Light-activated chemiluminescence immunoassay kit as well as preparation method and application thereof

Abstract

The invention relates to a light-activated chemiluminescence immunoassay kit and a preparation method and application thereof, the kit comprises: a component a1 comprising a specific pairing binding member and luminescent particles; the component b1 comprises a marker-long chain molecule-competitive small molecule compound; wherein the specific pairing binding force of the competitive small molecule and the specific pairing binding member is lower than the specific pairing binding force of the target molecule to be detected and the specific pairing binding member. When being used for detecting small molecular substances, the light-excited chemiluminescence immunoassay kit disclosed by the invention has relatively high signal value and distinction degree; in addition, the preparation method of the kit is simple, and the kit can be applied to detection of small molecular substances with the molecular weight smaller than 1000 Da.

Description

Technical Field

[0001] The present invention belongs to the technical field of light-initiated chemiluminescent immunoassay, and particularly relates to a light-initiated chemiluminescent immunoassay kit, a preparation method thereof, and an application thereof. Background Art

[0002] Light Initiated Chemiluminescent Assay is a homogeneous immunoassay technique and one of the common methods of chemiluminescent analysis technology. It can be used to study the interactions between biomolecules and is mainly used for disease detection clinically. This technology integrates research in related fields such as polymer particle technology, organic synthesis, protein chemistry, and clinical detection. It generates the transfer of ionic oxygen energy by the combination of photosensitive particles and luminescent particles within a certain range, emits a light signal, and thus detects the sample to be tested. Among them, the photosensitive particles are filled with a photosensitive compound inside, and the luminescent particles are filled with a luminescent compound and a lanthanide element inside. Under the excitation of red laser (600 - 700nm), the photosensitive particles release high-energy singlet oxygen ions (4μS), and the propagation distance is about 200nm. When the distance between the photosensitive particles and the luminescent particles is close enough, the singlet oxygen ions released by the photosensitive particles can reach the luminescent particles and emit light at a high energy level of 520 - 620nm through a series of chemical reactions, which is detected by the instrument. In the light-initiated chemiluminescent immunoassay reaction system, the concentration of the particles is very low, the collision probability is small, and the background signal is weak. Only after the photosensitive particles and the luminescent particles are combined through an immune reaction, obvious light will be emitted, so the detection sensitivity is very high. Compared with the traditional enzyme-linked immunosorbent assay method, it has the characteristics of being homogeneous, having high sensitivity, and being easy to operate and automate. Therefore, its application prospect is very broad.

[0003] A light-initiated chemiluminescent immunoassay kit is a product for detecting a target molecule to be detected prepared based on the detection principle of light-initiated chemiluminescent assay. Currently, research shows that when the existing kit is used to detect a target molecule with a large molecular weight, it has good accuracy and high sensitivity. However, when the kit is used to detect small molecule substances, due to the small molecular weight of the small molecule substances themselves, the molecular weight of the substance obtained after labeling the small molecule substances by common methods (such as biotin-labeled substances) is still small, and the detection signal value and discrimination degree are low; moreover, the molecular weight difference between the two substances before and after labeling is small, and it is difficult to obtain a relatively pure labeled substance using conventional dialysis purification methods. When preparing the kit for detection, more mixtures containing the labeled substance need to be added, and more impurities are introduced, which easily interfere with normal detection, resulting in a lower detection signal value and discrimination degree. Therefore, there is an urgent need to develop a light-initiated chemiluminescent immunoassay kit that can obtain a higher signal value and discrimination degree when applied to the detection of small molecule substances. Summary of the Invention

[0004] In view of the deficiencies of the prior art, the present invention provides a chemiluminescent immunoassay kit for photoexcitation, its preparation method and application. Using the chemiluminescent immunoassay kit for photoexcitation of the present invention to detect small molecules has a high signal value and discrimination; moreover, the preparation method of the kit is simple and can be applied to detect small molecules with a molecular weight less than 1000 Da.

[0005] For this reason, in the first aspect of the present invention, there is provided a chemiluminescent immunoassay kit for photoexcitation, comprising:

[0006] Component a1, which contains specific pairing binding members and luminescent particles;

[0007] Component b1, which contains a marker-long chain molecule-competitive small molecule complex;

[0008] Wherein, the specific pairing binding force of the competitive small molecule with the specific pairing binding member is lower than the specific pairing binding force of the target molecule to be detected with the specific pairing binding member.

[0009] In some embodiments of the present invention, the molecular weight of the marker-long chain molecule-competitive small molecule complex is 1500 Da to 20000 Da, preferably 2000 Da to 16000 Da, more preferably 3000 Da to 11000 Da, and further preferably 4000 Da to 6000 Da. For example, the molecular weight of the marker-long chain molecule-competitive small molecule complex can be 4000 Da, 4063 Da, 4500 Da, 5000 Da, 5500 Da, 6000 Da, etc.

