New method for intramolecular allosteric regulation of antibody activity

Abstract

The invention discloses a novel method for intramolecular allosteric regulation of antibody activity. The compound PEP-35 provided by the invention improves the antigen recognition activity of an antibody through a forward allosteric effect, has the advantages of simplicity and convenience in operation, low cost, strong universality and the like compared with a traditional antibody Fab region modification strategy, avoids complex molecular modification, is expected to lead a new trend of antibody activity optimization, and has a wide application prospect. The method has a wide application prospect in related fields of popularization of antibody detection technologies such as food safety detection and biological analysis.

Description

Technical Field

[0001] The present invention relates to the technical field of immunoassay, and particularly to a new method for allosteric regulation of antibody activity within a molecule. Background Art

[0002] Chemical contaminants in food usually have characteristics such as small molecular weight and diverse structures, and their bioanalysis methods are often relatively unique. Among various detection methods for small molecule harmful substances, immunoassay has become one of the preferred methods for rapid food safety detection due to its advantages of strong specificity, simple operation, low cost, etc., and is also widely used in the fields of environmental monitoring, clinical diagnosis, biological medicine, etc.

[0003] Immunoassay is based on the specific recognition of an antigen (analyte) by an antibody. As a core molecular component, the antibody is one of the key factors affecting the performance of the analysis method. The characteristics of the antibody (such as selectivity and affinity for the antigen) largely depend on the uniqueness of the immunogen used. In vitro mutagenesis of the antigen-binding fragment (Fab) of a wild-type antibody by genetic engineering technology is one of the strategies for improving antibody performance.

[0004] The functions of immunoglobulin G antibodies (IgG) are mediated by two regions, namely the Fab fragment and the crystallizable fragment (Fc). Among them, the variable region of the antibody Fab is the binding site of the antigen epitope, which is complementary to the spatial conformation of the antigen; the Fc segment of the antibody consists of a pair of C H 2 and C H 3 domains, has a relatively stable sequence and structure, does not have antigen-binding activity, but is the site for interaction with complement and cell surface IgG Fc receptors (FcR). Therefore, the use of genetic engineering technology for directed modification of antibodies mainly focuses on the Fab segment. However, since this operation involves cumbersome steps such as recombinant expression and site-directed mutagenesis, it has a long cycle, high cost, complex operation, and great uncertainty. Therefore, it is urgent to develop a simpler antibody modification method to improve its antigen-binding activity. Summary of the Invention

[0005] In order to solve the problems of complex operation, high cost and long cycle in the existing antibody activity regulation technology, the present invention provides a new method for allosteric regulation of antibody activity within a molecule.

[0006] The first object of the present invention is to provide the use of a compound having the structural formula shown in formula (Ⅰ) in improving antibody performance,

[0007]

[0008] The second object of the present invention is to provide the use of a compound having the structural formula shown in formula (Ⅰ) in preparing a positive allosteric regulator of an antibody,

[0009]

[0010] The third object of the present invention is to provide the use of the compound shown in the structural formula (I) in combination with an antibody in the preparation of a detection product.

[0011]

[0012] The fourth object of the present invention is to provide a positive allosteric modulator of an antibody.

[0013] The fifth object of the present invention is to provide the use of the positive allosteric modulator in combination with an antibody in the preparation of a detection product.

[0014] The sixth object of the present invention is to provide a composition.

[0015] In order to achieve the above objects, the present invention is realized by the following solutions:

[0016] In the prior art, the main functions played by the Fc segment of an antibody during the immune process are to exert antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cell-mediated phagocytosis (ADCP), complement-dependent cytotoxicity (CDC) and increase the half-life of IgG in vivo after binding to complement or cell surface Fc receptors. However, the applicant found in previous studies that although the Fc segment does not directly bind to the antigen, after certain polypeptides bind to the Fc, the sensitivity of the antibody to recognize small molecule haptens is significantly improved, indicating that the Fc-binding molecule may enhance the Fab-binding activity through a certain effect.

