Biotinylated antigen derivatives and related kits and uses

Abstract

The present invention relates to a biotinylated antigen derivative, wherein the biotinylated antigen derivative has a structure of the formula B-L-(Ag)2, wherein B is derived from a biotin PEG active ester, L represents a connecting arm, and Ag is derived from an antigen, wherein the connecting arm L is connected to one molecule of biotin PEG active ester and two molecules of antigen via an amide bond. The biotinylated antigen derivative of the present invention improves the binding competitiveness of the antigen derivative with a labeled antibody due to its specific structure, thereby improving the sensitivity of a detection kit.

Description

Technical Field

[0001] The present invention relates to the field of biological detection reagents, and specifically to biotinylated antigen derivatives, and in particular to biotinylated trans-triiodothyronine and related kits and uses. Background Art

[0002] At present, the determination of small molecules usually uses biotin-streptavidin amplification technology. The biotin-avidin system is a new type of biological reaction amplification system developed in the late 1970s. It is based on the fact that biotin and avidin can be coupled to antigen (or antibody) molecules and a series of tracer substances, and can bridge the antigen-antibody system and the tracer substance system. Therefore, due to its multi-stage amplification effect, it gives the traditional labeled immunoassay a higher detection sensitivity.

[0003] In traditional labeled immunoassays, chemiluminescence technology occupies an important position. In chemiluminescence technology, the competitive method is often used to determine small molecules. Usually, the biotinylated antigen is first coated on magnetic beads to form a solid-phase antigen, and the antigen to be tested competes with the solid-phase antigen to bind to the labeled antibody. Thus, the amount of antigen contained in the sample is determined by the color intensity or the intensity of the measured signal value, which is negatively correlated with the amount of antigen contained in the sample.

[0004] As we all know, the normal content of many hormones in the human body, such as reverse triiodothyronine (rT3), is at the picogram (pg) level. However, these trace hormones are of great significance in the diagnosis and treatment of related diseases. For example, the biotransformation characteristics of rT3 make it have high clinical value in the differential diagnosis of hypothyroidism (abbreviated as hypothyroidism)-related diseases, especially primary hypothyroidism and euthyroid sick syndrome (ESS), which can avoid unnecessary treatment of patients with primary diseases such as liver disease, kidney disease, heart disease, tumors, diabetes, etc. when their thyroid function is abnormal.

[0005] When detecting these trace amounts of small molecules in the body, once the content is below the detection limit, it is difficult to solve the problem from the perspective of the prepared antibodies. This is because: first, the preparation of small molecule antibodies is relatively difficult, and the development of new antibodies is time-consuming and costly; second, in chemiluminescence detection, the sensitivity of the reagent increases with the increase of dilution, but excessive dilution will lead to decreased stability of the reagent and poor precision; third, for trace amounts of small molecules in the body, the amount of biotin-labeled antigen cannot be adjusted, which limits the research on sensitivity.

[0006] Therefore, it is necessary to develop a test process that improves detection sensitivity while ensuring potency from the perspective of system sensitivity. Summary of the invention

[0007] In view of the deficiencies of the prior art, the present invention aims to provide a novel biotinylated antigen derivative, which comprises one molecule of biotin and two molecules of antigen (eg, rT3), thereby enhancing the binding competitiveness between the antigen and the labeled antibody, thereby improving the sensitivity of the detection kit.

[0008] Therefore, the present invention relates to a biotinylated antigen derivative having a structure of formula BL-(Ag)2. Wherein, B represents biotin PEG active ester, L represents a connecting arm, and Ag represents an antigen; wherein the connecting arm L is connected to one molecule of biotin PEG active ester and two molecules of antigen through an amide bond. In some embodiments, the connecting arm has one amino end and two carboxyl ends, thereby connecting to the biotin PEG active ester through the amino end, and connecting to two molecules of the antigen through the carboxyl end. In some embodiments, the antigen may have at least one amino group.

[0009] Preferably, the biotinylated antigen derivative of the present invention has a structure shown in the following formula I:

[0010]

[0011] Wherein, in formula I, n is 3-6, preferably 4;

[0012] The linker arm L is derived from an α-amino dibasic acid or a β-amino dibasic acid, wherein the dibasic acid is selected from succinic acid, glutaric acid and adipic acid;

[0013] The Ag represents an antigen having an amino group, and is preferably trans-triiodothyronine.

[0014] Preferably, the linker arm can be derived from DL-aminosuccinic acid, L-aminosuccinic acid, D-aminosuccinic acid, DL-glutamic acid, L-glutamic acid, D-glutamic acid, DL-α-aminoadipic acid, L-α-aminoadipic acid and D-α-aminoadipic acid; preferably derived from DL-glutamic acid, L-glutamic acid and D-glutamic acid; more preferably derived from L-glutamic acid.

[0015] In a particularly preferred embodiment, the biotinylated antigen derivative of the present invention uses glutamic acid containing two carboxyl ends and one amino end to connect with biotin-PEG4 activated ester to form a linker, which is then coupled with two molecules of antigen (rT3) to form a biotinylated antigen derivative having a structure shown in Formula II:

[0016]

[0017] Furthermore, the present invention relates to a kit, comprising:

[0018] Reagent 1, immunomagnetic particles-streptavidin magnetic particles;

[0019] Reagent 2, biotin end—biotinylated antigen derivative as described above; and

[0020] Reagent 3, label end - monoclonal antibody labeled with a label corresponding to the biotin-terminal antigen derivative.

[0021] Preferably, in the kit, the concentration of the magnetic particles may be 0.01 mg / mL to 1.00 mg / mL, and the magnetic particles have superparamagnetism and contain avidin groups on the surface.

[0022] Preferably, in the kit, the concentration of the monoclonal antibody labeled with a marker may be 0.01 μg / mL to 1.00 μg / mL, and the marker used may include enzymes (such as HRP, β-galactosidase, alkaline phosphatase, etc.), biotin, digoxin, colloidal metals (such as colloidal gold, etc.), fluorescent dyes (such as fluorescein, rhodamine, Texas Red, etc.), chemiluminescent compounds or bioluminescent compounds (such as dioxetane, luminol or acridinium, etc.). In addition, any labeling step well known in the art, such as covalent coupling of an enzyme or biotin group, iodination, phosphorylation, biotinylation, etc., may be used to obtain the marker end. Preferably, in some embodiments, the monoclonal antibody labeled with a marker may be acridinium ester-labeled mouse anti-triiodothyronine monoclonal antibody; the concentration of the biotinylated antigen derivative may be 0.2 ng / mL to 200 ng / mL, and preferably biotinylated anti-triiodothyronine.

[0023] Each reagent in the kit may include additives conventionally used in the art, such as buffers, inorganic salt ions, surfactants, etc., and conventionally used solvents, such as purified water. For example, reagent 1 may further include a buffer (about 0.5 g / L to 20 g / L), an inorganic salt ion (about 5.0 g / L to 20.0 g / L), a surfactant (about 0.01 mL to 5 mL / L), a stabilizer (about 5 g / L to 40 g / L), a preservative (about 0.4 mL / L to 5 mL / L), and a defoamer (about 0.01 μL / L to 1 μL / L), etc., and purified water is used as a solvent. In addition to the corresponding biotinylated antigen, reagent 2 may further include, for example, a buffer (about 0.5 g / L to 20 g / L), an inorganic salt ion (about 5.0 g / L to 20.0 g / L), a surfactant (about 0.01 mL / L to 5 mL / L), a stabilizer (about 5 g / L to 40 g / L), and a preservative (about 0.4 mL / L to 5 mL / L), etc., and purified water is used as a solvent. In addition to the corresponding antibody, reagent 3 may further include, for example, a buffer (about 0.5 g / L to 20 g / L), an inorganic salt ion (about 5.0 g / L to 20.0 g / L), a surfactant (about 0.01 mL to 5.0 mL / L), a stabilizer (about 10 g / L to 30 g / L), a dissociating agent (about 0.1 μg / mL to 10 μg / mL), and a preservative (about 0.4 mL / L to 5 mL / L), and purified water is used as a solvent.

[0024] The above-mentioned kit may further include calibrators, quality control products, etc. of the corresponding antigens. For calibrators and quality control products, etc., they can be prepared, for example, according to conventional techniques in the art. For example, in addition to the corresponding antigens, the calibrators may include a buffer (about 2.00 g / L to 20.00 g / L), an inorganic salt ion (about 1.00 g / L to 10.00 g / L), a pH regulator (about 3.00 mL / L to 20.00 mL / L), a stabilizer (about 3.00 g / L to 300.00 g / L), a surfactant (about 0.10 mL / L to 10.00 ml / L), and a preservative (about 0.30 mL / L to 30.00 mL / L), etc.

