Immunodetection kit and application thereof

Abstract

According to the immunodetection kit and the application thereof, the method is used for detecting the concentration of a sample to be detected, and the sample to be detected 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 to an immunoassay kit and its application. Background Art

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

[0003] The hook effect refers to the 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 analyzer generally does not give a data alarm. Except for combining clinical information for research and comparison with the results after dilution, it cannot be easily detected. If not screened and processed, false low-value reports will seriously mislead 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 samples, but the dilution operation is time-consuming and laborious, and occasionally it may not be possible to achieve 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 kit and its application.

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

[0007] Luminescence / sensitization microspheres coated with a first antibody / antigen;

[0008] A second antibody / antigen modified with a ligand;

[0009] Sensitization / luminescence microspheres A modified with a receptor; and

[0010] Sensitization / luminescence microspheres B coated with a third antibody / antigen.

[0011] In some embodiments, the luminescence microspheres A and the luminescence microspheres B contain the same lanthanide metal so that they can emit signals of the same wavelength.

[0012] In some embodiments, the lanthanide elements are selected from lanthanum, cerium, praseodymium, neodymium, promethium, samarium, europium, gadolinium, terbium, dysprosium, holmium, erbium, thulium, ytterbium, and lutetium.

[0013] In some embodiments, the ligand and the receptor specifically bind to each other.

[0014] In some embodiments, the ligand and the receptor include a combination of any of the following: an antigen or a fragment thereof / antibody, nanogold / iodoacetyl-mercapto, amino-aldehyde / carboxyl / isothiocyanato, silyl / acrylamide, avidin or streptavidin / biotin or biotin analog, antibody / anti-antibody, hapten / antibody, peptide tag / anti-tag antibody, genetically encodable polypeptide / protein reaction pair.

[0015] According to a second aspect of the present disclosure, there is provided an immunoassay method, characterized in that the method comprises the following steps:

[0016] 1) Performing an immune reaction on a test sample containing a target antigen / antibody to be tested;

[0017] 2) Adding a receptor-modified photosensitive / luminescent microsphere A to form a first luminescent complex M, irradiating with a laser, and detecting the luminescence signal RLU1 of the first luminescent complex M;

[0018] 3) Determining that the luminescence signal RLU1 ≤ a first threshold value, then terminating the reaction;

[0019] Determining that the luminescence signal RLU1 > the first threshold value, then adding a photosensitive / luminescent microsphere B coated with a third antibody / antigen to form a second luminescent complex N, irradiating with a laser, and detecting the luminescence signal RLU2 of the second luminescent complex N;

[0020] 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.

[0021] In some embodiments, the detection wavelengths of the luminescence signal RLU1 and the luminescence signal RLU2 are the same.

[0022] In some embodiments, step 1) includes: adding a luminescent / photosensitive microsphere 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;

[0023] Wherein, the first sandwich complex contains the luminescent / photosensitive microsphere coated with the first antibody / antigen, the target antigen / antibody, and the second antibody / antigen modified with a ligand, and the first complex contains the luminescent / photosensitive microsphere coated with the first antibody / antigen and the target antigen / antibody.

[0024] In some embodiments, the ligand / receptor includes a combination of an antigen or antigen fragment and an antibody, nanogold / iodoacetyl-mercapto, amino-aldehyde / carboxyl / isothiocyanato, silyl-acrylamide or a combination of streptavidin and biotin.

[0025] In some embodiments, the ligand / receptor comprises a combination of streptavidin and biotin.

[0026] In some embodiments, the receptor-modified photosensitive / light-emitting microsphere A binds to the first sandwich complex to form a first light-emitting complex M.

[0027] In some embodiments, the third antibody / antigen-coated photosensitive / light-emitting microsphere B and the first complex form a second light-emitting complex N.

[0028] In some embodiments, the first antibody / antigen and the second antibody / antigen bind to different sites on the target antigen / antibody, respectively.

[0029] In some embodiments, the first antibody / antigen and the third antibody / antigen bind to different sites on the target antigen / antibody, respectively.

[0030] In some embodiments, the second antibody / antigen and the third antibody / antigen bind to the same site on the target antigen / antibody, respectively.