[0017] The applicant proposed in the present invention the mechanism of "Fc allosterically regulates Fab activity": a certain region of C H 2 and C H 3 of the Fc serves as an allosteric site, and the variable region of the Fab serves as an orthosteric site. After the allosteric regulatory molecule binds to the allosteric site, it triggers an intramolecular long-range conformational conduction from the Fc to the Fab direction, which is conducted to the variable region of the Fab and causes a conformational change in it, thereby resulting in a change in the antigen-binding activity of the Fab.

[0018] In the present invention, the allosteric regulatory molecule refers to a compound having the function of an allosteric modulator, which is PEP-35, and its structural formula is shown in formula (I).

[0019]

[0020] The present invention aims to open up a new path for regulating the activity of Fab based on Fc in the fields of food safety immunoassay, antibody drug activity regulation, etc. Due to the high conservation of the antibody Fc sequence, the research results may have general methodological significance.

[0021] The present invention claims the following:

[0022] Use of a compound having a structural formula as shown in formula (I) in improving antibody performance

[0023]

[0024] Preferably, the antibody performance includes at least one of the affinity of the antibody, the detection sensitivity of the antibody, or the stability of the antibody.

[0025] Preferably, the compound uses the Fc segment of the antibody as an allosteric site.

[0026] Preferably, the antibody is an antibody against a small molecule compound.

[0027] More preferably, the small molecule compound is a small molecule organic compound.

[0028] More preferably, the antibody includes any one or several of anti-sulfonylurea antibody, anti-phenolphthalein antibody, or anti-bis(4-hydroxyphenyl)butane antibody.

[0029] Use of a compound having a structural formula as shown in formula (I) in preparing a positive allosteric modulator of an antibody

[0030]

[0031] Preferably, the compound uses the Fc segment of the antibody as an allosteric site.

[0032] Preferably, the antibody is an antibody against a small molecule compound.

[0033] More preferably, the small molecule compound is a small molecule organic compound.

[0034] More preferably, the antibody includes any one or several of anti-sulfonylurea antibody, anti-phenolphthalein antibody, or anti-bis(4-hydroxyphenyl)butane antibody.

[0035] Use of a compound having a structural formula as shown in formula (I) in combination with an antibody in preparing a detection product

[0036]

[0037] Preferably, the compound uses the Fc segment of the antibody as an allosteric site.

[0038] Preferably, the antibody is an antibody against a small molecule compound.

[0039] More preferably, the small molecule compound is a small molecule organic compound.

[0040] More preferably, the antibody includes any one or several of anti-sulfonylurea antibody, anti-phenolphthalein antibody, or anti-bis(4-hydroxyphenyl)butane antibody.

[0041] A positive allosteric modulator of an antibody, comprising a compound represented by the structural formula as shown in formula (I),

[0042]

[0043] Preferably, the allosteric site of the compound is the Fc segment of the antibody.

[0044] Preferably, the antibody is an antibody against a small molecule compound.

[0045] More preferably, the small molecule compound is a small molecule organic compound.

[0046] More preferably, the antibody comprises any one or more of an anti-sulfonylurea antibody, an anti-phenolphthalein antibody, or an anti-bisacodyl antibody.

[0047] Use of the positive allosteric modulator in combination with an antibody in the preparation of a detection product.

[0048] Preferably, the antibody is an antibody against a small molecule compound.

[0049] More preferably, the small molecule compound is a small molecule organic compound.

[0050] More preferably, the antibody comprises any one or more of an anti-sulfonylurea antibody, an anti-phenolphthalein antibody, or an anti-bisacodyl antibody.

[0051] A composition, comprising a compound represented by the structural formula as shown in formula (I) or the positive allosteric modulator, and an antibody;

[0052]

[0053] Preferably, the antibody is an antibody against a small molecule compound.

[0054] More preferably, the small molecule compound is a small molecule organic compound.

[0055] More preferably, the antibody comprises any one or more of an anti-sulfonylurea antibody, an anti-phenolphthalein antibody, or an anti-bisacodyl antibody.

[0056] Use of the composition in the preparation of a detection product.

[0057] A detection product, comprising a compound represented by the structural formula as shown in formula (I) or the positive allosteric modulator, and an antibody;

[0058]

[0059] Preferably, the antibody is an antibody against a small molecule compound.

[0060] More preferably, the small molecule compound is a small molecule organic compound.

[0061] More preferably, the antibody includes any one or more of anti-sulfonylurea antibody, anti-phenolphthalein antibody or anti-bis(4-hydroxyphenyl)butane antibody.

[0062] Preferably, the detection product further includes ELISA reagent.

[0063] A method for regulating the activity of antibody Fc allosterically modulating Fab, treating the antibody with the positive allosteric modulator.

[0064] Preferably, the antibody is an antibody against a small molecule compound.

[0065] More preferably, the small molecule compound is a small molecule organic compound.

[0066] More preferably, the antibody includes any one or more of anti-sulfonylurea antibody, anti-phenolphthalein antibody or anti-bis(4-hydroxyphenyl)butane antibody.

[0067] Compared with the prior art, the present invention has the following beneficial effects:

[0068] The compound PEP-35 provided by the present invention enhances the activity of the antibody recognizing the antigen through positive allosteric effect. Compared with the traditional antibody Fab region modification strategy, it has the advantages of simple operation, low cost and strong universality, avoiding complex molecular modification, and is expected to lead a new trend in antibody activity optimization, and has a wide application prospect in related fields where antibody detection technologies such as food safety detection and biological analysis are popularized. Description of the Drawings

[0069] Figure 1 It is the HPLC detection result diagram of PEP-35.

[0070] Figure 2 It is the mass spectrometry (MS) detection result diagram of PEP-35.

[0071] Figure 3 It is the schematic diagram of the binding region of the molecular docking of PEP-35 and anti-sulfonylurea antibody.

[0072] Figure 4 It is the schematic diagram of the interaction of the molecular docking of PEP-35 and anti-sulfonylurea antibody.

[0073] Figure 5 It is the detection result diagram of the influence of PEP-35 on the inhibition rate of anti-sulfonylurea antibody at different concentrations.

[0074] Figure 6 It is the detection result diagram of the influence of PEP-35 on the sensitivity of anti-sulfonylurea antibody at the concentrations of 0.7 mM and 1.5 mM.

[0075] Figure 7 It is a test result graph of the influence of PEP-35 on the stability of sulfonylurea antibodies.

[0076] Figure 8 It is a test result graph of the influence of PEP-35 on the sensitivity of other antibodies; A is anti-phenolphthalein antibody; B is anti-bis(4-hydroxyphenyl)butane antibody. Specific embodiments

[0077] The present invention will be further elaborated in detail below in conjunction with the accompanying drawings of the specification and specific embodiments. The embodiments are only used to explain the present invention and are not used to limit the scope of the present invention. The test methods used in the following embodiments are all conventional methods unless otherwise specified; the materials, reagents, etc. used are all reagents and materials that can be obtained from commercial channels unless otherwise specified.

[0078] Example 1 Synthesis, identification and functional analysis of PEP-35

[0079] 1. Synthesis and identification of PEP-35

[0080] The structural formula of PEP-35 is shown in formula (Ⅰ). It is a cyclic peptide structure as a whole, and the side chain contains various functional groups, including indole ring, benzene ring, N-propylguanidine, imidazole ring, phenol, amide group and thioether, etc.

[0081]

[0082] PEP-35 was synthesized by Shanghai Qiangyao Biotechnology Co., Ltd., and its structure was identified by HPLC and mass spectrometry (MS) techniques. The results are shown in Figure 1 and Figure 2 respectively. The identification results are consistent with its structural formula, indicating that the synthesis of PEP-35 was successful.

[0083] 2. Molecular docking

[0084] The structure of PEP-35 was optimized using Gaussian 09 software, and energy minimization was completed at the B3LYP / 6-31G(d) basis set level to obtain a ligand conformation for molecular docking.

