PAMAM-mediated labeled small molecule compound as well as synthesis method and application thereof

Abstract

The invention belongs to the technical field of immunochemiluminescence, and relates to a PAMAM-mediated labeled small molecule compound and a synthesis method and application thereof.The PAMAM-mediated labeled small molecule compound is composed of small molecules, PAMAM and a marker, and the small molecules are provided with active groups or provided with derivative groups capable of reacting with amino under certain activation conditions to form covalent binding; the marker is provided with a succinimide group. The difference between a coupling product and a small molecular structure is reduced to the maximum extent through PAMAM mediation, the PAMAM dosage is extremely small and stable, compared with PEG, the molecular weight is more uniform, non-specific binding caused by hydrophobic interaction can be greatly reduced in immunoreaction, and good signals and separation can be obtained without purification of the product.

Description

Technical Field

[0001] The present invention belongs to the technical field of immunochemiluminescence, and relates to a PAMAM (Polyamidoamine)-mediated labeled small molecule compound and a synthesis method and application thereof. Background Art

[0002] In the development of immunochemiluminescent reagents, the detection of small molecule antigens is often achieved by competitive methods. The relevant detection items include testosterone, progesterone, estradiol, vitamin D, vitamin B12, T3, T4 and other items. During the test, the small molecule antigens in the sample and the small molecule antigens in the reagent system compete for the binding sites of the antibodies in the reaction system. In some reagent systems, small molecule antigens can be linked to marker molecules, such as alkaline phosphatase, acridinium ester, terpyridine ruthenium, etc.; antibodies are connected to solid phase magnetic beads. Then, the reaction pathway of solid phase magnetic beads-antibody-small molecule + marker is realized to form a complex that can generate signals. The more small molecule antigens in the sample, the fewer complexes formed by the reaction of small molecule + marker and antibody-magnetic beads. Conversely, the fewer antigens, the more bound complexes; the concentration of small molecule antigens is detected by this quantitative relationship.

[0003] The connection between small molecules and markers can be simply divided into two categories. In the alkaline phosphatase system, small molecules can be labeled on the enzyme protein; in the acridinium ester or electrochemical system, small molecules can be directly connected to acridinium esters or terpyridine ruthenium. Taking acridinium ester as an example, generally speaking, small molecules can be directly connected to acridinium esters after deriving carboxyl -COOH or amino -NH2, but due to the structure of small molecules, there are basically not too many active groups on them, which ultimately limits the proportion of acridinium esters that can be coupled to small molecules; less acridinium esters will inevitably result in lower luminescence signal values, which cannot meet the requirements of reagent development. The solution derived from this is to first couple the small molecule to the protein and then couple the acridinium ester to the protein; however, this method will lead to structural imbalance between the small molecule in the sample and the small molecule conjugate in the reagent, mismatch in competitive kinetics, or require a higher antibody concentration to bind to the small molecule on the protein; and this structure requires optimization of the small molecule feed ratio and the acridinium ester feed ratio during the production process, because small molecules and acridinium esters are often connected using amino groups and the preparation process is relatively long.

[0004] The applicant has used the structure of multi-arm polyethylene glycol, each arm of which contains an amino group at the top. First, estradiol is connected to one of the arms, and then acridinium ester is connected to the other arms, thereby realizing a structure in which estradiol is coupled to multiple markers. This structure realizes the coupling of the small molecule to be tested and the marker, which is a certain improvement over the conventional protein-mediated coupling structure. However, the structure is still too large. Depending on the molecular weight of the multi-arm polyethylene glycol, the final marker molecular weight is between 5-12KD, and there are still problems with competitive kinetics, and the background is high in some projects. Summary of the invention

[0005] In view of the above technical problems, the object of the present invention is to provide a PAMAM-mediated labeled small molecule compound and a synthesis method and application thereof.

[0006] The technical solution adopted by the present invention to achieve the technical purpose is: The present invention provides a PAMAM-mediated labeled small molecule compound, which is composed of a small molecule, PAMAM and a label. The small molecule has an active group or a derivative group that can react with an amino group under certain activation conditions to form a covalent bond; the label has a succinimide group.