[0031] In some embodiments, step 4) further comprises the following steps:

[0032] When it is determined that the luminescence signal RLU1 ≤ the first threshold and the reaction is terminated, the luminescence signal RLU1 is substituted into the first calibration curve to calculate the concentration of the target antigen / antibody to be measured.

[0033] When it is determined that the second threshold ≤ RLU2 ≤ the third threshold, based on the luminescence signal RLU1 and the luminescence signal RLU2, the luminescence signal RLU3 is calculated, and RLU3 is substituted into the second calibration curve to calculate the concentration of the target antigen / antibody to be measured, where RLU3 = RLU2 - k × RLU1, and k is the signal attenuation coefficient of RLU1.

[0034] When it is determined that RLU2 > the third threshold, it is reported that the concentration of the target antigen / antibody to be measured exceeds the detection range.

[0035] In some embodiments, the establishment of the calibration curve comprises the following steps:

[0036] S1) Perform an immune reaction on the standard product, where the standard product comprises a plurality of target antigens / antibodies diluted in gradients.

[0037] S2) Add the receptor-modified photosensitive / light-emitting microsphere A to form a first light-emitting complex M', irradiate with a laser, and detect the luminescence signal RLU1' of the first light-emitting complex M'.

[0038] S3) Add the third antibody / antigen-coated photosensitive / light-emitting microspheres B to form the second light-emitting complex N'. After laser irradiation, detect the luminescence signal RLU2' of the second light-emitting complex N'.

[0039] S4) Calculate the Pearson correlation coefficient between the luminescence signal RLU1' and the target antigen / antibody concentration in the corresponding standard product. Perform linear fitting on the n luminescence signals RLU1' with a Pearson correlation coefficient ≥ 0.99 and the n target antigen / antibody concentrations in the corresponding standard products to obtain the first calibration curve.

[0040] S5) Calculate the decay coefficient k of the luminescence signal RLU1'.

[0041] S6) Based on the decay coefficient k, calculate the luminescence signal RLU3’, where RLU3' = RLU2' - k × RLU1'.

[0042] S7) Calculate the Pearson correlation coefficient between the luminescence signal RLU3' and the target antigen / antibody concentration in the corresponding standard product. Perform linear fitting on the m luminescence signals RLU3' with a Pearson correlation coefficient ≥ 0.99 and the m target antigen / antibody concentrations in the corresponding standard products to obtain the second calibration curve.

[0043] In some embodiments, the first calibration curve corresponds to Formula 1, and Formula 1 is as follows:

[0044] Formula 1: RLU1 = a1 × concentration of the target antigen / antibody + b1.

[0045] In some embodiments, the second calibration curve corresponds to Formula 2, and Formula 2 is as follows:

[0046] Formula 2: RLU3 = a2 × concentration of the target antigen / antibody + b2.

[0047] In some embodiments, the detection wavelengths of the luminescence signal RLU1' and the luminescence signal RLU2' are the same.

[0048] In some embodiments, the decay coefficient k is the average value of n decay coefficients Kn, and the nth decay coefficient Kn is calculated from the nth luminescence signal RLU1' and the nth luminescence signal RLU2' at the corresponding concentration.

[0049] In some embodiments, multiply the highest value among the n luminescence signals RLU1' by a preset multiple to determine the first threshold; multiply the lowest value among the m luminescence signals RLU3' by a preset multiple to determine the second threshold; multiply the highest value among the m luminescence signals RLU3' by a preset multiple to determine the third threshold.

[0050] In some embodiments, the preset multiple is selected from 1.0 to 1.5.

[0051] 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.

[0052] In some embodiments, the immunoassay is a photoactivated chemiluminescence assay.

[0053] In some embodiments, the luminescent microspheres contain lanthanide metals.

[0054] In some embodiments, the lanthanide metal is selected from at least one of lanthanum, cerium, praseodymium, neodymium, promethium, samarium, europium, gadolinium, terbium, dysprosium, holmium, erbium, thulium, ytterbium, and lutetium.

[0055] 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.

[0056] According to a third aspect of the present disclosure, there is provided an immunoassay system for implementing the method described in the second aspect. The system includes:

[0057] An immunoreaction element for forming a first luminescent complex M / M' and a second luminescent complex N / N' with the target antigen / antibody to be tested;

[0058] A signal acquisition element for exciting and recording signal values,

[0059] A processor for calculating and obtaining the concentration of the target antigen / antibody corresponding to the signal value in the sample to be tested.

[0060] According to a fourth aspect of the present disclosure, there is provided the use of the kit described in the first aspect, the method described in the second aspect, or the system described in the third aspect in detecting a target antigen / antibody.

[0061] In some embodiments, the target antigen / antibody includes a disease marker.

[0062] In some embodiments, the disease markers include tumor markers, myocardial markers, thyroid function markers, sex hormone markers, infectious disease markers, inflammatory markers, etc.

[0063] In some embodiments, the tumor markers include carbohydrate antigens.

[0064] In some embodiments, the tumor markers include CA19-9 antigen, CA15-3 antigen, CA72-4 antigen, etc.

[0065] 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

[0066] Figure 1 Exemplarily shows a schematic diagram of the reaction principle of single-wavelength double readings.

[0067] Figure 2 Exemplarily shows a dose-response curve of single-wavelength double readings.

[0068] Figure 3 Exemplarily shows a concentration-signal curve of CA72-4 detection. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0069] Based on the traditional homogeneous photochemiluminescence immunoassay, the present disclosure provides a method of adding single-wavelength double readings to improve the HOOK effect in immunoassays. The present disclosure can be used to detect antigens, antibodies, etc., and is based on the detection of antigens by double-antibody sandwich.

[0070] The immunoassay method includes the step of adding single-wavelength multiple readings.

[0071] Figure 1 Exemplarily shows the reaction principle of adding single-wavelength double readings. As Figure 1 shown, the first antibody / antigen (R1 reagent) coated with luminescent microspheres, the second antibody / antigen (R2 reagent) labeled with biotin, and the antigen form a first double-antibody sandwich complex, and the first antibody / antigen coated with binding luminescent microspheres binds to the antigen to form a single antibody-antigen complex. When binding to the streptavidin-modified photosensitive microspheres for the first reading, only the double-antibody sandwich complex will trigger the signal X1 (RLU1). When binding to the photosensitive microspheres conjugated with specific antibodies for the second reading, the signal (RLU2) read includes, in addition to the signal of the aforementioned double-antibody sandwich complex decaying from X1 to X2 over time, the signal Y (RLU3) triggered by the second double-antibody sandwich complex formed by the single antibody-antigen complex and the photosensitive microspheres conjugated with specific antibodies.

[0072] By taking multiple readings of signals X1, X2, and Y, the Figure 2 calibration curve shown can be obtained, where the abscissa is the antigen concentration and the ordinate is the signal value read. As Figure 2 shown:

[0073] ① When the antigen concentration is before inflection point 1, the signal X1 is triggered by the above-mentioned first double-antibody sandwich complex. The relationship between the fitted signal X1 and the antigen concentration C is X1 = a1C + b1. Theoretically, this curve is similar to the linear range curve and is suitable for using this curve to perform fitting calculations for the antigen concentration. The signal Y is triggered by the single antibody-antigen complex. As the antigen concentration increases, this signal Y rises slowly and almost approaches a horizontal level. At the same time, the signal X1 decays to the signal X2, and there is an attenuation coefficient k between the two.

[0074] ② When the antigen concentration is in the range from inflection point 1 to inflection point 2, as the antigen concentration increases, the signal X1 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 perform fitting calculations for the antigen concentration. At the same time, the signal X1 decays to the signal X2, and there is an attenuation coefficient k between the two.

[0075] ③ When the antigen concentration is after inflection point 2, as the antigen concentration increases, both the signal X1 and the signal Y first rise slowly, then tend to a plateau and then decline again (HOOK effect). At the same time, the signal X1 decays to the signal X2, and there is an attenuation coefficient k between the two.

[0076] In some embodiments, the attenuation coefficient k is the average value of the ratios of the second readings to the first readings of n target antigens / antibodies with continuous serial dilutions where the Pearson correlation coefficient between the first reading and the concentration is ≥0.99.

[0077] To make the objectives, technical solutions, and advantages of the present disclosure clearer and more understandable, the following further detailed description of the present disclosure is provided 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 descriptions of well-known structures and technologies are omitted to avoid unnecessarily confusing the concepts of the present disclosure. Such structures and technologies have also been described in many publications.

[0078] Definition

[0079] Unless otherwise defined, all technical terms and scientific and technical terms used in the present disclosure have the same meanings as those commonly used in the field to which the present disclosure belongs. For the purpose of explaining 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.

[0080] Unless the context clearly indicates otherwise, as used herein, the expressions "a" and "an" include plural referents.

[0081] As used herein, the expression "about" is as understood by one of ordinary skill in the art and varies within a certain range according to the context in which it is used. If one of ordinary skill in the art does not understand the use of this term according to the context in which it is used, "about" shall mean a specific value plus or minus 10%.

[0082] As used in the present disclosure, the term "antigen" 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.

[0083] As used in the present disclosure, the term "antibody" as used herein encompasses immunoglobulins (whether naturally occurring or partially or fully synthetically produced) and fragments thereof. 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, fragments thereof, 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 antibodies and multispecific antibodies, provided that they exhibit the desired biological activity or function.

[0084] As used in the present disclosure, the term "antigenic determinant" 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 antigenic determinants 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).

[0085] As used in the present disclosure, the term "sandwich immunoassay" refers to an immunoassay method well known to those skilled in the art. The conventional practice is to immobilize the first antibody on a solid-phase carrier, 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.

[0086] 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.

[0087] As used herein, the term "specific binding" refers to the discriminatory and selective binding reaction between two substances, which in terms of three-dimensional structure 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 ELISA, competitive method, neutralization competition method, indirect method, or capture method.

[0088] As used in the present invention, 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.

[0089] In any case as 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.

[0090] As used herein, the term "luminescent oxygen channeling immunoassay" refers to a method for detecting a sample to be tested by the combination of photosensitive microparticles and luminescent microparticles within a certain range to generate the transfer of ionic oxygen energy and emit a light signal. In some embodiments, the photosensitive microparticles are filled with a photosensitive compound inside, and the luminescent microparticles are filled with a luminescent compound and lanthanide elements inside. Under the excitation of a red laser, the photosensitive microparticles release singlet oxygen ions in a high-energy state. When the distance between the photosensitive microparticles and the luminescent microparticles is close enough, the singlet oxygen ions released by the photosensitive microparticles can reach the luminescent microparticles and emit high-energy light through a series of chemical reactions, which is detected by the instrument.

[0091] 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 body or natural environment, mainly an excited state oxygen molecule, including the one-electron reduction product superoxide anion (O2·□), two-electron reduction product hydrogen peroxide (H2O2), three-electron reduction product hydroxyl radical (·OH), as well as nitric oxide and singlet oxygen (1O2), etc.

[0092] The reactive oxygen species can be provided by "photosensitive microspheres", which are nano-microspheres capable of generating reactive oxygen species in an excited state. Preferably, the photosensitive microspheres can be polymer microspheres coated and bound to a matrix through functional groups and filled with a photosensitive compound, and 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.

[0093] The term "luminescent microspheres" as used in the present disclosure refers to nano-microspheres capable of reacting with reactive oxygen species to produce a detectable chemiluminescence 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.

[0094] In some embodiments, the luminescent microspheres contain a reagent capable of being triggered to emit light by reactive oxygen species.

[0095] In some embodiments, the reagent having the property of being triggered to emit light by 1O2 includes lanthanide metals and olefin compounds.

[0096] 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.

[0097] In some embodiments, the olefin compounds include dimethylthiophene, diketobutane compounds, dioxacyclohexene, enol ethers, enamines, 9-alkylideneacridanes, 9-alkylidene-N-9,10-dihydroacridines, aryl ethyl ether alkenes, aryl imidazoles, and lucigenin, as well as their derivatives.

[0098] In some embodiments, the photosensitive microspheres contain a reagent capable of activating oxygen molecules into reactive oxygen species.

[0099] In some embodiments, the reagent capable of activating oxygen molecules into reactive oxygen species includes phthalocyanine.

[0100] The term "ligand" as used in the present disclosure generally refers to any compound or molecule capable of covalently or otherwise chemically binding 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, and the interaction between the ligand and the receptor can result in a biochemical reaction, or can simply be a physical interaction or binding. In this context, "ligand" and "receptor" can be used interchangeably.

[0101] As used herein, the term "sample to be tested" refers to a mixture that contains or is suspected of containing a target molecule to be detected. Samples to be tested that can be used in the present disclosure include body fluids such as blood (which can be anticoagulated blood samples commonly seen), plasma, serum, urine, semen, saliva, cell cultures, tissue extracts, etc. Other types of samples to be tested include solvents, seawater, industrial water samples, food samples, environmental samples such as soil or water, plant materials, eukaryotic cells, bacteria, plasmids, viruses, fungi, and prokaryotic cells.

[0102] Examples and accompanying drawings are provided below to assist in understanding the present disclosure. It should be understood, however, that these examples and drawings are only for illustrative purposes and do not constitute any limitation. The actual scope of protection 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.

[0103] Example 1

[0104] 1. Main experimental materials and equipment

[0105] Homogeneous photochemiluminescence immunoassay system, luminescent microspheres with a wavelength of 620 nm (purchased from Shanghai Suoxin Biotechnology Co., Ltd.), photosensitive microspheres (purchased from Shanghai Suoxin Biotechnology Co., Ltd.), CA72-4 first antibody (XM7, batch number 1812CD2, purchased from XEMA), CA72-4 second antibody (CA724-McAb1#, batch number 20190401, purchased from FAPON), biotin (purchased from thermofisher), CA72-4 antigen (R244C, batch number 1711D100, purchased from XEMA).

[0106] 2 Coating of luminescent microspheres with the first antibody / coating of photosensitive microspheres with the third antibody:

[0107] 2.1 Antibody dialysis: Place the CA72-4 first antibody (the first antibody) / CA72-4 second antibody (the third antibody) in a dialysis bag with a molecular weight cut-off of 14000 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.

[0108] 2.2 Microsphere treatment: Add 0.05 M CB buffer (pH 9.6) to the microspheres, centrifuge (4°C, 16000 rpm, 30 min), discard the supernatant, and supplement with 0.05 M CB buffer (pH 9.6) for ultrasonic suspension.

[0109] 2.3 Mixing reaction: Mix the processed microspheres and the processed antibody at a ratio of 10:0.3 (mass ratio), and make the volume constant until the microsphere concentration is 25 mg / mL. Mix well overnight at 37°C.

[0110] 2.4 Reduction reaction: For every 10 mg of microspheres, add 0.02 mL of 8 mg / mL NaBH4 solution and mix well at 37°C for 2 hours.

[0111] 2.5 Blocking: Add 160 μL of 75 mg / mL Gly solution to every 10 mg of the prepared amount, mix well, and react at 4°C for 1 hour.

[0112] 3. Biotin-labeled secondary antibody:

[0113] 3.1 Antibody dialysis (changing the buffer system): Place the CA72-4 antibody 2 in a dialysis bag with a molecular weight cut-off of 14000D, and dialyze and change the solution twice with 0.1M NaHCO3 buffer (pH 8.5) 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.

[0114] 3.2 Labeling reaction: Dilute the dialyzed antibody to a reaction concentration of 1 mg / mL with 0.1M NaHCO3 buffer, and mix the processed antibody and Biotin (DMSO) at a ratio of 1:8. Mix well overnight at 4°C.

[0115] 3.3 Post-labeling dialysis: Place the Bio-CA72-4 antibody 2 in a dialysis bag with a molecular weight cut-off of 14000D, and dialyze and change the solution twice with the biotin-labeling dialysis buffer 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.

[0116] 4. Streptavidin-modified photosensitive microspheres:

[0117] 4.1 Activation: Centrifuge the stock solution of photosensitive microspheres to remove the preservation solution; add 0.01M phosphate buffer to wash twice, discard the supernatant; add freshly prepared 5 mg / mL NHS and 5 mg / mL BOP solution, and incubate with rotation at 37°C for 2 hours to activate the carboxyl groups on the surface of the photosensitive microspheres; after activation, centrifuge and add 0.01M phosphate buffer to wash the magnetic beads 3 times to remove the activator.

[0118] 4.2 Covalent coupling of photosensitive microspheres and streptavidin: Place the activated photosensitive microspheres in the coupling buffer, add streptavidin, and incubate with rotation at room temperature for at least 3 hours. Centrifuge to discard the supernatant; add 0.01M phosphate buffer to wash the magnetic beads three times, and add 500 μL of 0.01M phosphate buffer to resuspend the magnetic beads.

[0119] Among them, the coupling buffer was 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 was prepared from 10% of 0.05 M Na2B4O7 solution and 90%

[0120] 0.2 M H3BO3 solution, and the pH value was 7.4.