[0085] Using PEP-35 as a ligand and an anti-sulfonylurea antibody (i.e., anti-SUs mAb in the prior art "Broad-specific immunochromatography for simultaneous detection of various sulfonylureas in adulterated multi-herbal tea (DOI: 10.1016 / j.foodchem.2021.131055)") as the receptor of PEP-35, a full-length anti-sulfonylurea antibody model was constructed using homology modeling. The receptor and ligand were preprocessed using AutoDock4.2.6 software. The water was removed and hydrogen was added to the receptor structure, and the rotatable bonds of the ligand were defined to generate the corresponding pdbqt file. The global molecular docking of the full-length anti-SUs mAb and PEP-35 was performed using the Lamarckian genetic algorithm to generate multiple docking conformations, and the molecular docking model with the highest binding free energy was selected for analysis.

[0086] The binding region of PEP-35 and anti-SUs mAb in molecular docking is as Figure 3 shown. PEP-35 mainly binds to the lower hinge region of anti-SUs mAb, and its binding pocket is composed of four regions: C H 1, C H 2, CL, and the hinge region. PEP-35 is inserted into the binding cavity in a horizontal manner. Its phenol group is located on the left side of the binding cavity, and the hydroxyl group on the phenol points to the hinge region; the indole and thioether groups are located on the right side of the binding cavity. Among them, the benzene ring on the indole points to the CL region, and the sulfur atom on the thioether points to C H 1 region; the imidazole ring points to C H 2 region. In addition, the benzene ring on the side chain of PEP-35 is exposed outside the binding pocket and is in a vertically upward manner. Overall, the binding mode of PEP-35 is similar to the "T" binding type.

[0087] There is a weak interaction between PEP-35 and anti-SUs mAb. The interaction situation of their molecular docking is as Figure 4 shown. In the N-propylguanidine group of the side chain of PEP-35, a conventional hydrogen bond is formed between the amino group on the guanidine group and the L-Ser208 amino acid residue of the antibody, and the bond length is In addition, there is also a conventional hydrogen bond between the amino group in an amide group on the main chain of PEP-35 and the L-Asp213 amino acid residue of the antibody, and the bond length is These hydrogen bonds are one of the stronger types of weak interactions, indicating that the L-Ser208 and L-Asp213 residues involved in hydrogen bond formation are key sites for recognizing PEP-35. In addition to hydrogen bonds, aromatic ring-mediated hydrophobic interactions are another key interaction. The indole ring of the PEP-35 side chain forms a Pi-Alkyl interaction with the L-Leu209 residue of anti-SUs mAb; the benzene ring of the PEP-35 side chain forms a Pi-Sulfur interaction with the L-Cys214 residue; the imidazole ring of the PEP-35 side chain forms a Pi-Alkyl interaction with the H-Lys256 residue; the phenol of the PEP-35 side chain forms Pi-Alkyl interactions with the H-Cys215 and H-Pro217 residues and a Pi-Sulfur interaction with the L-Cys214 residue. This indicates that anti-SUs mAb stabilizes the binding conformation of PEP-35 from different directions mainly through hydrophobic interactions with the groups on the PEP-35 side chain.

[0088] Example 2 Effect of Allosteric Regulatory Molecule (PEP-35) on the Performance of Anti-Sulfonylurea Antibody

[0089] 1. Effect of Allosteric Regulatory Molecule (PEP-35) on the Inhibition Rate of Anti-Sulfonylurea Antibody

[0090] When using the indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) method to measure the interaction between an antibody and an antigen, the antibody inhibition rate is one of the key indicators for evaluating the effectiveness and reliability of antibody binding to the antigen. In the ic-ELISA system, there is a competitive binding relationship among the antibody, the coating antigen, and the antigen. The level of the inhibition rate directly reflects the affinity strength between the antibody and the antigen. A higher inhibition rate indicates that the antibody can effectively bind to the antigen in the competitive reaction, thereby preventing the antibody from binding to the coating antigen.

