit and preparation and use methods thereof
WO2000060357A1 (en) Tandem immuno-assay for cancer
CN107167595A (en) A kind of immunochromatography reagent bar of fluorogenic quantitative detection INHB and preparation method thereof
CN116539597A (en) cTnI homogeneous phase chemiluminescence detection kit, detection method and device
CN108169497A (en) Human prolactin detection kit and preparation method and application
CN104034892A (en) Magnetic particle chemiluminescence immune assay kit of tumor marker AFP (alpha fetal protein) and detection method thereof
CN103389375B (en) A kind of liquid phase chip reagent box for pulmonary cancer diagnosis
CN108445239B (en) Homogeneous phase immunoassay reagent kit for detecting beta human chorionic gonadotropin, and preparation method and application thereof
TW201802472A (en) Anti-human hemoglobin monoclonal antibody or antibody kit, insoluble carrier particle to which anti-human hemoglobin monoclonal antibody is immobilized, and measurement reagent and measurement method using same
CA2753873A1 (en) Method for detecting substance in biological sample
CN116381248A (en) Kit for detecting total IgE antibody and application thereof
CN112379092A (en) Autoantibody joint detection immunochemiluminescence kit for early lung cancer screening and preparation method thereof
CN111239404B (en) Detection kit capable of simultaneously detecting retinol binding protein in urine sample and serum sample
CN109633163B (en) procalcitonin/C reactive protein two-in-one detection kit
CN101910843A (en) Reagent for detecting HIV-1 antigen and detection method
CN111735946A (en) Serum ALDH1B1 autoantibody quantitative detection kit and application thereof
CN116381245A (en) Reagent combination, kit and application of reagent combination for detecting CEA by photo-excitation chemiluminescence
KR20010025027A (en) Immunoassay reagents and immunoassay method
CN111665238A (en) Application of chemiluminescence microarray chip
EP4119945A1 (en) Highly sensitive immunoconjugate, preparing method thereof, in vitro diagnostic reagent and in vitro diagnostic kit including the same
CN114994318A (en) Kit for detecting AKR1B10 and preparation method thereof
CN108445238A (en) Detect the homogeneous immunological detection reagent suit and its preparation method and application of interstitialcellstimulating hormone (ICSH)
CN111239403A (en) β 2 microglobulin latex enhanced immunoturbidimetry kit and application thereof
JP3663516B2 (en) Post-synthesis chemical modification of particle reagents
CN113125703B (en) Myoglobin homogeneous detection kit and application thereof

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination