Immunoassay method and application thereof

Abstract

The invention provides an immunoassay method and application thereof, the method is used for detecting the concentration of a to-be-detected sample, multiple wavelengths are added, and the concentration of the sample is detected through one-time reading. The to-be-detected sample does not need to be diluted, so that more time and labor are saved; compared with direct detection of a traditional method, the detection range is widened, and the aim of improving the HOOK effect can be achieved.

Description

Technical Field

[0001] The present disclosure relates to the field of photochemiluminescence technology, and particularly relates to an immunoassay method and its application. Background Art

[0002] Chemiluminescence immunoassay is currently the world-recognized advanced in vitro immuno-diagnosis technology, which is widely used in the medical diagnosis field. Among them, enzyme-catalyzed chemiluminescence, direct chemiluminescence, and electrochemiluminescence are the mainstream chemiluminescence technologies.

[0003] The hook effect is a phenomenon that in the double antibody sandwich method of immunoassay, within a certain concentration range, the analyte concentration is proportional to the detection signal, but beyond a certain defined concentration, the signal value decreases instead. When the hook effect occurs, the chemiluminescence instrument generally does not give a data alarm. Except for combining clinical information for research and judgment and comparing the results after dilution, it is not easy to detect. If not screened and processed, the false low-value report will seriously mislead the clinical diagnosis.

[0004] In chemiluminescence immunoassay, the detection range of traditional detection methods is limited. The common solution is to use a suitable dilution method to dilute the suspicious sample, but the dilution operation is time-consuming and laborious, and occasionally it cannot reach the detection requirements in one dilution. Summary of the Invention

[0005] To solve at least one of the above problems, the present disclosure provides an immunoassay method and its application. The method can improve the hook effect.

[0006] According to a first aspect of the present disclosure, there is provided an immunoassay method, characterized in that the method comprises the following steps:

[0007] 1) Performing an immune reaction on a test sample containing a target antigen / antibody to be tested;

[0008] 2) Adding a first luminescent reagent and a second luminescent reagent to form a first luminescent complex A and a second luminescent complex B respectively;

[0009] 3) Irradiating with a laser and detecting the luminescence signal RLU1 of the first luminescent complex A and the luminescence signal RLU2 of the second luminescent complex B; and

[0010] 4) Calculating the concentration of the target antigen / antibody to be tested in the test sample based on the luminescence signal RLU1 or the luminescence signal RLU2.

[0011] In some embodiments, the wavelengths of the luminescence signals RLU1 and RLU2 are different.

[0012] In some embodiments, the step of performing an immune reaction on a test sample containing a target antigen / antibody to be detected includes: adding photosensitive microspheres coated with a first antibody / antigen and a second antibody / antigen modified with a ligand to form a first sandwich complex and a first complex with the target antigen / antibody;

[0013] Wherein, the first sandwich complex contains photosensitive microspheres coated with a first antibody / antigen, the target antigen / antibody, and a second antibody / antigen modified with a ligand, and the first complex contains photosensitive microspheres coated with a first antibody / antigen and the target antigen / antibody.

[0014] In some embodiments, the first luminescent reagent includes a luminescent microsphere FG1 modified with a receptor, and the luminescent microsphere FG1 modified with a receptor binds to the first sandwich complex to form a first luminescent complex A; and / or,

[0015] The second luminescent reagent includes a luminescent microsphere FG2 coated with a third antibody / antigen, and the luminescent microsphere FG2 coated with a third antibody / antigen forms a second luminescent complex B with the first complex.

[0016] In some embodiments, the step of calculating the concentration of the target antigen / antibody to be detected in the test sample based on the luminescence signal RLU1 or the luminescence signal RLU2 includes:

[0017] Determine that RLU1 ≤ the first threshold, and substitute RLU1 into the first calibration curve to calculate the concentration of the target antigen / antibody to be detected;

[0018] Determine that RLU1 > the first threshold and the second threshold ≤ RLU2 ≤ the third threshold, and substitute RLU2 into the second calibration curve to calculate the concentration of the target antigen / antibody to be detected;

[0019] Determine that RLU1 > the first threshold and RLU2 > the third threshold, and report that the concentration of the target antigen / antibody to be detected exceeds the detection range.

[0020] In some embodiments, the method further includes a method for establishing the calibration curve, which includes:

[0021] S1) Perform an immune reaction on a standard product, where the standard product includes a plurality of target antigens / antibodies diluted in gradients;

[0022] S2) Add the first luminescent reagent and the second luminescent reagent to form a first luminescent complex A' and a second luminescent complex B' respectively; and

[0023] S3) After laser irradiation, detect the luminescence signal RLU1' of the first luminescent complex A' and the luminescence signal RLU2' of the second luminescent complex B';

[0024] S4) Linearly fit the luminescence signal RLU1' with the target antigen / antibody concentration in the corresponding standard product to obtain a first calibration curve, and linearly fit the luminescence signal RLU2' with the target antigen / antibody concentration in the corresponding standard product to obtain a second calibration curve.

[0025] In some embodiments, the first calibration curve corresponds to Formula 1, and Formula 1 is as follows:

[0026] Formula 1: RLU1 = a1 × concentration of the target antigen / antibody + b1.

[0027] In some embodiments, the second calibration curve corresponds to Formula 2, and Formula 2 is as follows:

[0028] Formula 2: RLU2 = a2 × concentration of the target antigen / antibody + b2.

[0029] In some embodiments, the method further includes: calculating the Pearson correlation coefficients between the luminescence signal RLU1' and the luminescence signal RLU2' and the target antigen / antibody concentration in the corresponding standard products respectively to obtain a first threshold, a second threshold, and a third threshold.

[0030] In some embodiments, the highest value in the luminescence signals RLU1' with a Pearson correlation coefficient ≥ 0.99 with the concentration of the target antigen / antibody is multiplied by a preset multiple to determine the first threshold.

[0031] In some embodiments, the lowest value in the luminescence signals RLU2' with a Pearson correlation coefficient ≥ 0.99 with the concentration of the target antigen / antibody is multiplied by a preset multiple to determine the second threshold.

[0032] In some embodiments, the highest value in the luminescence signals RLU2' with a Pearson correlation coefficient ≥ 0.99 with the concentration of the target antigen / antibody is multiplied by a preset multiple to determine the third threshold.

[0033] In some embodiments, the preset multiple is selected from 1.0 to 1.5.

[0034] In some embodiments, the preset multiple is selected from 1.0, 1.01, 1.02, 1.03, 1.04, 1.05, 1.06, 1.07, 1.08, 1.09, 1.1, 1.11, 1.12, 1.13, 1.14, 1.15, 1.16, 1.17, 1.18, 1.19, 1.2, 1.21, 1.22, 1.23, 1.24, 1.25, 1.26, 1.27, 1.28, 1.29, 1.3, 1.31, 1.32, 1.33, 1.34, 1.35, 1.36, 1.37, 1.38, 1.39, 1.4, 1.41, 1.42, 1.43, 1.44, 1.45, 1.46, 1.47, 1.48, 1.49 or 1.5.

[0035] In some embodiments, the addition of the first antibody / antigen-coated photosensitive / light-emitting microspheres is in excess relative to the target antigen / antibody.

[0036] In some embodiments, the ligand and the receptor specifically bind.

[0037] In some embodiments, the ligand / receptor includes a combination of nanogold / iodacetyl-mercapto, amino-aldehyde / carboxyl / isothiocyanato, silane-acrylamide, or streptavidin and biotin.

[0038] In some embodiments, the ligand / receptor includes a combination of streptavidin and biotin.

[0039] In some embodiments, the ligand-modified second antibody / antigen is a biotin-modified second antibody / antigen.

[0040] In some embodiments, the receptor-modified light-emitting microspheres are streptavidin-modified light-emitting microspheres.

[0041] In some embodiments, the light-emitting microspheres contain lanthanide metals.

[0042] In some embodiments, the lanthanide metal may be at least one of lanthanum, cerium, praseodymium, neodymium, promethium, samarium, europium, gadolinium, terbium, dysprosium, holmium, erbium, thulium, ytterbium, lutetium.

[0043] In some embodiments, the light-emitting microspheres FG1 and the light-emitting microspheres FG2 contain different lanthanide metals so that they can emit signals of different wavelengths.

[0044] In some embodiments, the first antibody / antigen and the second antibody / antigen bind to different sites on the target antigen / antibody.

[0045] In some embodiments, the first antibody / antigen and the third antibody / antigen bind to different sites on the target antigen / antibody.

[0046] In some embodiments, the second antibody / antigen and the third antibody / antigen bind to the same site on the target antigen / antibody.

[0047] In some embodiments, the sample to be tested is selected from one or more of blood, plasma, serum, urine, semen, saliva, cell culture, and tissue samples.

[0048] According to a second aspect of the present disclosure, there is provided an immunoassay system for implementing the method described in the first aspect. The system includes:

[0049] An immunoreaction element for forming a first luminescent complex A / A' and a second luminescent complex B / B' with the target antigen / antibody to be tested;

[0050] A signal acquisition element for exciting and recording signal values,

[0051] A processor for calculating and obtaining the concentration of the target antigen / antibody to be tested corresponding to the signal value in the sample to be tested.

[0052] According to a third aspect of the present disclosure, there is provided an immunoassay kit, which includes: a photosensitive reagent, a first luminescent reagent, and a second luminescent reagent. The photosensitive reagent can react with the first luminescent reagent and the second luminescent reagent respectively to emit signals of different wavelengths.

[0053] In some embodiments, the photosensitive reagent includes photosensitive microspheres coated with the first antibody / antigen.

[0054] In some embodiments, the first luminescent reagent includes luminescent microspheres FG1 modified with a receptor.

[0055] In some embodiments, the second luminescent reagent includes luminescent microspheres FG2 coated with the third antibody / antigen.

[0056] In some embodiments, the luminescent microspheres FG1 and the luminescent microspheres FG2 contain different lanthanide metal elements so that they can emit signals of different wavelengths.

[0057] In some embodiments, the lanthanide metal element is selected from at least one of lanthanum, cerium, praseodymium, neodymium, promethium, samarium, europium, gadolinium, terbium, dysprosium, holmium, erbium, thulium, ytterbium, and lutetium.