[0007] Preferably, the molecular weight of the PAMAM is 517D, 1430D, 3256D and 6909D, corresponding to different surface amino groups of 4, 8, 16 and 32. If constructing a small molecule marker, a PAMAM with a smaller molecular weight is preferred to retain competition kinetics similar to that of the small molecule itself.

[0008] Preferably, the small molecules with active groups include but are not limited to progesterone, testosterone, estradiol, vitamin D or vitamin B12, etc.

[0009] Preferably, the label includes but is not limited to acridinium ester, acridinium sulfonamide, terpyridine ruthenium or FITC.

[0010] The present invention also provides a method for synthesizing a PAMAM-mediated labeled small molecule compound, comprising: first reacting a small molecule with PAMAM in a certain ratio, and then reacting the marker with the remaining amino group to obtain the PAMAM-mediated labeled small molecule compound.

[0011] Preferably, when the small molecule is estradiol E2-NHS carrying an NHS group activation structure, the synthesis method comprises: reacting at a molar ratio of E2-NHS:PAMAM-4NH2=0.9:1, after reacting for 1 hour, adding a marker acridinium ester or terpyridine ruthenium AE / Ru at a molar ratio of AE / Ru:PAMAM=3:1, and using a glycine buffer to provide free amino -NH2 with a molar molecular weight of 4 times that of PAMAM to terminate the reaction.

[0012] More preferably, during the reaction, PAMAM-4NH2 is diluted to 1 mg / ml with PBS buffer at pH 7.5-8.5, E2-NHS is dissolved to a concentration of 5 mg / ml with DMF (dimethylformamide), and acridinium ester or terpyridine ruthenium AE / Ru is dissolved to 5 mg / ml with DMF (dimethylformamide).

[0013] The present invention further provides a method for characterizing any of the above-mentioned PAMAM-mediated labeled small molecule compounds or PAMAM-mediated labeled small molecule compounds synthesized by any of the above-mentioned synthesis methods in immunochemiluminescence detection.

[0014] The present invention further provides an immunochemiluminescence detection kit, comprising any of the above-mentioned PAMAM-mediated labeled small molecule compounds or a PAMAM-mediated labeled small molecule compound synthesized by any of the above-mentioned synthesis methods.

[0015] Compared with existing methods using inert proteins (such as BSA) or PEG as mediators, PAMAM mediation has the following advantages: 1. Minimize the difference between the coupling product (small molecule-PAMAM-luminescent molecule) and the small molecule structure. The molecular weight of BSA is 67000D, PEG has different molecular weights but is roughly between 5000-10000D and cannot provide too many active groups, but PAMAM can provide 4 amino groups for subsequent reactions with a minimum molecular weight of 517D.

[0016] 2.PAMAM is a relatively cheap, inactive synthetic compound that is used in very small amounts and is stable. Its molecular weight is more uniform than that of PEG.

[0017] 3.PAMAM contains a large number of highly polar chemical bonds such as -NH and C=O bonds, which can easily form hydrogen bonds with water to maintain hydrophilicity, which will greatly reduce the nonspecific binding caused by hydrophobic effects in immune responses.

[0018] 4. The small molecular weight required is less, because it is necessary to maintain the advantage of the number of amino groups to ensure that most molecules can be coupled, so the product does not need to be purified to obtain better signals and separation. Of course, in theory, purification can obtain better signals and separation and more uniform structures. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 The relationship between the dilution ratio of E2-PAMAM-AE and the signal value in Test Example 1 is shown. DETAILED DESCRIPTION

[0020] In order to explain the present invention more clearly, the present invention is further described in detail below in conjunction with the embodiments and drawings. It should be understood by those skilled in the art that the content described below is illustrative rather than restrictive, and should not be used to limit the scope of protection of the present invention. Example

[0021] 1. Taking estradiol (E2) as an example, the structures that can be selected include β-estradiol-6-one 6-(O-carboxymethyloxime) (CAS: 35048-47-6), the carboxyl group on which can be converted into an NHS group through reaction; or the activated structure (Steraloids, E0982) directly carrying the NHS group can directly react with the primary amine group of PAMAM; this application selects the activated form directly carrying the NHS group (hereinafter abbreviated as E2-NHS); this small molecule structure can be extended to other possible detection targets, such as progesterone, testosterone, vitamin D, vitamin B12, etc.; 2.PAMAM: Different test items need to consider different numbers of amino groups. For estradiol, we choose PAMAM containing 4 amino groups, which is abbreviated as PAMAM-4NH2 below. The structural formula is as follows: ; 3. Labels include acridinium ester (AE, Cas 199293-83-9) and terpyridine ruthenium (Ru, CAS: 136724-73-7), both of which carry NHS groups that can directly react with amino groups; 4. The synthesis of E2-PAMAM-AE starts with the reaction of E2-NHS and PAMAM-4NH2. Use PBS (phosphate buffer, pH 7.5-8.5) to dilute a certain amount of PAMAM-4NH2 to 1 mg / ml; E2-NHS is dissolved in DMF (dimethylformamide) at a concentration of 5 mg / ml; the reaction ratio of the two is the molar ratio of E2-NHS:PAMAM-4NH2=0.9:1. Considering that there are 4 NH2 on PAMAM, this ratio ensures that most of E2-NHS can be coupled to PAMAM-4NH2; 5. After 1 hour of reaction, add the marker. Dissolve acridinium ester (AE) in DMF (dimethylamino) to 5 mg / ml. The reaction ratio is AE:PAMAM = 3:1. This ratio takes into account that most markers are coupled to reduce background adsorption. 6. Use glycine buffer to provide 4 times the molar molecular weight of PAMAM free amino groups -NH2 to terminate the reaction.

[0022] It is worth noting that the overall reaction ensures that most of the added E2 and markers are captured by the amino groups on the PAMAM, but there is still a portion of free matter, and the reaction product will be a hybrid. The number of E2 or markers on each PAMAM is not fixed.

[0023] To verify the effect of this structure, we designed a simple reaction model to demonstrate its binding efficiency.

[0024] The test was performed using an immunochemiluminescence device (Shenzhen Yingkai, FP2910).

[0025] Test Example 1 The kit consists of: 2.8um magnetic beads coated with streptavidin (SA magnetic beads, Dynal, 0.70mg / ml), Mes buffer (25mM Mes, 1% BSA, pH6.5), anti-estradiol antibody labeled with biotin (Bioventix, 2C7, 0.1ug / ml). E2-PAMAM-AE was diluted with Mes buffer at an appropriate ratio (dilution method see Table 1) and added to the reaction system as a sample. The reaction parameters are 50ul E2-PAMAM-AE diluent + 50ul Mes buffer, incubate for 9min, then add 50ul biotinylated antibody and 50ul SA magnetic beads, incubate again for 9min, a solid phase magnetic bead-SA-biotin + antibody-E2-PAMAM-AE structure will be formed, and the luminescence value is positively correlated with the E2-PAMAM-AE concentration; data see Table 1.

[0026] Table 1. Luminescence value detection results at different E2-PAMAM-AE concentrations when adding antibodies

[0027] like Figure 1 As shown in the figure, we can roughly see that between dilutions 2-5, the dilution ratio and the signal value show a roughly linear relationship, indicating that the signal is indeed related to the synthesized structure. Dilution 1 has a high concentration and there are kinetic reasons, so it does not show a linear relationship with the subsequent dilutions.

[0028] Test Example 2 If we remove the antibody (replace the antibody component with buffer), and the other configurations are the same as in Test Example 1, then theoretically the same sample will not produce a meaningful signal or only a background signal under the same system. The data are shown in Table 2.

[0029] Table 2. Luminescence value detection results at different E2-PAMAM-AE concentrations without adding antibody

[0030] It can be seen that the signal value of the same sample is greatly reduced after the antibody is removed from the reaction system, indicating that the antibody is indeed connected to E2-PAMAM-AE and magnetic beads to generate a signal; the signal generated in the absence of antibodies is presumably caused by the nonspecific adsorption of E2-PAMAM-AE and magnetic beads.

[0031] Based on the above two tests, we determined that the structure of E2-PAMAM-AE was successful and had the potential to be applied to actual sample testing. We constructed a serum estradiol detection system based on this molecular structure.