[0010] In some embodiments of the present invention, the molecular weight of the long chain molecule is not less than 1000 Da, preferably 1500 Da to 15000 Da, more preferably 2000 Da to 10000 Da, and further preferably 3000 Da to 5000 Da.

[0011] In some embodiments of the present invention, the long chain molecule is a hydrophilic long chain polymer molecule.

[0012] In some embodiments of the present invention, the long chain molecule is selected from polyethylene glycol or dextran.

[0013] In some embodiments of the present invention, the polyethylene glycol is selected from at least one of PEG25 to PEG200; preferably, selected from at least one of PEG30 to PEG180; more preferably, selected from at least one of PEG40 to PEG150; further preferably, selected from at least one of PEG70 to PEG120.

[0014] In some embodiments of the present invention, the dextran [C6H 10 O5]n is selected from at least one of dextrans with n = 7 to 55; preferably, selected from at least one of dextrans with n = 10 to 50; more preferably, selected from at least one of dextrans with n = 16 to 40; further preferably, selected from at least one of dextrans with n = 20 to 30.

[0015] In some embodiments of the present invention, the molecular weight of the competing small molecule is less than 1000 Da, preferably less than 800 Da, and more preferably less than 600 Da.

[0016] In some embodiments of the present invention, the kit further includes component c1, and the component c1 contains a labeled ligand and a photosensitive particle.

[0017] In some embodiments of the present invention, the label and the labeled ligand are selected from the biotin and streptavidin and their analog systems, and the FITC-anti-FITC system.

[0018] In some embodiments of the present invention, the analog of streptavidin is selected from any one of avidin, vitelline avidin, neutravidin, and streptavidin-like substances, etc.

[0019] In some embodiments of the present invention, the target molecule to be detected is a small molecule substance with a molecular weight less than 1000 Da.

[0020] In some embodiments of the present invention, the target molecule to be detected is a hapten.

[0021] In some embodiments of the present invention, the target molecule to be detected is selected from hormones.

[0022] In some embodiments of the present invention, the target molecule to be detected is selected from free tetraiodothyronine (FT4, molecular weight 776.93), tetraiodothyronine (T4, molecular weight 776.93), triiodothyronine (T3, molecular weight 650.97), free triiodothyronine (FT3, molecular weight 650.97), progesterone (PROG, molecular weight 314.46), testosterone (TESTO, molecular weight 288.42), estradiol (E2, molecular weight 274.39), etc.

[0023] In some embodiments of the present invention, the target molecule to be detected is free tetraiodothyronine, the competing small molecule is free triiodothyronine, and the specific pairing binding member is a tetraiodothyronine antibody.

[0024] In some embodiments of the present invention, the target molecule to be detected is thyroxine, the competitive small molecule is triiodothyronine, and the specific pairing binding member is thyroxine antibody.

[0025] In some embodiments of the present invention, the target molecule to be detected is free triiodothyronine, the competitive small molecule is free 3,5-diiodo-L-thyronine, and the specific pairing binding member is triiodothyronine antibody.

[0026] In some embodiments of the present invention, the target molecule to be detected is triiodothyronine, the competitive small molecule is 3,5-diiodo-L-thyronine, and the specific pairing binding member is triiodothyronine antibody.

[0027] In some embodiments of the present invention, in component a1, the concentration of the luminescent microparticles coated with the specific pairing binding member is 20 μg / mL to 40 μg / mL.

[0028] In some embodiments of the present invention, in component b1, the concentration of the complex of the label-long chain molecule-competitive small molecule is 0.5 μg / mL to 2 μg / mL.

[0029] In some embodiments of the present invention, in component c1, the concentration of the photosensitive microparticles coated with the label ligand is 20 μg / mL to 40 μg / mL.

[0030] The second aspect of the present invention provides an application of the photoactivated chemiluminescence immunoassay kit as described in the first aspect in the photoactivated chemiluminescence immunoassay for detecting whether a sample contains a target molecule to be detected.

[0031] The third aspect of the present invention provides an application of the photoactivated chemiluminescence immunoassay kit as described in the first aspect in the concentration of the target molecule to be detected in a photoactivated chemiluminescence immunoassay sample.

[0032] The fourth aspect of the present invention provides a preparation method of the photoactivated chemiluminescence immunoassay kit as described in the first aspect, and the preparation method includes preparing the complex of the label-long chain molecule-competitive small molecule, which specifically includes the following steps:

[0033] (1) Activate the competitive small molecule with DSS to obtain competitive small molecule-LC-NHS;

[0034] (2) Mix and react the competitive small molecule-LC-NHS obtained in step (1) with the label-long chain molecule-NHS to obtain a mixture containing the complex of the label-long chain molecule-competitive small molecule;

[0035] (3) Desalt and purify the mixture containing the labeled molecule-long chain molecule-competitive small molecule complex obtained in step (2) using a desalting column.