[0058] In some embodiments, the kit further includes a second antibody / antigen modified with a ligand.

[0059] In some embodiments, the ligand and the receptor specifically bind to each other.

[0060] In some embodiments, the ligand / receptor includes a combination of nanogold / iodacetyl-mercapto, amino-aldehyde / carboxyl / isothiocyanato, silyl-acrylamide, avidin / streptavidin and biotin / biotin analog, antibody-anti-antibody, hapten-antibody, peptide tag-anti-tag antibody, and a combination of gene-encodable polypeptides-protein reaction pairs.

[0061] In some embodiments, the first antibody / antigen and the second antibody / antigen bind to different sites on the target antigen / antibody.

[0062] In some embodiments, the first antibody / antigen and the third antibody / antigen bind to different sites on the target antigen / antibody.

[0063] In some embodiments, the second antibody / antigen and the third antibody / antigen bind to the same site on the target antigen / antibody.

[0064] According to the fourth aspect of the present disclosure, there is provided an application of the method described in the first aspect, the system described in the second aspect, or the kit described in the third aspect in detecting a target antigen / antibody.

[0065] In some embodiments, the target antigen / antibody includes a disease marker.

[0066] In some embodiments, the disease marker includes a tumor marker, a myocardial marker, a thyroid function marker, a sex hormone marker, an infectious disease marker, an inflammatory marker, and the like.

[0067] In some embodiments, the tumor marker includes CA19-9 antigen, CA15-3 antigen, CA72-4 antigen, and the like.

[0068] The detection method described in the present disclosure is more time-saving and labor-saving because the sample to be tested does not need to be diluted; compared with the direct detection of traditional methods, the detection range is improved, and the goal of improving the HOOK effect can be achieved. BRIEF DESCRIPTION OF THE DRAWINGS

[0069] Figure 1 Exemplarily shows a schematic diagram of the reaction principle of dual-wavelength single reading.

[0070] Figure 2 Exemplarily shows a dose-response curve of dual-wavelength single reading.

[0071] Figure 3 Exemplarily shows a concentration-signal curve of CA72-4 detection. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0072] Based on the traditional homogeneous photochemiluminescence immunoassay, the present disclosure provides a method of adding multi-wavelength single reading to improve the HOOK effect in immunoassays. The present disclosure can be used for detecting antigens, antibodies, etc., and is based on the detection of antigens by double antibody sandwich detection.

[0073] The immunoassay method may include the step of adding multi-wavelength single reading, taking dual wavelengths as an example.

[0074] As Figure 1 As shown in the reaction principle of dual-wavelength single reading, the first antibody / antigen (R1 reagent) coated with photosensitive microspheres, the second antibody / antigen (R2 reagent) labeled with biotin, and the antigen form a double antibody sandwich complex, and the first antibody / antigen coated with luminescent microspheres binds to the antigen to form a single antibody-antigen complex. After binding to the luminescent microspheres modified with streptavidin, when reading at a certain wavelength, only the double antibody sandwich complex will trigger signal X (RLU1); at the same time, after binding to the luminescent microspheres conjugated with specific antibodies and reading at another wavelength, the second double antibody sandwich complex formed by the single antibody-antigen complex and the luminescent microspheres conjugated with specific antibodies triggers signal Y (RLU2).

[0075] By performing a single reading on signals X and Y, Figure 2 the calibration curve shown can be obtained, where the abscissa is the antigen concentration and the ordinate is the read signal value. As Figure 2 shown:

[0076] ① When the antigen concentration is before inflection point 1, signal X is triggered by the above-mentioned double antibody sandwich complex, and the relationship between the fitted signal X and the antigen concentration C is X = a1C + b1. Theoretically, this curve is similar to the linear range curve and is suitable for using this curve to fit and calculate the antigen concentration; for the signal Y triggered by the above-mentioned second double antibody sandwich complex, as the antigen concentration increases, this signal Y rises slowly and is almost close to horizontal.

[0077] ② When the antigen concentration is in the range from inflection point 1 to inflection point 2, as the antigen concentration increases, signal X rises slowly with a certain slope; at this time, the relationship between the fitted signal Y and the antigen concentration C is Y = a2C + b2. Theoretically, this curve is similar to the linear range curve and is suitable for using this curve to fit and calculate the antigen concentration.

[0078] ③ When the antigen concentration is after inflection point 2, as the antigen concentration increases, both signal X and signal Y first rise slowly, then tend to a plateau and then decline again (HOOK effect).

[0079] Design a mathematical model according to the multi-wavelength single reading situation to fit the antigen concentration-signal curve.

[0080] To make the objectives, technical solutions and advantages of the present disclosure more clear and understandable, the present disclosure will be further described in detail below in conjunction with embodiments. The specific embodiments described herein are only used to explain the present disclosure and do not constitute any limitation to the present disclosure. In addition, in the following description, the description of well-known structures and technologies is omitted to avoid unnecessarily confusing the concepts of the present disclosure. Such structures and technologies have also been described in many publications.

[0081] Definition

[0082] Unless otherwise defined, all technical and scientific terms used in the present disclosure have the same meaning as commonly used in the field to which the present disclosure belongs. For the purpose of interpreting this specification, the following definitions will be applied, and where appropriate, terms used in the singular form will also include the plural form and vice versa.

[0083] Unless the context clearly indicates otherwise, the expressions "a" and "an" used herein include plural referents.

[0084] The expression "about" used herein is as understood by those of ordinary skill in the art and varies within a certain range according to the context in which it is used. If those of ordinary skill in the art do not understand the use of the term according to the context in which it is used, "about" will mean a particular value plus or minus 10%.

[0085] The term "antigen" as used in the present disclosure refers to a substance that can stimulate the body to produce an immune response and can bind to the immune response products, antibodies and sensitized lymphocytes, both in vivo and in vitro, to produce an immune effect.

[0086] The term "antibody" as used in the present disclosure covers immunoglobulins (whether naturally produced or partially or fully synthetically produced) and their fragments. The term also covers any protein having a binding domain homologous to the immunoglobulin binding domain. "Antibody" also includes polypeptides that contain complementarity-determining regions (CDRs) from immunoglobulin genes or fragments thereof that specifically bind and recognize antigens. The use of the term antibody is intended to include whole antibodies, polyclonal antibodies, monoclonal antibodies and recombinant antibodies, their fragments, and also includes single-chain antibodies, humanized antibodies, murine antibodies, chimeric monoclonal antibodies, mouse-human monoclonal antibodies, mouse-primate monoclonal antibodies, primate-human monoclonal antibodies, anti-idiotypic antibodies, antibody fragments (such as, for example, scFv, (scFv)2, Fab, Fab', and F(ab')2, F(ab1)2, Fv, dAb and Fd fragments, bispecific antibodies and antibody-related polypeptides. Antibodies include bispecific and multispecific antibodies as long as they exhibit the desired biological activity or function.

[0087] As used herein, the term "epitope" is synonymous with "antigen" and "epitope", and refers to the site on a polypeptide macromolecule where the antigen-binding portion binds to form an antigen-binding portion-antigen complex (e.g., a continuous segment of amino acids or a conformational configuration consisting of different regions of non-contiguous amino acids). Useful epitopes can be found, for example, on the surface of tumor cells, virus-infected cells, other diseased cells, immune cells, in serum (free) and / or in the extracellular matrix (ECM).

[0088] As used herein, the term "sandwich immunoassay" refers to an immunoassay method well known to those skilled in the art. The conventional procedure is to immobilize the first antibody on a solid-phase support, then react the first antibody with the antigen, then react with the labeled second antibody, and finally detect the signal by chemiluminescence, photochemiluminescence or enzyme-linked colorimetric reaction.

[0089] As used herein, the term "binding" refers to the direct association between two molecules caused by interactions such as covalent, electrostatic, hydrophobic, ionic and / or hydrogen bonding, including but not limited to interactions such as salt bridges and water bridges.

[0090] As used herein, the term "specific binding" refers to the mutual discrimination and selective binding reaction between two substances. From a three-dimensional structural perspective, it is the conformational correspondence between the corresponding reactants. Under the technical concept disclosed herein, the detection methods for specific binding reactions include but are not limited to: sandwich immunoassay, competitive assay, neutralization competition assay, indirect assay or capture assay.

[0091] As used herein, the term "biotin" is widely present in animal and plant tissues. Its molecule has two ring structures, namely an imidazolone ring and a thiophene ring, and the imidazolone ring is the main site for binding to streptavidin. Activated biotin can be conjugated to almost all known biological macromolecules, including proteins, nucleic acids, polysaccharides and lipids, etc., under the mediation of a protein cross-linking agent; while "streptavidin" is a protein secreted by Streptomyces, with a molecular weight of 65 kD. The "streptavidin" molecule is composed of 4 identical peptide chains, and each peptide chain can bind one biotin. Therefore, each antigen or antibody can be conjugated with multiple biotin molecules simultaneously, thereby producing a "tentacle effect" to improve the analysis sensitivity.

[0092] In any case where needed, any reagent used in the present invention, including antigens, antibodies, photosensitive microspheres or luminescent microspheres, etc., can be conjugated with biotin or streptavidin according to actual needs.

[0093] As used herein, the term "photoactivated chemiluminescence assay" refers to a method for detecting a sample to be tested by generating a transfer of ionic oxygen energy through the binding of photosensitive particles and luminescent particles within a certain range, thereby emitting a light signal. In some embodiments, the photosensitive particles are filled with a photosensitive compound, and the luminescent particles are filled with a luminescent compound and a lanthanide element. Under the excitation of a red laser, the photosensitive particles release singlet oxygen ions in a high-energy state. 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 high-energy light through a series of chemical reactions, which is detected by the instrument.

[0094] As used herein, the term "reactive oxygen species" refers to the general term for substances composed of oxygen, containing oxygen and having active properties in the organism or natural environment, mainly an excited state oxygen molecule, including the one-electron reduction product superoxide anion (O2·-), the two-electron reduction product hydrogen peroxide (H2O2), the three-electron reduction product hydroxyl radical (·OH), as well as nitric oxide and singlet oxygen ( 1 O2), etc.

[0095] The reactive oxygen species can be provided by "photosensitive microspheres", which are nanoscale microspheres capable of generating reactive oxygen species in an excited state. Preferably, the photosensitive microspheres can be polymer microspheres coated on a matrix through functional groups and filled with a photosensitive compound, which can generate singlet oxygen under light excitation. At this time, the photosensitive microspheres can also be referred to as oxygen-supplying microspheres or photosensitive microspheres.

[0096] As used herein, the term "luminescent microspheres" refers to nanoscale microspheres capable of reacting with reactive oxygen species to generate a detectable chemiluminescent signal, which can also be referred to as oxygen-receiving microspheres or luminescent microspheres. Preferably, the luminescent microspheres can be polymer microspheres filled in a matrix through functional groups and filled with a luminescent composition, and the luminescent composition contains a chemiluminescent compound capable of reacting with reactive oxygen species. In some specific embodiments of the present disclosure, the chemiluminescent compound undergoes a chemical reaction with reactive oxygen species to form an unstable metastable intermediate, and the metastable intermediate can decompose, emitting light simultaneously or subsequently.

[0097] In some embodiments, the luminescent microspheres contain a reagent capable of being triggered to emit light by reactive oxygen species.

[0098] In some embodiments, the 1 reagent with O2-triggered luminescence characteristics includes a lanthanide metal and an olefin compound.

[0099] In some embodiments, the lanthanide metal includes at least one of lanthanum, cerium, praseodymium, neodymium, promethium, samarium, europium, gadolinium, terbium, dysprosium, holmium, erbium, thulium, ytterbium, and lutetium.

[0100] In some embodiments, the olefin compounds include dimethylthiophene, diketone compounds, dioxacyclohexene, enol ethers, enamines, 9-alkylidenestrychnane, 9-alkylidene-N-9,10-dihydroacridine, aryl vinyl ether, aryl imidazole, and lucigenin, as well as their derivatives.

[0101] In some embodiments, the photosensitive microspheres contain a reagent capable of activating oxygen molecules into reactive oxygen species.

[0102] In some embodiments, the reagent capable of activating oxygen molecules into reactive oxygen species includes phthalocyanine.

[0103] As used herein, the term "ligand" generally refers to any compound or molecule that can bind covalently or otherwise chemically to a bioactive substance, such as an oligonucleotide. In certain embodiments, a ligand can interact directly or indirectly with another compound, such as a receptor. The interaction between the ligand and the receptor can result in a biochemical reaction or can be merely a physical interaction or binding. As used herein, "ligand" and "receptor" can be used interchangeably.

[0104] As used herein, the term "test sample" refers to a mixture that contains or is suspected of containing a target molecule to be detected. Test samples that can be used in the present disclosure include body fluids such as blood (which can be anticoagulated blood as commonly seen in collected blood samples), plasma, serum, urine, semen, saliva, cell cultures, tissue extracts, etc. Other types of test samples include solvents, seawater, industrial water samples, food samples, environmental samples such as soil or water, plant materials, eukaryotic cells, bacteria, plasmids, viruses, fungi, and cells from prokaryotes.

[0105] Examples and drawings are provided below to assist in understanding the present disclosure. However, it should be understood that these examples and drawings are only used to illustrate the present disclosure and do not constitute any limitation. The actual protection scope of the present disclosure is set forth in the claims. It should be understood that any modifications and changes can be made without departing from the spirit of the present disclosure.

[0106] Example 1

[0107] 1. Main experimental raw materials and equipment

[0108] Homogeneous photochemiluminescence immunoassay system, luminescent microspheres with a wavelength of 620 nm (luminescent microspheres 1) (purchased from PerkinElmer), luminescent microspheres with a wavelength of 545 nm (luminescent microspheres 2) (purchased from PerkinElmer), photosensitive microspheres (purchased from Shanghai Suoxin Biotechnology Co., Ltd.), CA72-4 primary antibody (XM7, lot number 1812CD2, purchased from XEMA), CA72-4 secondary antibody (CA724-McAb1#, lot number 20190401, purchased from FAPON), biotin (purchased from thermofisher), CA72-4 antigen (R244C, lot number 1711D100, purchased from XEMA).

[0109] 2. Coating of photosensitive microspheres with CA72-4 antibody 1 / Coating of luminescent microspheres 1 with CA72-4 antibody 2:

[0110] 2.1 Antibody dialysis: Place CA72-4 antibody 1 or CA72-4 antibody 2 in a dialysis bag with a molecular weight cut-off of 14,000 D, and dialyze and change the solution twice with 0.05 M CB buffer (pH 9.6) at 4°C. Each dialysis time is at least 4 hours, and the volume of each dialysis solution is not less than 100 times the volume of the object to be dialyzed. Aspirate the dialyzed protein and transfer it to a clean centrifuge tube, and take a sample to measure the protein concentration.

[0111] 2.2 Microsphere treatment: Add 0.05 M CB buffer (pH 9.6) to the microspheres, centrifuge (4°C, 16,000 rpm, 30 min), discard the supernatant, and supplement with 0.05 M CB buffer (pH 9.6) and ultrasonically suspend.