[0025] The specific selection of the above additives can be determined by those skilled in the art according to needs, and will not be elaborated here.

[0026] Furthermore, the present invention relates to the use of the above-mentioned biotinylated antigen derivatives in improving the sensitivity of detecting corresponding antigens. The biotinylated antigen derivatives of the present invention contain a 1:2 ratio of biotin moiety to antigen molecule, thereby improving the binding competitiveness between the antigen derivatives and antibodies, thereby improving the sensitivity of the detection kit.

[0027] By designing a linker arm for connecting biotin to an antigen, the present invention can greatly increase the sensitivity of the detection system without affecting the titer of the reagent. Thus, in view of the fact that the products currently on the market for detecting trace amounts (pg level) of hormones cannot meet the market demand due to their low sensitivity, the magnetic particle chemiluminescence technology is used to measure trace amounts of hormones in the body in a more sensitive manner using a competitive method. In addition, it also provides a solution for small molecule detection, which is difficult to prepare antibodies for small molecule antigens, has poor antibody affinity, but requires high sensitivity. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 shows the fitting curve obtained from the assay reagents of kit 1 in the test example of the present invention;

[0029] Figure 2 shows a fitting curve obtained from the assay reagents of kit 2 in the test example of the present invention; and

[0030] Figure 3 The figure shows the fitting curve obtained from the measuring reagent of the kit 3 in the test example of the present invention. DETAILED DESCRIPTION

[0031] The present invention will be specifically described below in conjunction with specific embodiments and accompanying drawings, and the advantages and various effects of the present invention will be more clearly presented. It should be understood by those skilled in the art that the described embodiments are only partial embodiments of the present application, rather than all embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without making creative work, for example, the selection of ingredients and the selection of dosage range of conventional reagents (such as pH adjusting agents, inorganic salt ions, surfactants and stabilizers, etc.) in the kit should all belong to the scope of protection of the present application.

[0032] Unless otherwise specified, the technical terms used in the following examples have the same meanings as those generally understood by those skilled in the art to which the present invention belongs. Unless otherwise specified, the reagents used in the following examples are all conventional biochemical reagents and are commercially available.

[0033] Example 1. Preparation of biotinylated antigen derivatives represented by formula II

[0034]

[0035] Specifically, the compound shown in formula II is prepared according to the reaction scheme 1 shown below.

[0036]

[0037] Reaction Scheme 1

[0038] Wherein, in the above reaction scheme 1, the structures of compound a, compound b and group R are as follows:

[0039]

[0040]

[0041] Step 1: 3.6 mg of compound 1 (0.024 mmol) and 14.4 mg of compound a (0.024 mmol) were dissolved in 2 mL of DMF (dimethylformamide) in a 10 mL brown borosilicate glass bottle, and then placed on a roller mixer at room temperature to mix and react for 24 h.

[0042] Step 2: Dissolve 20.2mg DCC (dicyclohexylcarbodiimide) (0.097mmol) in 0.5mL DMF, and dissolve 11.28mg NHS (N-hydroxysuccinimide) (0.097mmol) in another 0.5mL DMF. Then, the DMF solution containing NHS and DCC is added to the reaction system after fully reacting for 24h in step 1, and it is placed on a roller mixer to mix and fully react for 24h. Then, 2mL DMF solution containing 40mg compound b (0.061mmol) is added to the reaction system and fully reacted for 24h. Subsequently, 26mg of light yellow oily compound is obtained by separation and purification by chromatography column (eluent: ethyl acetate-n-butanol = 8:2), which is identified as the compound of formula II (0.014mol, reaction yield 46%).

[0043] HRMS(ESI): m / z 1886.8394(M+H).

[0044] Example 2. Preparation of biotinylated antigen derivatives represented by formula III using lysine as a linker

[0045]

[0046]

[0047] Specifically, the compound represented by Formula III is prepared according to the following reaction scheme 2. Wherein, compound a, compound b and group R have the same definitions as in Example 1.

