PAMAM-mediated antibody marker as well as synthesis method and application thereof

Abstract

The invention belongs to the technical field of immunochemiluminescence, and relates to a PAMAM-mediated antibody marker and a synthesis method and application thereof, the PAMAM-mediated antibody marker is composed of an antibody, PAMAM and a marker, amino groups on the surface of the antibody can be converted into sulfydryl groups, and the marker is provided with succinimide groups which can react with the amino groups. The steric hindrance of antigen-antibody reaction is reduced through PAMAM mediation, the labeling proportion is increased to the maximum extent, and a high coupling effect can be achieved with a small feeding proportion.

Description

Technical Field

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

[0002] In the development of immunochemiluminescent reagents, small molecule luminescent substances mainly include direct chemiluminescence based on acridinium ester / acridine sulfonamide (AE) and electrochemiluminescence based on terpyridine ruthenium (Ru). In both immunochemiluminescent systems, the detection antibody needs to be labeled with acridinium ester / acridine sulfonamide (AE) or terpyridine ruthenium (Ru) to achieve chemiluminescence.

[0003] The labeling and coupling of antibodies usually utilizes the primary amine group (-NH2) on the lysine side chain of the antibody. Of course, the primary amine group also exists at the N-terminus of the peptide chain. The primary amine group can form a covalent bond with the succinimidyl group (NHS) under mild conditions. Now acridinium ester / acridine sulfonamide (AE) or terpyridine ruthenium (Ru) have been succinimidized and carry NHS groups, which can quickly and easily achieve antibody labeling and coupling.

[0004] Generally speaking, antibodies contain multiple primary amine sites. An antibody molecule contains 30 to 50 lysines, and coupling may occur on nearly 20 different lysine residues. Therefore, most conventional coupling products are structurally hybrid products, with different numbers and positions of labeled molecules on each antibody; in most cases, they only meet the needs of conventional testing projects. However, with the changes in clinical needs, the concentrations that need to be detected are getting lower and lower, and the reliability is getting higher and higher. The requirements for detecting antibodies themselves and their derived coupling markers are also getting higher and higher.

[0005] As mentioned above, conventional antibody labeling techniques often involve mixing labeling molecules with antibodies, and reacting the NHS on the labeling molecules with the primary amines on the antibodies. The number of labeling molecules that are ultimately coupled to the antibody can be achieved by adjusting the molar ratio of the labeling molecules to the antibody during the reaction. The more labeling molecules there are, the more molecules that should be coupled, and vice versa. However, the ratio of labeling molecules is not the more the better, because it is limited by the number of active sites that can react on the antibody. If all active sites are occupied, no matter how many labeling molecules there are, they will not be able to produce a coupling reaction with the antibody. At the same time, more labeling molecules may bring about antigen-antibody binding steric hindrance, nonspecific reactions caused by the increased hydrophobicity brought by the labeling molecules, and so on. Therefore, a suitable ratio should be found for the detection antibodies used in each reagent, which can ensure the necessary signal strength and tolerate certain nonspecific reactions, etc.

[0006] In addition to the conventional method of improving antibody affinity to detect molecules at lower concentrations, the labeling technology for detecting antibodies is also developing in a more controllable direction. For example, in the field of antibody drugs (antibody-drug conjugate, ADC), site-specific coupling technology can be considered. The relevant technology in this field can achieve coupling at designated sites of antibodies; but most of the current site-specific coupling technologies basically involve molecular cloning methods to modify antibody sequences, and antibody sequence modification may also be the main way to achieve site-specific coupling in the future. Glycosylation site coupling can also achieve site-specific coupling, but the efficiency is low and affects the antibody structure. At the same time, the site-specific coupling method often brings quantity restrictions, and the labeling of detection antibodies often requires that an antibody can carry as many markers as possible to provide higher detection sensitivity. Summary of the invention

[0007] In view of the above technical problems, the purpose of the present invention is to provide a PAMAM-mediated antibody marker and a synthesis method and application thereof. The core idea is to try to concentrate the coupled molecules on the antibody toward one site, and at the same time, to increase the labeling ratio on one antibody as much as possible in the process, so as to improve the sensitivity of the detection reagent based on the currently fully verified antibodies, while avoiding sequence modification of the antibody.

[0008] The technical solution adopted by the present invention to achieve the technical purpose is: The present invention provides a PAMAM-mediated antibody marker, which is composed of an antibody, PAMAM and a marker, the core of which is to add more amino groups to the antibody, and the marker has a succinimide group that can react with the amino group.

[0009] Preferably, the PAMAM is a dendrimer containing multiple amino groups; the molecular weight of the PAMAM is 6909D and 14215D, corresponding to different numbers of surface amino groups of 32 and 64.