[0121] 5. Assemble the kit:

[0122] Reagent 1: Luminescent microsphere solution (FG-Ab1) coated with 1 package of CA72-4 antibody, with a working concentration of 150 μg / mL at a wavelength of 620 nm;

[0123] Reagent 2: Biotin-labeled CA72-4 antibody 2 (bio-Ab2), with a working concentration of 3 μg / mL;

[0124] Reagent 3: Photosensitive microsphere solution (GG-Ab2) coated with CA72-4 antibody 2, with a working concentration of 100 μg / mL;

[0125] Reagent 4: Photosensitive microsphere solution (GG-SA) modified with streptavidin, with a working concentration of 100 μg / mL.

[0126] Example 2

[0127] The reagent prepared by the present disclosure and the control experimental reagent were used to synchronously detect CA72-4 antigen samples with known gradient concentrations, and the experimental results are shown in Table 1.

[0128] The CA72-4 antigen was formulated into a series of antigen samples with concentration gradients using a calibration buffer in a PBS system.

[0129] Control experimental reagent: Cancer antigen 72-4 (CA72-4) detection kit (photoactivated chemiluminescence method), purchased from Kemi Boyang.

[0130] 1. The present disclosure mode

[0131] Step ①: Take 25 μL of each sample and mix it with 25 μL of the luminescent 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;

[0132] Step ②: For the first reading, 88 μL of the photosensitive microspheres modified with streptavidin was added to the mixed solution in Step ①. After incubating for 15 minutes, the excitation light was irradiated, and the emitted light amount of the reaction solution was detected. The detection wavelength was 620 nm, and the photon counter was read, denoted as RLU1;

[0133] Step ③: Second reading. After incubating the reaction solution after the first reading in Step ② with 88 μL of photosensitive microspheres conjugated with specific antibodies for 15 minutes, irradiate with excitation light and detect the emitted light intensity at 620 nm. The reading of the photon counter is recorded as RLU2. The experimental results are shown in Table 1.

[0134] 2. Control experiment

[0135] Using the reagent instruction manual method of the cancer antigen 72-4 (CA72-4) detection kit (photoactivated chemiluminescence method) (Keme Boyang), the signal value was obtained by direct detection or dilution detection. The experimental results are shown in Table 1.

[0136] 3. Experimental data

[0137] Table 1. Experimental detection results of the single-wavelength multiple-reading mode

[0138]

[0139] 4. Explanation, description and analysis of the experimental data

[0140] According to the experimental data, a suitable functional relationship was designed 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.

[0141] This method mainly involves the following formula:

[0142]

[0143] k n is the signal attenuation coefficient of the Sn-numbered sample. In this method, the standard is set such that the RLU1 of the Sn-numbered sample from S1 to Sn should satisfy the correlation with the concentration ≥ 0.99 to ensure better linearity in the range of S1 - Sn.

[0144]

[0145] k is k n the mean value of the signal attenuation coefficient.

[0146] Table 2. Statistical results of the experimental data of the single-wavelength multiple-reading mode

[0147]

[0148] *Correlation: Calculate the correlation coefficient between the signal value and the concentration using a function

[0149] 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. According to the above formula, the average attenuation coefficient k = 0.894 is obtained. Substitute different concentrations into X2 = k * X1 to calculate X2, and further calculate the signal Y through Y = RLU2 - X2. The correlation of the signal Y from S8 to S15 is ≥ 0.99, and the signal Y has a good linearity in this section. The concentration-signal curve is shown in Figure 3 。

[0150] Further establish the standard curve of this experiment:

[0151] ① When RLU1 ≤ 191570, relax it to 1.05 times, that is, when RLU1 ≤ 201149, 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 are shown in Table 3.

[0152] Table 3. Data of Calibration Curve 1 in Single-Wavelength Multiple-Reading Mode

[0153]

[0154] ② When 171540 < RLU2 < 462860, relax it to 0.95 times and 1.05 times respectively, that is, when 162963 < RLU2 < 486003, substitute the signal Y of a sample with an unknown concentration into the calibration curve 2 to calculate the sample concentration. The data of the calibration curve 2 are shown in Table 4.