[0112] 2.3 Mixing reaction: Mix the treated microspheres and the treated antibody in a ratio of 10:0.3 (mass ratio), and make up the volume to a microsphere concentration of 25 mg / mL, and mix well overnight at 37°C.

[0113] 2.4 Reduction reaction: Mix and react with 0.02 mL of 8 mg / mL NaBH4 solution at 37°C for 2 hours according to every 10 mg of microspheres.

[0114] 2.5 Blocking: Mix with 160 μL of 75 mg / mL Gly solution according to every 10 mg of the prepared amount, and mix well at 4°C for 1 hour.

[0115] 3. Biotin labeling of CA72-4 antibody 2:

[0116] 3.1 Antibody dialysis (changing buffer system): Place the CA72-4 antibody 2 in a dialysis bag with a molecular weight cut-off of 14,000 D. Dialyze and change the buffer twice with 0.1 M NaHCO3 buffer (pH 8.5) at 4 °C. Each dialysis time is at least 4 hours, and the volume of the dialysis buffer each time is not less than 100 times the volume of the object to be dialyzed. Aspirate the dialyzed protein and transfer it to a clean centrifuge tube, and take a sample to measure the protein concentration.

[0117] 3.2 Labeling reaction: Dilute the dialyzed antibody to a reaction concentration of 1 mg / mL with 0.1 M NaHCO3 buffer. Mix the treated antibody and Biotin (DMSO) in a ratio of 1:8 and mix well at 4 °C overnight.

[0118] 3.3 Post-labeling dialysis: Place the Bio-CA72-4 antibody 2 in a dialysis bag with a molecular weight cut-off of 14,000 D. Dialyze and change the buffer twice with the biotin-labeling dialysis buffer at 4 °C. Each dialysis time is at least 4 hours, and the volume of the dialysis buffer each time is not less than 100 times the volume of the object to be dialyzed. Aspirate the dialyzed protein and transfer it to a clean centrifuge tube, and take a sample to measure the protein concentration.

[0119] 4. Streptavidin-modified luminescent microspheres 2:

[0120] 4.1 Activation: Centrifuge the stock solution of the luminescent microspheres 2 to remove the preservation solution; add 0.01 M phosphate buffer to wash twice and discard the supernatant; add freshly prepared 5 mg / mL NHS and 5 mg / mL BOP solutions and incubate with rotation at 37 °C for 2 hours to activate the carboxyl groups on the surface of the luminescent microspheres 2; after activation, centrifuge and add 0.01 M phosphate buffer to wash 3 times to remove the activator.

[0121] 4.2 Covalent coupling of the luminescent microspheres 2 with streptavidin: Place the activated luminescent microspheres 2 in the coupling buffer, add streptavidin, and incubate with rotation at room temperature for at least 3 hours. Centrifuge to discard the supernatant; add 0.01 M phosphate buffer to wash the magnetic beads three times, and resuspend the magnetic beads with 500 μL of 0.01 M phosphate buffer.

[0122] Among them, the coupling buffer is prepared by adding 100 μL of 1 mg / mL streptavidin and 10 μL of 100 mg / mL BOP reagent to 390 μL of borate buffer. The borate buffer is prepared from 10% 0.05 M Na2B4O7 solution and 90% 0.2 M H3BO3 solution, and the pH value is 7.4.

[0123] 5. Assembly of the kit:

[0124] Reagent 1: Photosensitive microsphere solution (GG-Ab1) coated with CA72-4 antibody 1, concentration 150 μg / mL;

[0125] Reagent 2: Biotin-labeled CA72-4 antibody 2 (bio-Ab2), concentration 3 μg / mL;

[0126] Reagent 3: Luminescent microsphere solution with wavelength 620 nm coated with CA72-4 antibody 2 (FG-Ab2), concentration 100 μg / mL;

[0127] Reagent 4: Luminescent microsphere solution with wavelength 545 nm modified with streptavidin (FG-SA), concentration 100 μg / mL.

[0128] Example 2

[0129] Synchronously detect CA72-4 antigen samples with known gradient concentrations using the reagents prepared in the present disclosure and the control experimental reagents. The experimental results are shown in Table 1.

[0130] Prepare antigen samples with a series of concentration gradients of CA72-4 antigen using the calibration buffer in the PBS system.

[0131] Control experimental reagent: Cancer antigen 72-4 (CA72-4) detection kit (photoactivated chemiluminescence method), purchased from Kemi Boyang.

[0132] 1. The present disclosure mode

[0133] Step ①: Take 25 μL of each sample and mix it with 25 μL of photosensitive microspheres coated with the first antibody and 25 μL of biotin-labeled second antibody, and incubate for 17 min to obtain a mixture;

[0134] Step ②: Read once. Then add 88 μL of streptavidin-modified luminescent microspheres and 88 μL of luminescent microspheres conjugated with specific antibodies to the mixture in Step ① respectively. After incubating for 15 min, irradiate with excitation light, detect the emission light amount of the reaction solution, and the detection wavelengths are 620 nm (Eu) and 545 nm (Tb) respectively. Read with a photon counter, and record it as RLU1 at 620 nm and RLU2 at 545 nm; the experimental results are shown as "the method of the present disclosure" in Table 1.