[0036] In some embodiments of the present invention, the desalting and purification is carried out multiple times, such as 2 to 5 times.

[0037] In some embodiments of the present invention, the retention efficiency of the desalting column for substances with a molecular weight less than 1000 Da is ≥95%.

[0038] In some embodiments of the present invention, the desalting column is a Zeba desalting column.

[0039] In some embodiments of the present invention, in step (2), when the competitive small molecule-LC-NHS is mixed with the labeled molecule-long chain molecule-NHS, the amount of substance of the competitive small molecule-LC-NHS is greater than or equal to the amount of substance of the labeled molecule-long chain molecule-NHS.

[0040] The fifth aspect of the present invention provides an application of a photochemiluminescence immunoassay kit prepared by the preparation method described in the fourth aspect in detecting whether a sample for photochemiluminescence immunoassay contains a target molecule to be detected.

[0041] The sixth aspect of the present invention provides an application of a photochemiluminescence immunoassay kit prepared by the preparation method described in the fourth aspect in the concentration of the target molecule to be detected in a sample for photochemiluminescence immunoassay.

[0042] The beneficial effects of the present invention are as follows:

[0043] The present invention provides a kit based on the principle of the competitive method and capable of detecting small molecule substances on a photochemiluminescence immunoassay platform. The kit contains component b1, which contains a labeled molecule-long chain molecule-competitive small molecule complex. This complex has a relatively large molecular weight. When the competitive small molecule undergoes specific pairing binding with a specific pairing binding member, the formed specific pairing binding product also has a relatively large molecular weight, effectively overcoming the defects of low detection signal value and low discrimination degree caused by the small molecular weight of the specific pairing binding product in the existing kits. Secondly, the long chain molecule between the labeled molecule and the competitive small molecule increases the distance between the competitive small molecule and the labeled molecule, effectively reducing the steric hindrance for the specific pairing binding between the specific pairing binding member and the competitive small molecule, thereby increasing the success rate of the specific pairing binding between the specific pairing binding member and the competitive small molecule, increasing the detected signal value and the discrimination degree between the sample containing the target molecule to be detected and the sample not containing the target molecule to be detected; ultimately improving the sensitivity and accuracy of the detection.

[0044] The long-chain molecules of the present invention are selected from hydrophilic long-chain polymer molecules. Firstly, hydrophilicity can meet the requirements of the label for labeling competing small molecules in the aqueous phase. Secondly, the molecular weight of the long-chain polymer molecules is relatively easy to control, and long-chain polymer molecules with different molecular weights can be selected according to the needs of detection to prepare the label-long-chain molecule-competing small molecule complex. The long-chain molecules are selected from polyethylene glycol or dextran. The methods for obtaining polyethylene glycol and dextran with different molecular weights are simple and low-cost. Different molecular weights of polyethylene glycol and dextran can be flexibly selected according to the molecular weight of the selected competing small molecule to form a label-long-chain molecule-competing small molecule complex with a suitable molecular weight, so as to ensure that the luminescent immunoassay of the specific binding conjugate has high accuracy and sensitivity.

[0045] The present invention defines that the molecular weight of the long-chain molecule is not less than 1000 Da. The molecular weight of the label-long-chain molecule-competing small molecule complex formed by the label labeling the competing small molecule through the long-chain molecule is quite different from the molecular weight of the competing small molecule, which is beneficial to obtaining a relatively pure label-long-chain molecule-competing small molecule complex through conventional dialysis purification methods. The retention efficiency of the conventional desalting column for salts and small molecules (with a molecular weight less than 1000 Da) is ≥95%, and substances with a molecular weight slightly greater than 1000 Da are also easily retained in the desalting column. The molecular weight of the label-long-chain molecule-competing small molecule complex of the present invention is 1500 Da to 20000 Da, which is much larger than 1000 Da and is not easily retained in the desalting column when using the desalting column for desalting purification. Therefore, the desalting purification yield of the label-long-chain molecule-competing small molecule complex is also relatively high.