[0032] Application Examples The kit structure is as follows: 2.8um magnetic beads coated with streptavidin (SA magnetic beads, Dynal, M280, 0.70mg / ml), E2-PAMAM-AE diluted in Mes buffer (25mM Mes, 1% BSA, pH6.5) at an appropriate concentration, anti-estradiol antibody labeled with biotin (Bioventix, 2C7, 0.1ug / ml, with 100ng / ml of norgestrel as a dissociator). Serum samples with different concentrations of estradiol were measured using the Roche e411 electrochemical detection method and added to the reaction system as samples. The reaction parameters are as follows: 50ul sample is first incubated with 50ul antibody for 9 minutes, and then 50ul E2-PAMAM-AE and 50ul SA magnetic beads are added and incubated again for 9 minutes. E2-PAMAM-AE will bind to the sites on the antibody that are not bound by E2, and the biotin on the antibody will form a structure of solid phase magnetic beads-SA-biotin + antibody-E2-PAMAM-AE. The luminescence value is negatively correlated with the concentration of estradiol in the serum sample; the data are shown in Table 3.

[0033] Table 3. Luminescence value detection results of serum samples with different concentrations of estradiol

[0034] The signal-to-noise ratio was calculated by dividing the signal at 3.2 pg / ml by the signal value at each concentration point.

[0035] It can be seen from the data in the above table that E2 in the sample and E2-PAMAM-AE in the reagent compete with each other. The development of competitive detection reagents for estradiol can be further carried out on this basis. E2-PAMAM-AE is an effective structure for constructing competitive detection reagents for estradiol.

[0036] Obviously, the above embodiments of the present invention are merely examples to more clearly illustrate the present invention, and are not limitations on the implementation methods of the present invention. For ordinary technicians in the relevant field, other different forms of changes or modifications can be made on the basis of the above description. It is impossible to list all the implementation methods here. All obvious changes or modifications derived from the technical solution of the present invention are still within the protection scope of the present invention.

Claims

1. PAMAM-mediated labeling of small molecule compounds, characterized in that: It consists of a small molecule, PAMAM and a marker. The small molecule has an active group or a derivative group that can react with an amino group under certain activation conditions to form a covalent bond; the marker has a succinimide group.

2. The PAMAM-mediated labeled small molecule compound according to claim 1, characterized in that: The molecular weights of the PAMAM are 517D, 1430D, 3256D and 6909D, corresponding to different surface amino group numbers of 4, 8, 16 and 32.

3. The PAMAM-mediated labeled small molecule compound according to claim 1, characterized in that: Small molecules with active groups include progesterone, testosterone, estradiol, vitamin D or vitamin B12.

4. The PAMAM-mediated labeled small molecule compound according to claim 1, characterized in that: The label includes acridinium ester, acridinium sulfonamide, terpyridine ruthenium or FITC.

5. A method for synthesizing a PAMAM-mediated labeled small molecule compound, characterized in that: include: First, a small molecule is reacted with PAMAM in a certain ratio, and then the label is reacted with the remaining amino group to obtain a PAMAM-mediated labeled small molecule compound.

6. The method for synthesizing a PAMAM-mediated labeled small molecule compound according to claim 5, characterized in that: When the small molecule is estradiol E2-NHS carrying an NHS group activation structure, the synthesis method comprises: reacting according to a molar ratio of E2-NHS:PAMAM-4NH2=0.9:1, reacting for 1 hour, adding a marker acridinium ester or terpyridine ruthenium AE / Ru according to a molar ratio of AE / Ru:PAMAM=3:1, and using a glycine buffer to provide free amino groups -NH2 with a molecular weight of 4 times that of PAMAM to terminate the reaction.

7. The method for synthesizing a PAMAM-mediated labeled small molecule compound according to claim 6, characterized in that: During the reaction, PAMAM-4NH2 was diluted to 1 mg / ml with PBS buffer at pH 7.5-8.5, E2-NHS was dissolved to a concentration of 5 mg / ml with DMF, and acridinium ester or terpyridine ruthenium AE / Ru was dissolved to 5 mg / ml with DMF.

8. A method for characterizing a PAMAM-mediated labeled small molecule compound according to any one of claims 1 to 4 or a PAMAM-mediated labeled small molecule compound synthesized by the synthesis method according to any one of claims 5 to 7 in an immunochemiluminescence assay.

9. An immunochemiluminescence detection kit, characterized in that: It comprises the PAMAM-mediated labeled small molecule compound described in any one of claims 1 to 4 or the PAMAM-mediated labeled small molecule compound synthesized by the synthesis method described in any one of claims 5 to 7.

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