[0010] Preferably, the label includes but is not limited to acridinium ester, acridinium sulfonamide, terpyridine ruthenium or FITC.

[0011] The present invention provides a method for synthesizing a PAMAM-mediated antibody marker, comprising: Method 1: 1) After a certain proportion of amino groups on PAMAM are modified to maleimide groups (MI), a marker is added to react with the remaining amino groups to generate MI-PAMAM-markers; 2) Aby-SH, an antibody whose surface amino groups are converted to thiol groups, is mixed with MI-PAMAM-markers to form Aby-PAMAM-markers; or Method 2: First, the antibody and PAMAM are coupled to form an Aby-PAMAM structure, and then Aby-PAMAM is coupled to a marker to form an Aby-PAMAM-marker.

[0012] Preferably, PAMAM reacts with a certain proportion of chemical modification reagents so that a certain proportion of amino groups on PAMAM are modified into maleimide groups (Maleimide, MI), and the chemical modification reagents include but are not limited to sulfosuccinimide-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (Sulfo-SMCC) or succinimide-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC).

[0013] More preferably, step 1) of method 1 further comprises: removing unreacted chemical modification reagents and markers by Sephaxdex G-25 gel filtration.

[0014] Preferably, the conversion into thiol groups is carried out by reacting a thiolating reagent with amino groups on the antibody surface, wherein the thiolating reagent includes SATA or Traut's reagent.

[0015] Preferably, when the target detection substance is thyroid stimulating hormone TSH, method 1 specifically comprises: ① SATA was added to TSH antibody at 9 times the molar amount of the antibody to perform TSH antibody thiolation; SMCC was added at 10 times the molar amount of PAMAM G4-NH2, and AE was added at 30 times the molar amount of PAMAM G4-NH2, and Sephaxdex G-25 gel filtration was used with phosphate buffer as the mobile phase to remove unreacted SMCC and AE to obtain MI-PAMAM-AE; ② Calculate the amount of MI-PAMAM-AE according to 10 times the molar amount of the antibody, and add the corresponding MI-PAMAM-AE to react; after the reaction is completed, collect the product and filter it with an ultrafiltration tube with a molecular weight cutoff of 30KD. The Aby-AE structure will be retained and the unreacted MI-PAMAM-AE will be eluted; ③ Resuspend the product with PBS and repeat the centrifugation washing process 3 times to completely remove the unreacted MI-PAMAM-AE and complete the Aby-PAMAM-AE coupling.

[0016] Method 2 is to first couple the antibody and PAMAM to form an Aby-PAMAM structure, which greatly increases the number of amino groups on the antibody. Then, Aby-PAMAM is coupled using a conventional coupling method, which can also increase the number of markers coupled to the antibody.

[0017] The present invention further provides an immunochemiluminescence detection kit, comprising any of the above-mentioned PAMAM-mediated antibody markers or a PAMAM-mediated antibody marker synthesized by any of the above-mentioned synthesis methods.

[0018] Compared with the existing simple antibody and marker molecule mixing scheme: Maximize the labeling ratio: In theory, this scheme is equivalent to converting one amino group on the antibody into multiple amino groups. For PAMAM-32NH2, even if some groups need to react with chemical modification reagents, there are still quite a few amino groups that react with the markers. By increasing the ratio of MI-PAMAM-marker and antibody Aby-SH, it can be ensured that a certain proportion of the antibody amino groups are amplified in this way.

[0019] Because PAMAM is a molecule with a relatively small molecular weight and the amino group is on the surface, it reacts relatively efficiently with the chemical modification reagent and the NHS group on the marker; a smaller feed ratio can be achieved to produce a higher coupling effect.

[0020] 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.

[0021] 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.

[0022] As described in reaction principle 1), the labeled molecules will be concentrated on the PAMAM instead of being spread throughout the antibody as in existing solutions, thus reducing the steric hindrance of the antigen-antibody reaction. DETAILED DESCRIPTION

[0023] In order to more clearly illustrate the present invention, the present invention is further described in detail below in conjunction with embodiments. It should be understood by those skilled in the art that the content specifically described below is illustrative rather than restrictive, and should not be used to limit the scope of protection of the present invention.

[0024] Due to the clinical need for a high-sensitivity thyroid-stimulating hormone (hs-TSH) reagent, we used this scheme to construct the structure of TSH-PAMAM-AE and compared it with the TSH-AE structure constructed by the classic scheme in order to improve the sensitivity of the TSH reagent. The specific experimental scheme is as follows: Example PAMAM-mediated construction of TSH-PAMAM-AE 1. Thiolation of Aby-SH Antibody 1. Dialyze 1 mg, 2 mg / ml TSH antibody (Roche, 11367978103) in advance with 0.1 M phosphate buffer (PBS, pH 7.2-7.5); 2. Dissolve SATA (N-succinimidyl S-acetylthioacetate) in DMF to a concentration of 55 mM; 3. Add SATA to TSH antibody at 9 times the antibody molar amount and react at 37°C for 30 minutes; 4. Desalt the antibody using gel filtration to remove excess SATA; 5. Add 100ul of deacetylation reagent to 1ml Aby-SH to deacetylate the thiol groups (40mM NaOH, 25mM EDTA) to activate the thiol groups on the antibody.

[0025] 2. Synthesis of MI-PAMAM-AE PAMAM dendrimer G3-NH2 (PAMAM Dendrimer G3-NH2, 32NH2, Sigma, 41242-2, MW 6909D) or PAMAM dendrimer G4-NH2 (PAMAM Dendrimer G4-NH2, 64NH2, Sigma, 41,244-9, MW 14215D) can be used. The TSH antibody in this example uses PAMAM G4-NH2, which has 64 amino groups on the surface.

[0026] 1. Dilute 1 mg PAMAM G4-NH2 with 0.1 M phosphate buffer (PBS, pH 7.2-7.5) to a concentration of 1 mg / ml; dissolve SMCC with DMF to a concentration of 5 mg / ml; 2. Add SMCC at 10 times the molar amount of PAMAM G4-NH2 and react in a water bath at 30°C for 30 minutes; 3. Dissolve acridinium ester (AE, Helison) in DMF to a concentration of 5 mg / ml; 4. Add AE at 30 times the molar amount of PAMAM G4-NH2 and react in a water bath at 30°C for 30 minutes; 5. Use Sephadex G-25 gel filtration and phosphate buffer as the mobile phase to remove unreacted SMCC and AE.

[0027] 3. Coupling of Aby-PAMAM-AE 1. Quantify Aby-SH using a UV spectrophotometer and measure the absorbance at a wavelength of 280nm for protein quantification; 2. Quantification of MI-PAMAM-AE was performed by estimation, based on a PAMAM recovery rate of 75%; 3. Calculate the antibody molar mass based on the antibody molecular weight of 150kD; 4. Calculate the amount of MI-PAMAM-AE according to 10 times the molar amount of the antibody, add the corresponding MI-PAMAM-AE for reaction; react in a water bath at 30°C for 30 minutes; 5. After the reaction is completed, collect the product and filter it with an ultrafiltration tube with a molecular weight cutoff (MWCO) of 30KD. Centrifuge at a relative centrifugal force (RCF) of 4000g for 15 minutes. The Aby-AE structure will be retained, while the unreacted MI-PAMAM-AE will be eluted. 6. Resuspend the product with PBS and repeat the centrifugation washing process 3 times to completely remove unreacted MI-PAMAM-AE; 7. Aby-PAMAM-AE coupling is completed.

[0028] Comparative Example Traditional Aby-AE Conjugation to Construct TSH-AE 1. Dialyze 1 mg, 2 mg / ml TSH antibody (Roche, 11367978103) in advance with 0.1 M phosphate buffer (PBS, pH 7.2-7.5); 2. Quantify Aby-SH using a UV spectrophotometer and measure the absorbance at a wavelength of 280nm for protein quantification; 3. Dissolve acridinium ester (AE, Helison) in DMF to a concentration of 2 mg / ml; 4. Calculate the amount of AE according to 15 times the molar amount of the antibody, add the corresponding AE for reaction; react in a 30°C water bath for 30 minutes; 5. After the reaction is completed, collect the product and filter it with an ultrafiltration tube with a molecular weight cutoff (MWCO) of 30KD. Centrifuge at a relative centrifugal force (RCF) of 4000g for 15 minutes. The Aby-AE structure will be retained, while the unreacted AE will be eluted. 6. Resuspend the product with PBS and repeat the centrifugation washing process 3 times to completely remove unreacted MI-PAMAM-AE; 7. Aby-AE coupling is completed.

[0029] Test Case We constructed a TSH detection system to verify the effect of the coupling product. The detection method is carried out by immunochemiluminescence. TSH uses the sandwich method principle. The capture antibody and the detection antibody are combined with the TSH antigen in the sample; the acridinium ester and hydrogen peroxide coupled with the detection antibody generate light signals in an alkaline environment and are detected by the detector. The number of photons is positively correlated with the antigen concentration in the sample.

[0030] The test was performed on the Yingkai Shine FP2910 device. The components involved were: streptavidin 2.8um magnetic beads (Dynal M280, 0.7mg / ml, 50mM HEPES buffer, 1% BSA, pH7.0), the capture antibody was a biotin-labeled anti-TSH antibody (Roche, 10767778103, 0.5ug / ml, 100mM PBS buffer, 1% BSA, pH7.0); the detection antibody was the coupled product TSH Aby-PAMAM-AE or TSH Aby-AE complex, and the same PBS buffer was diluted to 0.3ug / ml as the working solution; the three components were 35μL, 50μL, and 50μL respectively; six samples with different TSH concentrations, the concentrations are shown in Table 1, and the sample volume was 30ul. After incubating the sample with the two antibody working solutions for 8 minutes, streptavidin magnetic beads were added, and the test was performed after incubation for another 8 minutes. The results are as follows: Table 1. Signal value detection results of different detection antibodies at different TSH concentrations

[0031] It can be seen that while maintaining the same background signal, TSH-PAMAM-AE exhibits a higher signal value than the traditional coupling method, providing a higher separation ratio and laying the foundation for a more sensitive TSH detection reagent.

[0032] 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. A PAMAM-mediated antibody marker, characterized in that The invention is composed of an antibody, PAMAM and a label. The amino group on the surface of the antibody can be converted into a thiol group, and the label has a succinimide group which can react with the amino group.

2. The PAMAM-mediated antibody marker according to claim 1, characterized in that The PAMAM is a dendrimer containing multiple amino groups; the molecular weight of the PAMAM is 6909D and 14215D, corresponding to different surface amino group numbers of 32 and 64.

3. The PAMAM-mediated antibody marker according to claim 1, characterized in that The label includes acridinium ester, acridinium sulfonamide, terpyridine ruthenium or FITC.

4. A method for synthesizing a PAMAM-mediated antibody marker, characterized in that: include: Method 1: 1) After a certain proportion of amino groups on PAMAM are modified to maleimide groups MI, a marker is added to react with the remaining amino groups to generate MI-PAMAM-markers; 2) Antibodies Aby-SH, which convert surface amino groups to thiol groups, are mixed with MI-PAMAM-markers to form Aby-PAMAM-markers; or Method 2: First, the antibody and PAMAM are coupled to form an Aby-PAMAM structure, and then Aby-PAMAM is coupled to a marker to form an Aby-PAMAM-marker.

5. The method for synthesizing a PAMAM-mediated antibody marker according to claim 4, characterized in that: In step 1) of method 1, a certain proportion of amino groups on PAMAM are modified into maleimide groups MI by reacting PAMAM with a certain proportion of chemical modification reagents, wherein the chemical modification reagents include sulfosuccinimide-4-(N-maleimidomethyl)cyclohexane-1-carboxylate or succinimide-4-(N-maleimidomethyl)cyclohexane-1-carboxylate.

6. The method for synthesizing a PAMAM-mediated antibody marker according to claim 4, characterized in that: Step 1) of method 1 also includes: removing unreacted chemical modification reagents and markers by Sephaxdex G-25 gel filtration.

7. The method for synthesizing a PAMAM-mediated antibody marker according to claim 4, characterized in that: In step 2) of method 1, the sulfhydryl groups are converted into sulfhydryl groups by reacting a sulfhydrylation reagent with amino groups on the antibody surface. The sulfhydrylation reagent includes SATA or Traut reagent.

8. The method for synthesizing a PAMAM-mediated antibody marker according to claim 4, characterized in that: When the target test substance is thyroid stimulating hormone (TSH), method 1 specifically includes: ① SATA was added to TSH antibody at 9 times the molar amount of the antibody to perform TSH antibody thiolation; SMCC was added at 10 times the molar amount of PAMAM G4-NH2, and AE was added at 30 times the molar amount of PAMAM G4-NH2, and Sephaxdex G-25 gel filtration was used with phosphate buffer as the mobile phase to remove unreacted SMCC and AE to obtain MI-PAMAM-AE; ② Calculate the amount of MI-PAMAM-AE according to 10 times the molar amount of the antibody, and add the corresponding MI-PAMAM-AE to react; after the reaction is completed, collect the product and filter it with an ultrafiltration tube with a molecular weight cutoff of 30KD. The Aby-AE structure will be retained and the unreacted MI-PAMAM-AE will be eluted; ③ Resuspend the product with PBS and repeat the centrifugation washing process 3 times to completely remove the unreacted MI-PAMAM-AE and complete the Aby-PAMAM-AE coupling.

9. Use of the PAMAM-mediated antibody marker according to any one of claims 1 to 3 or the PAMAM-mediated antibody marker synthesized by the synthesis method according to any one of claims 4 to 8 in immunochemiluminescence detection.

10. An immunochemiluminescent detection kit comprising the PAMAM-mediated antibody marker according to any one of claims 1 to 3 or the PAMAM-mediated antibody marker synthesized by the synthesis method according to any one of claims 4 to 8.

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