[0046] In addition, the inventors found that within a certain range, appropriately increasing the molecular weight of the long-chain molecule to prepare the labeled substance-long-chain molecule-competitive small molecule complex can increase the molecular weight difference between the labeled substance-long-chain molecule-competitive small molecule complex and substances with small molecular weights such as the competitive small molecule, thereby making it easier to remove substances with small molecular weights such as the competitive small molecule from the mixture obtained after the reaction and obtaining a labeled substance-long-chain molecule-competitive small molecule complex with higher purity. However, when the molecular weight of the long-chain molecule reaches a certain value, further increasing the molecular weight of the long-chain molecule does not result in a significant increase in the purity of the labeled substance-long-chain molecule-competitive small molecule complex. Similarly, within a certain range, the further the molecular weight of the labeled substance-long-chain molecule-competitive small molecule complex is from 1000 Da, the less the amount of the labeled substance-long-chain molecule-competitive small molecule complex retained in the desalting column during desalting purification using a conventional desalting column, and the higher the yield of the labeled substance-long-chain molecule-competitive small molecule complex. However, when the molecular weight of the labeled substance-long-chain molecule-competitive small molecule complex reaches a certain value, further increasing the molecular weight of the labeled substance-long-chain molecule-competitive small molecule complex does not result in a significant increase in the yield. Moreover, when the molecular weight of the labeled substance-long-chain molecule-competitive small molecule complex is too high, the immune complex formed during the detection process (such as: luminescent particle-specific pairing binding member-competitive small molecule-long-chain small molecule-labeled substance-labeled substance ligand-photosensitive particle) will have too large a distance between the luminescent particle and the photosensitive particle due to the overly long chain, which will reduce the signal transmission efficiency between the two or even cause the luminescent particle to not receive the singlet oxygen ions released by the photosensitive particle and thus unable to emit light, resulting in a long detection time or errors in the detection results. Controlling the molecular weight of the labeled substance-long-chain molecule-competitive small molecule complex within 20000 Da can ensure the detection efficiency and the accuracy of the detection results.

[0047] The kit of the present invention can be used to detect small molecule substances with a molecular weight less than 1000 Da, especially can be used to detect hormones with relatively small molecular weights, solve the problems of low detection signal values and low discrimination when existing kits detect hormones with relatively small molecular weights, and has good application prospects. Detailed implementation manners

[0048] To make the present invention easy to understand, the present invention will be described in detail below. However, before describing the present invention in detail, it should be understood that the present invention is not limited to the specific implementation manners described. It should also be understood that the terms used herein are only for describing the specific implementation manners and do not represent any limitation.

[0049] Where a numerical range is provided, it should be understood that each intermediate value between the upper and lower limits of the range and any other specified or intermediate value in the specified range is encompassed within the present invention. The upper and lower limits of these smaller ranges can be independently included in the smaller ranges and are also encompassed within the present invention, subject to any explicitly excluded limits in the specified range. Where the specified range includes one or both of the limits, ranges excluding either or both of the included limits are also included in the present invention.

[0050] Unless otherwise defined, all terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, the preferred methods and materials are now described.

[0051] In the present invention, "label-long chain molecule-competitive small molecule complex" means that the label is linked to the competitive small molecule through the long chain molecule.

[0052] In the present invention, "competitive small molecule-LC-NHS" means a conjugate formed by linking the competitive small molecule to NHS through LC.

[0053] In the present invention, "label-long chain molecule-NHS" means a conjugate formed by linking the label to NHS through the long chain molecule.

[0054] In the present invention, the molecule of "biotin (abbreviation in English: Biotin)" 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. The "avidin" 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, thus producing a "tentacle effect" to improve the analysis sensitivity.

[0055] In the present invention, "polyethylene glycol" (abbreviation in English: "PEG"), chemical formula is HO(CH2CH2O) n H, has no irritation, tastes slightly bitter, has good water solubility, and has good compatibility with many organic components.

[0056] In the present invention, "dextran" (chemical formula is [C6H 10"O5]n)" refers to a homopolysaccharide composed of glucose as the monosaccharide. Dextran has a relatively high molecular weight, and the glucose units are linked by glycosidic bonds. It is mainly composed of D-glucopyranose linked by α,1→6 bonds, and the branch points have 1→2, 1→3, and 1→4 linkages.

[0057] In the present invention, "DSS" is dithiobis(succinimidyl propionate), and its molecular formula is C 16 H 20 N2O8. DSS is an uncleavable membrane-permeable crosslinking agent with an amine-reactive N-hydroxysuccinimide (NHS) ester at each end of an 8-carbon atom spacer arm. The NHS ester reacts with primary amines under the condition of pH = 7-9 to form stable amide bonds, while releasing the N-hydroxysuccinimide leaving group. Proteins (including antibodies) usually have several primary amines on the side chain of lysine (K) residues and the N-terminus of each polypeptide, and these primary amines can be used as targets for NHS ester crosslinking reagents. DSS is first dissolved in an organic solvent (such as DMF or DMSO), and then added to the aqueous crosslinking reaction.

[0058] In the present invention, "NHS" is N-hydroxysuccinimide, and its chemical formula is C4H5NO3. It is a white crystal, soluble in water, and easily soluble in acetone, alcohol, and ethyl acetate; it can be used for bioconjugation, crosslinking, labeling, and immobilization.