[0135] 2. Control experiment

[0136] Conduct a control experiment according to the method described in the control experimental reagent instructions, and obtain the signal value through direct detection or dilution detection; the experimental results are shown as "dilution detection" and "direct detection" in Table 1.

[0137] 3. Experimental data

[0138] Table 1. Experimental detection results of the dual-wavelength one-time reading mode

[0139]

[0140] 4. Interpretation, explanation and analysis of the experimental data

[0141] Design an appropriate functional relationship based on the experimental data to finally convert RLU1 and RLU2 into the concentration-signal standard curve of the antigen. Subsequently, it can be used to compare the readings RLU1 and RLU2 of the test sample containing the target antigen to be detected with the standard curve to determine the concentration of the sample.

[0142] Table 2. Statistical results of experimental data

[0143]

[0144] As can be seen from Table 2, the correlation between RLU1 and concentration from S1 to S8 is ≥0.99, and <0.99 from S9 onwards. The signal X has a better linearity within the section of S1 to S8. The correlation between the signal RLU2 and concentration from S8 to S15 is ≥0.99, and the signal Y has a better linearity within this section. The concentration-signal curve is shown in Figure 3 。

[0145] Further establish the standard curve of this experiment:

[0146] ① When RLU1 ≤ 176237, appropriately relax it to 1.05 times, that is, when RLU1 ≤ 185049, substitute the signal RLU1 of a sample with an unknown concentration into the calibration curve 1 to calculate the sample concentration. The data of the calibration curve 1 is shown in Table 3.

[0147] Table 3. Data of calibration curve 1 in the dual-wavelength single-reading mode

[0148]

[0149] ② When 677 < RLU2 < 168258, appropriately relax it to the signal near the detection limit and 1.05 times respectively, that is, 1000 < RLU2 < 176671. Substitute the signal RLU2 of a sample with an unknown concentration into the calibration curve 2 to calculate the sample concentration. The data of the calibration curve 2 is shown in Table 4.

[0150] Table 4. Data of calibration curve 2 in the dual-wavelength single-reading mode

[0151]

[0152] Example 3

[0153] Detection and comparison verification of the test sample:

[0154] Step 1: Mix 25 μL of a sample with a known concentration, 25 μL of photosensitive microspheres coated with the first antibody, and 25 μL of the biotin-labeled second antibody, and incubate for 17 min to obtain a mixed solution;

[0155] Step 2: Add 88 uL of streptavidin-modified luminescent microspheres and 88 uL of luminescent microspheres conjugated with specific antibodies, incubate for 15 min, then irradiate with excitation light. Read the photon counter at a wavelength of 620 nm to obtain the luminescence value RLU1; read the photon counter at a wavelength of 545 nm to obtain the luminescence value RLU2;

[0156] Step 3: For samples with RLU1 ≤ 185049, substitute them into calibration curve 1 to calculate the sample concentration and output the result; for samples with RLU1 > 185049, if they meet the condition of 1000 < RLU2 < 176671, calculate the signal Y, use calibration curve 2 to calculate the sample concentration and output the result; if they do not meet the condition, report that the concentration is too high and out of range, and the test results are shown in Table 5.

[0157] Table 5. Test results of samples to be tested according to the calibration curve of the dual-wavelength single-reading mode

[0158] Sample to be tested A B C D E Detected concentration U / mL 598.84 114.97 669.36 281.01 478.59 True concentration U / mL 600 115 670 280 480

[0159] Experimental conclusion:

[0160] Compared with the dilution test of the control method, the method of the present disclosure is more time-saving and labor-saving because the sample to be tested does not need to be diluted, and it avoids the situation where the dilution cannot meet the detection requirements at one time; compared with the direct detection of the on-line method, it expands the detection range and can achieve the goal of improving the HOOK effect.

[0161] The technical solution of the present disclosure is not limited to the limitations of the above specific embodiments. Any technical deformation made according to the technical solution of the present disclosure falls within the protection scope of the present disclosure.

Claims

1. An immunoassay method, characterized in that, The method includes the following steps: 1) Perform an immune reaction on a test sample containing a target antigen / antibody to be detected; 2) Add a first luminescent reagent and a second luminescent reagent to form a first luminescent complex A and a second luminescent complex B respectively; 3) Detect the luminescence signal RLU1 of the first luminescent complex A and the luminescence signal RLU2 of the second luminescent complex B by laser irradiation; and 4) Calculate the concentration of the target antigen / antibody to be detected in the test sample based on the luminescence signal RLU1 or the luminescence signal RLU2.

2. The method according to claim 1, characterized in that, The step of performing an immune reaction on a test sample containing a target antigen / antibody to be detected includes: adding photosensitive microspheres coated with a first antibody / antigen and a second antibody / antigen modified with a ligand to form a first sandwich complex and a first complex with the target antigen / antibody; Wherein, the first sandwich complex contains photosensitive microspheres coated with a first antibody / antigen, the target antigen / antibody, and a second antibody / antigen modified with a ligand, and the first complex contains photosensitive microspheres coated with a first antibody / antigen and the target antigen / antibody.