[0155] Table 4. Data of Calibration Curve 2 in Single-Wavelength Multiple-Reading Mode

[0156]

[0157] Example 3

[0158] Detection and Comparative Verification of Samples to be Tested:

[0159] Step 1: Mix 25 μL of the sample to be tested with 25 μL of luminescent microspheres coated with the first antibody and 25 μL of the biotin-labeled second antibody, and incubate for 17 min to obtain a mixture;

[0160] Step 2: Add 88 μL of streptavidin-modified photosensitive microspheres, irradiate with excitation light after incubating for 15 minutes, detect the emitted light amount of the reaction solution, the detection wavelength is 620 nm, and take the first reading with a photon counter to obtain RLU1; Samples with RLU1 ≤ 201149 terminate the detection, calculate the sample concentration using the calibration curve 1 and output the result; Samples with RLU1 > 201149 continue to the detection step 3;

[0161] Step 3: Add 88 uL of specific antibody-conjugated photosensitive microspheres to the sample reaction cups with RLU1 > 201149, incubate for 15 minutes, then perform excitation light irradiation and detect the emitted light intensity at 620 nm, take the second reading of the photon counter to obtain RLU2; determine whether 162963 < RLU2 < 486003 is satisfied. If it is satisfied, calculate to obtain signal Y, use the calibration curve 2 to calculate the sample concentration and output the result; if it is not satisfied, report that the concentration is too high and out of range, and the test results are shown in Table 5.

[0162] Table 5. Test Results of Detecting Samples to Be Tested According to the Calibration Curve of the Single-Wavelength Multiple-Reading Mode

[0163] Sample to be measured A B C D E Detection concentration of this method U / mL 599.17 115.09 668.88 280.46 477.92 True concentration U / mL 600 115 670 280 480

[0164] Experimental conclusion:

[0165] Compared with the dilution detection 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, avoiding the situation where the dilution cannot meet the detection requirements at one time; compared with the direct detection of the control method, the detection range is improved, and the goal of improving the HOOK effect can be achieved.

[0166] Compared with the dilution detection 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, avoiding the situation where the dilution cannot meet the detection requirements at one time; compared with the direct detection of the online method, the detection range is improved, and the goal of improving the HOOK effect can be achieved.

[0167] 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 kit, characterized in that, The kit includes: Luminescent / sensitized microspheres coated with a first antibody / antigen; A second antibody / antigen modified with a ligand; Sensitized / luminescent microsphere A modified with a receptor; and Sensitized / luminescent microsphere B coated with a third antibody / antigen.

2. The kit according to claim 1, wherein the luminescent microsphere A and the luminescent microsphere B contain the same lanthanide element; preferably, the lanthanide element is selected from lanthanum, cerium, praseodymium, neodymium, promethium, samarium, europium, gadolinium, terbium, dysprosium, holmium, erbium, thulium, ytterbium, and lutetium; preferably, the ligand and the receptor specifically bind to each other, and more preferably, the ligand and the receptor include any one of the following combinations: antigen or its fragment / antibody, nanogold / iodoacetyl-thiol, amino-aldehyde group / carboxyl group / isothiocyanato group, silyl group / acrylamide, avidin or streptavidin / biotin or biotin analog, antibody / anti-antibody, hapten / antibody, peptide tag / anti-tag antibody, gene-encodable polypeptide / protein reaction pair.

3. An immunoassay method, characterized in that, The method uses the kit according to claim 1 or 2 for immunoassay.

4. The method according to claim 3, characterized in that, The method includes the following steps: 1) Performing an immune reaction on a test sample containing a target antigen / antibody to be detected; 2) Adding sensitized / luminescent microsphere A modified with a receptor to form a first luminescent complex M, irradiating with a laser, and detecting the luminescence signal RLU1 of the first luminescent complex M; 3) Determining that if the luminescence signal RLU1 ≤ the first threshold, the reaction is terminated; Determining that if the luminescence signal RLU1 > the first threshold, adding sensitized / luminescent microsphere B coated with a third antibody / antigen to form a second luminescent complex N, irradiating with a laser, and detecting the luminescence signal RLU2 of the second luminescent complex N; 4) 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, preferably, the detection wavelengths of the luminescence signal RLU1 and the luminescence signal RLU2 are the same.