[0059] In the present invention, "DMSO" is dimethyl sulfoxide, and its chemical formula is C2H6OS. It is a sulfur-containing organic compound, which is a colorless, odorless, and transparent liquid at room temperature, and is a hygroscopic and flammable liquid; DMSO has the characteristics of high polarity, high boiling point, good thermal stability, aprotic, and miscible with water, and can dissolve in most organic substances such as ethanol, propanol, benzene, and chloroform, and is known as the "universal solvent".

[0060] In the present invention, "DIPEA" is N,N-diisopropylethylamine, and its chemical formula is C8H 19 N. It is a colorless or light yellow transparent liquid and is used in organic synthesis.

[0061] In the present invention, the luminescent particles can react with reactive oxygen species to produce detectable chemiluminescence signals. The photosensitive particles can generate reactive oxygen species in the excited state.

[0062] Examples

[0063] To make the present invention easier to understand, the following will further illustrate the present invention in detail with reference to examples. These examples are only illustrative and are not limited to the application scope of the present invention. The raw materials or components used in the present invention can be obtained through commercial channels or conventional methods without special instructions.

[0064] Example 1: Preparation of T2-PEG-Biotin

[0065] 1.1 Experimental raw materials and equipment

[0066] The raw materials and equipment used in the preparation process are shown in Table 1 and Table 2 respectively.

[0067] Table 1: Raw materials

[0068] 3,5-Diiodo-L-thyroxine (T2) DSS DMSO DIPEA Biotin-PEG-NHS (588Da) Biotin-PEG-NHS (1000Da) Biotin-PEG-NHS (3400Da) Biotin-PEG-NHS (10000Da) Biotin-PEG-NHS (20000Da) <![CDATA[0.1M NaHCO3 (pH = 8.5)]]> Zeta desalting column

[0069] Table 2: Equipment

[0070] LCD digital control rotary mixer Freezing centrifuge

[0071] 1.2 Preparation process

[0072] (1) Weigh 8 μmol of T2 and dissolve it in 600 μL of DMSO. Take a centrifuge tube and transfer the prepared solution into the centrifuge tube.

[0073] (2) Weigh 8 μmol of DSS and dissolve it in 114.25 μL of DMSO, and then add the prepared DSS solution into the centrifuge tube in step (1).

[0074] (3) Sequentially add 174 μL of DMSO and 100 μL of DIPEA into the centrifuge tube obtained in step (2), vortex and mix well, then place it on a rotary mixer and react overnight at room temperature and 60 rpm to obtain T2-LC-NHS with a concentration of 8 μmol / mL.

[0075] (4) Take 5 centrifuge tubes, add 0.4 μmol of Biotin-PEG-NHS (588 Da), Biotin-PEG-NHS (1000 Da), Biotin-PEG-NHS (3400 Da), Biotin-PEG-NHS (10000 Da), and Biotin-PEG-NHS (20000 Da) respectively, and then add 0.4 μmol of the T2-LC-NHS obtained in step (3) to each tube. Supplement with 0.1 M NaHCO3 buffer solution until the concentration of T2-LC-NHS is 0.4 μmol / mL, and react at room temperature and 60 rpm for 3 - 6 h to obtain a mixed solution containing T2-PEG-Biotin.

[0076] (5) Desalt and purify the mixed solution containing T2-PEG-Biotin obtained in step (4) using a Zeba desalting column, and thus obtain purifications of T2-PEG-Biotin (1250Da), purifications of T2-PEG-Biotin (1663Da), purifications of T2-PEG-Biotin (4063Da), purifications of T2-PEG-Biotin (10663Da), and purifications of T2-PEG-Biotin (20663Da).

[0077] Among them, the desalting and purification process in step (5) includes the following steps:

[0078] First centrifugation: Take a desalting column, remove the tail of the desalting column, and loosen the cap of the desalting column. Place it in a suitable centrifuge tube and weigh it on an electronic balance for balance. Centrifuge at 2 - 8°C (set at 4°C), 1000g, for 5 minutes.

[0079] Second centrifugation: Replace with a new centrifuge tube, add 0.1M NaHCO3 (pH = 8.5) buffer solution to the desalting column, and weigh it on an electronic balance for balance. Centrifuge at 2 - 8°C (set at 4°C), 1000g, for 5 minutes.

[0080] Third centrifugation: Replace with a new centrifuge tube, add 0.1M NaHCO3 (pH = 8.5) buffer solution to the desalting column, and weigh it on an electronic balance for balance. Centrifuge at 2 - 8°C (set at 4°C), 1000g, for 5 minutes.

[0081] Fourth centrifugation: Replace with a new centrifuge tube, add the mixed solution containing T2-PEG-Biotin obtained in step (4) to the desalting column, and weigh it on an electronic balance for balance. Centrifuge at 2 - 8°C (set at 4°C), 1000g, for 5 minutes. Collect the liquid in the centrifuge tube, which is the purified reagent.