3. The method according to claim 2, characterized in that The first luminescent reagent includes luminescent microspheres FG1 modified with a receptor, and the luminescent microspheres FG1 modified with a receptor bind to the first sandwich complex to form a first luminescent complex A; and / or, The second luminescent reagent includes luminescent microspheres FG2 coated with a third antibody / antigen, and the luminescent microspheres FG2 coated with a third antibody / antigen form a second luminescent complex B with the first complex.

4. The method according to claim 1, characterized in that The step of calculating the concentration of the target antigen / antibody to be detected in the test sample based on the luminescence signal RLU1 or the luminescence signal RLU2 includes: Determine that RLU1 ≤ the first threshold, and substitute RLU1 into the first calibration curve to calculate the concentration of the target antigen / antibody to be detected; Determine that RLU1 > the first threshold and the second threshold ≤ RLU2 ≤ the third threshold, and substitute RLU2 into the second calibration curve to calculate the concentration of the target antigen / antibody to be detected; Determine that RLU1 > the first threshold and RLU2 > the third threshold, and report that the concentration of the target antigen / antibody to be detected exceeds the detection range.

5. The method according to claim 4, wherein The establishment of the calibration curve includes the following steps: S1) Perform an immune reaction on a standard product, where the standard product includes a plurality of target antigens / antibodies diluted in gradients; S2) Add the first luminescent reagent and the second luminescent reagent to form a first luminescent complex A' and a second luminescent complex B' respectively; and S3) Detect the luminescence signal RLU1' of the first luminescent complex A' and the luminescence signal RLU2' of the second luminescent complex B' by laser irradiation; S4) Perform linear fitting on the luminescence signal RLU1' and the concentration of the target antigen / antibody in the corresponding standard product to obtain a first calibration curve, and perform linear fitting on the luminescence signal RLU2' and the concentration of the target antigen / antibody in the corresponding standard product to obtain a second calibration curve.

6. The method according to claim 5, characterized in that, The method further includes: calculating the Pearson correlation coefficients between the luminescence signal RLU1' and the luminescence signal RLU2' and the concentrations of the target antigens / antibodies in the corresponding standard products respectively to obtain the first threshold, the second threshold, and the third threshold; Preferably, the highest value in the luminescence signal RLU1' with a Pearson correlation coefficient ≥ 0.99 with the concentration of the target antigen / antibody is multiplied by a preset multiple to determine the first threshold; Preferably, the lowest value in the luminescence signal RLU2' with a Pearson correlation coefficient ≥ 0.99 with the concentration of the target antigen / antibody is multiplied by a preset multiple to determine the second threshold; Preferably, the highest value in the luminescence signal RLU2' with a Pearson correlation coefficient ≥ 0.99 with the concentration of the target antigen / antibody is multiplied by a preset multiple to determine the third threshold; Preferably, the preset multiple is selected from 1.0 to 1.

5.

7. The method according to any one of claims 1 to 6, characterized in that The addition of the photosensitive / luminescent microspheres coated with the first antibody / antigen is in excess relative to the target antigen / antibody; Preferably, the first antibody / antigen and the second antibody / antigen bind to different sites on the target antigen / antibody; Preferably, the first antibody / antigen and the third antibody / antigen bind to different sites on the target antigen / antibody; Preferably, the second antibody / antigen and the third antibody / antigen bind to the same site on the target antigen / antibody.

8. An immunoassay system for implementing the method according to any one of claims 1 to 7, the system comprising: An immunoreaction element for forming a first luminescent complex A / A' and a second luminescent complex B / B' with the target antigen / antibody to be detected; A signal acquisition element for exciting and recording signal values; A processor for calculating and obtaining the concentration of the target antigen / antibody to be detected in the test sample corresponding to the signal value.

9. An immunoassay kit, characterized in that, The kit includes: a photosensitive reagent, a first luminescent reagent, and a second luminescent reagent, and the photosensitive reagent can react with the first luminescent reagent and the second luminescent reagent respectively to emit signals of different wavelengths; Preferably, the photosensitive reagent includes photosensitive microspheres coated with the first antibody / antigen; Preferably, the first luminescent reagent includes luminescent microspheres FG1 modified with a receptor; Preferably, the second luminescent reagent includes luminescent microspheres FG2 coated with the third antibody / antigen; Preferably, the luminescent microspheres FG1 and the luminescent microspheres FG2 contain different lanthanide metals so that they can emit signals of different wavelengths; Preferably, the lanthanide metal is selected from at least one of lanthanum, cerium, praseodymium, neodymium, promethium, samarium, europium, gadolinium, terbium, dysprosium, holmium, erbium, thulium, ytterbium, lutetium; Preferably, the kit further includes a second antibody / antigen modified with a ligand; Preferably, the ligand and the receptor specifically bind; Preferably, the ligand / receptor includes a combination of nano gold / iodacetyl-mercapto, amino-aldehyde / carboxyl / isothiocyanato, silyl-acrylamide, avidin / streptavidin and biotin / biotin analog, antibody-anti-antibody, hapten-antibody, peptide tag-anti-tag antibody, combinable gene-encoded polypeptide-protein reaction pair.

10. Use of the method according to any one of claims 1 to 7, the system according to claim 8 or the kit according to claim 9 for detecting a target antigen / antibody, preferably the target antigen / antibody includes a disease marker.

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