[0048]

[0049] Reaction Scheme 2

[0050] Step 1: 3.45 mg of compound 4 (0.023 mmol) and 27.8 mg of compound a (0.047 mmol) were dissolved in 2 mL of DMF in a 10 mL brown borosilicate glass bottle, and then placed on a roller mixer to mix at room temperature and react for 24 h.

[0051] Step 2: 9.7mg DCC (0.047mmol) was dissolved in 0.5mL DMF, and 5.4mg NHS (0.047mmol) was dissolved in another 0.5mL DMF. Then, the DMF solution containing NHS and DCC was added to the reaction system after fully reacting for 24h in step 1, and it was placed on a roller mixer to mix and fully react for 24h. Then, 2mL DMF solution containing 20mg compound b (0.03mmol) was added to the reaction system and fully reacted for 24h. Subsequently, 20mg of light yellow oily compound was obtained by separation and purification by chromatography column (eluent: ethyl acetate-n-butanol = 8:2), which was identified as the compound of formula III (0.012mol, reaction yield 40%).

[0052] HRMS(ESI): m / z 1726.3315(M+H).

[0053] Example 3. Preparation of biotinylated antigen derivatives of Formula IV without a linker

[0054]

[0055] Specifically, the compound represented by formula IV is prepared according to the reaction scheme 3 shown below.

[0056]

[0057] Reaction Scheme 3

[0058] 40 mg of compound b (0.061 mmol) was dissolved in 2 mL of DMF, and then 35.3 mg of compound a (0.061 mmol) was added, and the mixture was mixed on a roller mixer at room temperature and reacted for 24 h. Subsequently, 44 mg of a light yellow oily compound was obtained by separation and purification by chromatography (eluent: ethyl acetate-n-butanol = 8:2), which was identified as a compound of formula IV (0.044 mol, reaction yield 72%).

[0059] HRMS(ESI): m / z 1125.0173(M+H).

[0060] Test Case

[0061] Preparation of Calibrators

[0062] First, the calibrator dilutions were prepared using the composition and ratio shown in Table 1 below, and then testosterone antigen was added to the 5 calibrator dilutions to obtain calibrators A to E. The concentrations of the antigens in calibrators A to E were: CalA: 0.0 ng / mL; CalB: 0.2 ng / mL; CalC: 1 ng / mL; CalD: 5 ng / mL; CalE: 10 ng / mL.

[0063] Table 1. Preparation of Calibrator Dilutions

[0064] Reagent name Calibrator diluent Tris 12.11g / L Sodium chloride 8.5g / L hydrochloric acid About 5-7mL / L Trehalose 20.00g / L BSA 10.00g / L TWeen-20 0.55ml / L Biopreservative PC-950 3.00mL / L Purified water Make up to 1L

[0065] Kit Configuration

[0066] The compounds obtained in Examples 1 to 3 are used to prepare corresponding kits 1 to 3 according to the compositions and proportions in Tables 2 to 4 below.

[0067] Table 2. Preparation of Kit 1

[0068]

[0069] Table 3, Preparation of Kit 2

[0070]

[0071]

[0072] Table 4, Preparation of Kit 3

[0073]

[0074] Determination method

[0075] The obtained reagents R1 to R3 are respectively used as the biotin ends of the corresponding kits, and then the calibrators AE are measured, wherein the antigen at the biotin end and the antigen of the calibrator to be measured are competitively bound to the antibody at the marker end, and the antigen in the sample bound to the antibody is washed away.

[0076] The luminescence signal value was determined using a fully automatic chemiluminescence immunoassay analyzer (i3000), and the mean value (AVE) and coefficient of variation (CV) were calculated from the luminescence signal value, which was inversely proportional to the concentration of the calibrator.

[0077] According to the measurement results, the determination data of the main calibrators of the above-mentioned kits 1 to 3 were obtained, and the corresponding specific data are shown in the following Table 5.

[0078] Table 5

[0079]

[0080] Sensitivity assessment

[0081] Sensitivity refers to the degree of change in the response of a method to a change in the unit concentration or unit amount of the substance to be tested. According to the data in Table 5, the calibration curve gradient of Kit 1 is better than that of Kits 2 and 3, thus confirming that Kit 1 has a better sensitivity.