[0082] Example 2: Preparation of the kit

[0083] Prepare component a1: Mix T3 and luminescent microparticles in HEPES buffer solution to prepare a reagent with a concentration of 30 μg / mL of luminescent microparticles coated with T3 as component a1.

[0084] Prepare component b1: Dilute the purification of T2-PEG-Biotin obtained after desalting and purification in step (5) of Example 1 with Tris buffer solution to a concentration of 1 μg / mL of the purification as component b1.

[0085] Prepare component c1: Mix streptavidin and photosensitive microparticles in HEPES buffer solution to prepare a reagent with a concentration of 30 μg / mL of photosensitive microparticles coated with streptavidin as component c1.

[0086] The prepared kits were used to detect six kinds of samples containing FT3 at concentrations of 0, 1.53, 4.00, 7.96, 13.73, and 52.14 pg / mL respectively; 25 μL of each of component a1, component b1, and the sample to be tested were mixed, incubated at 37 °C for 15 min, then 175 μL of component c1 was added, incubated at 37 °C for 10 min, and then the mixture was placed on a photochemiluminescence immunoassay instrument ( 500 Chemiluminescence Detection System, from Kemi Boyang Diagnostic Technology (Shanghai) Co., Ltd.) for detection. The signal values and inhibition rates obtained by the photochemiluminescence immunoassay instrument after the detection are shown in Table 3.

[0087] Table 3 Detection signal values and inhibition rate data of samples containing FT3

[0088]

[0089] In Table 3:

[0090] 1. Inhibition rate = current signal value / zero-point signal value, and the zero-point signal value is the signal value of the sample with a concentration of 0 pg / mL. The inhibition rate can illustrate the sensitivity of the detected mixture to the change in FT3 concentration. A low inhibition rate indicates high sensitivity.

[0091] 2. Overall discrimination = multiple between the signal value of the lowest concentration and the signal value of the highest concentration.

[0092] It can be seen from the results in Table 3 that:

[0093] When the molecular weight of Biotin-PEG-NHS is 588 Da (corresponding to the molecular weight of T2-PEG-Biotin being 1250 Da), the overall sample signal value is relatively low, and the overall discrimination of the detected mixture is 17.7;

[0094] When the molecular weight of Biotin-PEG-NHS increases from 588 Da to 1000 Da (corresponding to the molecular weight of T2-PEG-Biotin increasing from 1250 Da to 1663 Da), based on the data when the molecular weight of Biotin-PEG-NHS is 588 Da, the average signal value of samples with different FT3 concentrations increases by 12 times, the inhibition rate of the highest concentration increases by 2 times, and the overall discrimination of the detected mixture is 36.2;

[0095] When the molecular weight of Biotin-PEG-NHS increases from 1000 Da to 3400 Da (corresponding to the molecular weight of T2-PEG-Biotin increasing from 1663 Da to 4063 Da), based on the data when the molecular weight of Biotin-PEG-NHS is 588 Da, the signal value increases by an average of 24 times, the inhibition rate at the highest concentration increases by 14 times, and the overall discrimination of the detected mixture is 243.2;

[0096] When the molecular weight of Biotin-PEG-NHS increases from 3400 Da to 10000 Da (corresponding to the molecular weight of T2-PEG-Biotin increasing from 4063 Da to 10663 Da), based on the data when the molecular weight of Biotin-PEG-NHS is 588 Da, the signal value increases by an average of 24 times, the inhibition rate at the highest concentration increases by 2 times, and the overall discrimination of the detected mixture is 39.6;

[0097] When the molecular weight of Biotin-PEG-NHS increases from 10000 Da to 20000 Da (corresponding to the molecular weight of T2-PEG-Biotin increasing from 10663 Da to 20663 Da), based on the data when the molecular weight of Biotin-PEG-NHS is 588 Da, the signal value increases by an average of 4 times, the inhibition rate at the highest concentration is the same, and the overall discrimination of the detected mixture is 15.0.

[0098] Example 3: Preparation of T3-Dextran-Biotin

[0099] 3.1 Experimental Raw Materials and Equipment

[0100] The raw materials and equipment used in the preparation process are shown in Table 4 and Table 5 respectively.

[0101] Table 4: Raw Materials

[0102] Triiodothyronine (T3) DSS DMSO DIPEA Biotin-dextran-NHS (3000Da) Biotin-dextran-NHS (10000Da) Zeta desalting column <![CDATA[0.1M NaHCO3 (pH = 8.5)]]>

[0103] Table 5: Equipment

[0104] LCD digital control rotary mixer Freezing centrifuge

[0105] 3.2 Preparation Process

[0106] (1) Weigh 7 μmol of T3 and dissolve it in 600 μL of DMSO. Take a centrifuge tube and transfer the prepared solution into the centrifuge tube.