5. The method according to claim 4, characterized in that, Step 1) includes: adding luminescent / sensitized 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 the luminescent / sensitized microspheres coated with the first antibody / antigen, the target antigen / antibody, and the second antibody / antigen modified with a ligand, and the first complex contains the luminescent / sensitized microspheres coated with the first antibody / antigen and the target antigen / antibody; preferably, the sensitized / luminescent microsphere A modified with a receptor binds to the first sandwich complex to form a first luminescent complex M; preferably, the sensitized / luminescent microsphere B coated with a third antibody / antigen and the first complex form a second luminescent complex N; preferably, the first antibody / antigen and the second antibody / antigen bind to different sites on the target antigen / antibody respectively; preferably, the first antibody / antigen and the third antibody / antigen bind to different sites on the target antigen / antibody respectively; preferably, the second antibody / antigen and the third antibody / antigen bind to the same site on the target antigen / antibody respectively.

6. The method according to claim 4, characterized in that, Step 4) further includes the following steps: After determining that the luminescence signal RLU1 ≤ the first threshold and terminating the reaction, substituting the luminescence signal RLU1 into the first calibration curve to calculate the concentration of the target antigen / antibody to be detected; When it is determined that the second threshold ≤ RLU2 ≤ the third threshold, based on the luminescence signal RLU1 and the luminescence signal RLU2, calculate the luminescence signal RLU3, and substitute RLU3 into the second calibration curve to calculate the concentration of the target antigen / antibody to be detected, where RLU3 = RLU2 - k × RLU1, and k is the signal attenuation coefficient of RLU1; When it is determined that RLU2 > the third threshold, report that the concentration of the target antigen / antibody to be detected exceeds the detection range; Preferably, the establishment of the calibration curve includes the following steps: S1) Perform an immune reaction on the standard product, where the standard product includes multiple target antigens / antibodies diluted in gradients; S2) Add the receptor-modified photosensitive / luminescent microsphere A to form the first luminescent complex M', irradiate with a laser, and detect the luminescence signal RLU1' of the first luminescent complex M'; S3) Add the photosensitive / luminescent microsphere B coated with the third antibody / antigen to form the second luminescent complex N', irradiate with a laser, and detect the luminescence signal RLU2' of the second luminescent complex N'; S4) Calculate the Pearson correlation coefficient between the luminescence signal RLU1' and the concentration of the target antigen / antibody in the corresponding standard product, and perform linear fitting on the n luminescence signals RLU1' with Pearson correlation coefficient ≥ 0.99 and the n concentrations of the target antigen / antibody in the corresponding standard products to obtain the first calibration curve; S5) Calculate the attenuation coefficient k of the luminescence signal RLU1'; S6) Based on the attenuation coefficient k, calculate the luminescence signal RLU3’, where RLU3' = RLU2' - k × RLU1'; S7) Calculate the Pearson correlation coefficient between the luminescence signal RLU3' and the concentration of the target antigen / antibody in the corresponding standard product, and perform linear fitting on the m luminescence signals RLU3' with Pearson correlation coefficient ≥ 0.99 and the m concentrations of the target antigen / antibody in the corresponding standard products to obtain the second calibration curve, Preferably, the detection wavelengths of the luminescence signal RLU1' and the luminescence signal RLU2' are the same.

7. The method according to claim 6, wherein The attenuation coefficient k is the average value of n attenuation coefficients Kn, and the nth attenuation coefficient Kn is calculated by the nth luminescence signal RLU1' and the nth luminescence signal RLU2' at the corresponding concentration; Preferably, multiply the highest value among the n luminescence signals RLU1' by a preset multiple to determine the first threshold; multiply the lowest value among the m luminescence signals RLU3' by a preset multiple to determine the second threshold; multiply the highest value among the m luminescence signals RLU3' by a preset multiple to determine the third threshold; Preferably, the preset multiple is selected from 1.0 to 1.

5.

8. The method according to claim 3, wherein The immunoassay is a photoactivated chemiluminescence detection.

9. An immunoassay system, which is used to implement the method according to any one of claims 3 to 8, and the system includes: An immune response element for forming a first luminescent complex M / M' and a second luminescent complex N / N' with a 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 corresponding to the signal value in the sample to be detected.

10. Use of the kit according to claim 1 or 2, the method according to any one of claims 3 to 8, or the system according to claim 9 in detecting a target antigen / antibody, preferably the target antigen / antibody includes a disease marker.

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