[0082] Furthermore, the zero value calibrator (i.e., calibrator A) corresponding to the reagent was continuously measured 20 times using the above kits 1 to 3, and the signal values ​​were recorded and the mean of the signal values ​​was calculated. and standard deviation (SD), and calculate Signal value. A linear equation is obtained by performing a two-point regression fit based on the concentration-RLU value results between the zero-value calibrator and the adjacent calibrator (i.e., calibrator B). The corresponding RLU value is substituted into the above equation to calculate the corresponding concentration, which is the blank limit (LoB) of the kit and is used to evaluate the sensitivity of the kit.

[0083] According to the above calculation method, the sensitivity of kit 1 is 0.034, the sensitivity of kit 2 is 0.199, and the sensitivity of kit 3 is 0.072. The specific data are shown in Table 6 below.

[0084] Table 6

[0085]

[0086] Actual sample measurement

[0087] 10 serum samples were measured using kits 1 to 3 obtained as above. The luminescent signal values ​​were obtained by using a fully automatic chemiluminescent immunoassay analyzer (i3000), and the antigen concentration (unit ng / mL) in the samples was obtained through the corresponding standard curve. The results are shown in Table 7.

[0088] At the same time, these 10 serum samples were measured using a high performance liquid chromatography-mass spectrometer (API5500-Shimadzu 30A), and the results obtained are shown in Table 7 as comparison results.

[0089] Table 7

[0090] Unit: ng / mL

[0091]

[0092] The results obtained from kits 1 to 3 were fitted to the comparison results and the correlation coefficients were calculated. Figure 1As shown, the fitting curve of kit 1 is y=0.0023+1.0441x, and the correlation coefficient R 2 is 0.9944. Figure 2 As shown, the fitting curve of kit 2 is y=-0.0656+1.2445x, and the correlation coefficient R 2 is 0.9294. Figure 3 As shown, the fitting curve of kit 3 is y=0.0059+1.1526x, and the correlation coefficient R 2 It is 0.9834. It can be confirmed that the use of kit 1 to measure the sample does not affect the measurement results, and the correlation coefficient of the measurement results is larger, which is better than kits 2 and 3.

Claims

1. A biotinylated antigen derivative having a structure of formula BL-(Ag)2, wherein: B is derived from biotin PEG active ester, L represents a linker arm, and Ag is derived from an antigen; and the antigen is trans-triiodothyronine; Wherein, the linker arm L is connected to one molecule of biotin PEG active ester and two molecules of antigen via an amide bond; Wherein, the connecting arm has one amino terminus and two carboxyl termini, so as to be connected to the biotin PEG active ester via the amino terminus, and to two molecules of the antigen via the carboxyl terminus; The biotinylated antigen derivative has the structure of the following formula I: Wherein, in Formula I, n is 4; The linker arm is derived from an α-amino dibasic acid or a β-amino dibasic acid, wherein the dibasic acid is selected from succinic acid, glutaric acid or adipic acid.

2. The biotinylated antigen derivative according to claim 1, wherein The linker arm is derived from DL-aminosuccinic acid, L-aminosuccinic acid, D-aminosuccinic acid, DL-glutamic acid, L-glutamic acid, D-glutamic acid, DL-α-aminoadipic acid, L-α-aminoadipic acid, or D-α-aminoadipic acid.

3. The biotinylated antigen derivative according to claim 2, wherein The linker arm is derived from DL-glutamate, L-glutamate or D-glutamate.

4. The biotinylated antigen derivative according to claim 3, wherein The linker arm is derived from L-glutamic acid.

5. The biotinylated antigen derivative according to claim 1 or 2, wherein The biotinylated antigen derivative has a structure shown in Formula II:

6. A kit, comprising: Immunomagnetic particles-streptavidin magnetic particles; Biotin end—a biotinylated antigen derivative according to any one of claims 1 to 5; as well as Label end - monoclonal antibody labeled with a label corresponding to the biotin-terminal antigen derivative.

7. The kit according to claim 6, wherein The concentration of the magnetic particles is 0.01 mg / mL to 1.00 mg / mL; the concentration of the biotinylated antigen derivative is 0.2 ng / mL to 200 ng / mL; and the concentration of the monoclonal antibody labeled with the marker is 0.01 μg / mL to 1.00 μg / mL.

8. The kit according to claim 6 or 7, wherein The biotin end is biotinylated trans-triiodothyronine, and the marker end is acridinium ester-labeled mouse anti-trans-triiodothyronine monoclonal antibody.

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