[0107] (2) Weigh 7 μmol of DSS and dissolve it in 114.25 μL of DMSO, and then add the prepared DSS solution to the centrifuge tube in step (1).

[0108] (3) Add 174 μL of DMSO and 100 μL of DIPEA to the centrifuge tube obtained in step (2) in sequence. After vortexing and mixing evenly, place it on a rotary mixer and react overnight at room temperature and 60 rpm to obtain T3-LC-NHS with a concentration of 7 μmol / mL.

[0109] (4) Take 2 centrifuge tubes, add 0.4 μmol of Biotin-dextran-NHS (3000 Da) and Biotin-dextran-NHS (10000 Da) respectively, and then add 0.45 μmol of T3-LC-NHS obtained in step (3) to each tube. Add 0.1 M NaHCO3 buffer to make the concentration of T3-LC-NHS 0.45 μmol / mL, and react at room temperature and 60 rpm for 3 - 6 h to obtain a mixed solution containing T3-dextran-Biotin.

[0110] (5) Use a Zeba desalting column to desalt and purify the mixed solution containing T3-dextran-Biotin obtained in step (4), and two pure products containing T3-dextran-Biotin are obtained.

[0111] Among them, the desalting and purification process in step (5) includes the following steps:

[0112] The first centrifugation: Take the desalting column, remove the tail of the desalting column, and loosen the lid of the desalting column. Place it in a suitable centrifuge tube and weigh it on an electronic balance for balance. Centrifuge at 2 - 8 °C (set at 4 °C), 1000 g, for 5 min.

[0113] The second centrifugation: Replace with a new centrifuge tube, add 0.1 M NaHCO3 (pH = 8.5) buffer to the desalting column, and weigh it on an electronic balance for balance. Centrifuge at 2 - 8 °C (set at 4 °C), 1000 g, for 5 min.

[0114] The third centrifugation: Replace with a new centrifuge tube, add 0.1 M NaHCO3 (pH = 8.5) buffer to the desalting column, and weigh it on an electronic balance for balance. Centrifuge at 2 - 8 °C (set at 4 °C), 1000 g, for 5 min.

[0115] The fourth centrifugation: Replace with a new centrifuge tube, add the mixed solution containing T3-dextran-Biotin obtained in step (4) to the desalting column, and weigh it on an electronic balance for balance. Centrifuge at 2 - 8 °C (set at 4 °C), 1000 g, for 5 min. Collect the liquid in the centrifuge tube, which is the purified reagent.

[0116] Example 4: Preparation of the kit

[0117] Prepare component a1: Mix T4 and luminescent particles in HEPES buffer to prepare a reagent with a concentration of 30 μg / mL of T4-coated luminescent particles as component a1.

[0118] Preparation of Component b1: The purified product containing T3-glucan-Biotin obtained after desalting and purification in step (5) of Example 3 was diluted with Tris buffer to a concentration of 1 μg / mL of the purified product, which was used as Component b1.

[0119] Preparation of Component c1: Avidin and photosensitive microparticles were mixed in HEPES buffer to prepare a reagent with a concentration of 30 μg / mL of streptavidin-coated photosensitive microparticles, which was used as Component c1.

[0120] Six kinds of samples with concentrations of FT4 being 0, 4.55, 7.46, 15.45, 27.9, and 85.55 pg / mL were detected respectively using the kit prepared above; 25 μL of each of Component a1, Component b1, and the sample to be detected were mixed and incubated at 37 °C for 15 min, then 175 μL of Component c1 was added and incubated at 37 °C for 10 min, and then the mixture was placed on a photochemiluminescence immunoassay instrument ( 500 Chemiluminescence Detection System, from Kemi Boyang Diagnostic Technology (Shanghai) Co., Ltd.) for detection. The signal values and inhibition rates of the photochemiluminescence immunoassay instrument obtained after the detection are shown in Table 6.

[0121] Table 6 Detection Signal Values and Inhibition Rate Data of Samples Containing FT4

[0122]

[0123] From the results of Example 2 and Example 4, it can be seen that the marker-long chain molecule-competitive small molecule complex of the present invention has universality. Using PEG or glucan as the long chain molecule, the marker-long chain molecule-competitive small molecule complex formed by connecting the marker to the competitive small molecule through the long chain molecule is applied to the detection of small molecule substances, and relatively high signal values and discrimination degrees can be obtained.

[0124] It should be noted that the above-described embodiments are only used to explain the present invention and do not constitute any limitation to the present invention. The present invention has been described by referring to typical embodiments, but it should be understood that the words used therein are descriptive and explanatory words rather than limiting words. Modifications can be made to the present invention within the scope of the claims of the present invention as provided, and the present invention can be revised without departing from the scope and spirit of the present invention. Although the present invention described therein relates to specific methods, materials, and embodiments, it does not mean that the present invention is limited to the specific examples disclosed therein. On the contrary, the present invention can be extended to all other methods and applications with the same functions.

Claims

1. A chemiluminescent immunoassay kit based on photochemical excitation, characterized in that, The kit includes: Component a1, which contains specific binding members and luminescent microparticles; Component b1, which contains a marker-long chain molecule-competitive small molecule complex; Wherein, the specific binding force of the competitive small molecule to the specific binding member is lower than the specific binding force of the target molecule to be detected to the specific binding member.

2. The opto-chemiluminescent immunoassay kit according to claim 1, wherein The molecular weight of the marker-long chain molecule-competitive small molecule complex is 1500 Da to 20000 Da, preferably 2000 Da to 16000 Da, more preferably 3000 Da to 11000 Da, and further preferably 4000 Da to 6000 Da; and / or The molecular weight of the long chain molecule is not less than 1000 Da, preferably 1500 Da to 15000 Da, more preferably 2000 Da to 10000 Da, and further preferably 3000 Da to 5000 Da; and / or The molecular weight of the competitive small molecule is less than 1000 Da, preferably less than 800 Da, and more preferably less than 600 Da.

3. The amplified luminescent proximity homogeneous assay immunoassay kit according to claim 1 or 2, wherein The long chain molecule is a hydrophilic long chain polymer molecule.

4. The amplified luminescent proximity homogeneous assay (ALPHA) immunoassay kit according to any one of claims 1-3, characterized in that, The long chain molecule is selected from polyethylene glycol or dextran; preferably, The polyethylene glycol is selected from at least one of PEG25 to PEG200; more preferably, selected from at least one of PEG30 to PEG180; further preferably, selected from at least one of PEG40 to PEG150; even more preferably, selected from at least one of PEG70 to PEG120; and / or The dextran [C6H 10 O5]n is selected from at least one of dextrans with n = 7 to 55; more preferably, selected from at least one of dextrans with n = 10 to 50; further preferably, selected from at least one of dextrans with n = 16 to 40; still more preferably, selected from at least one of dextrans with n = 20 to 30.

5. The amplified luminescent proximity homogeneous assay immunoassay kit according to any one of claims 1-4, characterized in that, The kit further includes component c1, and the component c1 contains a marker ligand and photosensitive microparticles; preferably, the marker and the marker ligand are selected from the biotin and streptavidin and their analog systems, and the FITC-anti-FITC system.

6. The amplified luminescent proximity homogeneous assay immunoassay kit according to any one of claims 1-5, characterized in that The target molecule to be detected is a small molecule substance with a molecular weight less than 1000 Da; preferably, the target molecule to be detected is a hapten; more preferably, the target molecule to be detected is selected from hormones; further preferably, the target molecule to be detected is selected from any one of free thyroxine, thyroxine, triiodothyronine, free triiodothyronine, progesterone, testosterone, and estradiol.

7. The amplified luminescent proximity homogeneous assay immunodetection kit according to any one of claims 1-6, characterized in that, In component a1, the concentration of the luminescent microparticles coated with the specific binding member is 20 μg / mL to 40 μg / mL; In component b1, the concentration of the marker-long chain molecule-competitive small molecule complex is 0.5 μg / mL to 2 μg / mL; Preferably, in component c1, the concentration of the photosensitive microparticles coated with the marker ligand is 20 μg / mL to 40 μg / mL.

8. The preparation method of the photochemiluminescence immunoassay kit according to any one of claims 1-7, characterized in that, The preparation method includes preparing the marker-long chain molecule-competitive small molecule complex, which specifically includes the following steps: (1) Activate the competitive small molecule with DSS to obtain competitive small molecule-LC-NHS; (2) Mix and react the competitive small molecule-LC-NHS obtained in step (1) with the marker-long chain molecule-NHS to obtain a mixture containing the marker-long chain molecule-competitive small molecule complex; (3) Desalting purification is carried out on the mixture containing the complex of the labeled substance-long-chain molecule-competitive small molecule obtained in step (2) by using a desalting column.

9. The preparation method according to claim 8, wherein When the competitive small molecule-LC-NHS is mixed with the labeled substance-long-chain molecule-NHS in step (2), the amount of substance of the competitive small molecule-LC-NHS is greater than or equal to the amount of substance of the labeled substance-long-chain molecule-NHS.

10. Use of a photochemiluminescence immunoassay kit according to any one of claims 1-7 or a photochemiluminescence immunoassay kit prepared by the preparation method according to claim 8 or 9 in detecting whether a target molecule to be detected is contained in a photochemiluminescence immunoassay sample or in detecting the concentration of the target molecule to be detected in a photochemiluminescence immunoassay sample.

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