[0091] In this example, the ic-ELISA method was used to detect the change in the inhibition rate of anti-SUs mAb in the presence of the allosteric regulatory molecule (PEP-35). The specific operation steps are as follows:

[0092] (1) Coating: Dilute the coating antigen (i.e., ACBT-OVA in the prior art "Broad-specific immunochromatography for simultaneous detection of various sulfonylureas in adulterated multi-herbal tea (DOI: 10.1016 / j.foodchem.2021.131055)") to the required concentration with coating buffer (1.69 g of Na2CO3 and 2.93 g of NaHCO3 dissolved in primary water, made up to 1 L, pH 9.6) to obtain the coating antigen dilution. Add 100 μL per well to a 96-well ELISA plate and incubate in a water bath at 37 °C for 12 h.

[0093] (2) Blocking: Discard the coating antigen dilution, wash the plate twice with PBST and pat dry. Add 120 μL of blocking solution (PBST containing 6% w / v skim milk powder) per well, incubate and block in a water bath at 37 °C for 3 h, pour out the liquid in the wells, and dry in an oven at 37 °C.

[0094] (3) Preparation of allosteric regulatory molecule-antibody complex: Centrifuge the dry powder of the allosteric regulatory molecule (PEP-35) at 4000 rpm at 4 °C for 5 min, collect the dry powder at the bottom, add PBST and mix well by shaking, sonicate for 5 min to completely dissolve it to obtain the PEP-35 mother liquor, and then dilute it with PBST buffer to obtain PEP-35 dilutions at different concentrations. Dilute anti-SUsmAb with PBST buffer to a concentration of 20 ng / mL to obtain the antibody dilution. Take 50 μL of PEP-35 dilutions at different concentrations and 50 μL of the antibody dilution, mix well by shaking, and incubate at 37 °C for 20 min to obtain the allosteric regulatory molecule-antibody complex, where the molar concentrations of PEP-35 are 17.21 μM, 86.06 μM, 172.11 μM, 430.3 μM, 860.59 μM, 1721.2 μM, and 3442.3 μM, respectively.

[0095] (4) Sample addition: Dilute the antigen drug (chlorpropamide, CAS#: 94-20-2) to 1 μg / mL with PBST buffer to obtain the drug standard dilution. Add 50 μL / well of the drug standard dilution to the even columns of a 96-well ELISA plate, add 50 μL / well of PBST buffer to the odd columns, and then add 100 μL / well of the allosteric regulatory molecule-antibody complex to each column. Use the addition of 100 μL / well of the antibody dilution as a negative control. After incubating at 37 °C for 40 min, wash the plate 5 times with the washing solution (6 g of Na2HPO4·12H2O, 16 g of NaCl, and 1.2 mL of Tween-20 dissolved in ultrapure water, made up to 2 L) and pat dry.

[0096] (5) Adding secondary antibody: Dilute the HRP-goat anti-mouse antibody (manufacturer: TransGen Biotech, product number: HS201) 5000 times with PBST, and add 100 μL to each well of the 96-well enzyme-linked immunosorbent assay (ELISA) plate. Incubate in a water bath at 37 °C for 30 min, then wash the plate 5 times with the washing solution and pat dry.

[0097] (6) Color development: Add 100 μL of the color developing solution to each well and incubate in a water bath at 37 °C for 10 min.

[0098] (7) Termination: Add 50 μL of 10% H2SO4 termination solution to each well to terminate the reaction.

[0099] (8) Reading: Use a multi-functional microplate reader to measure the absorbance value of each well at 450 nm (A 450 ).

[0100] (9) Curve fitting: Let B be the A 450 of each well with the addition of the allosteric regulatory molecule-antibody complex, and B0 be the A 450 of each well in the negative control. Use the logarithm of the PEP-35 molar concentration as the abscissa and B / B0 as the ordinate, and perform linear fitting using the Logistic function.

[0101] As Figure 5 shown, when the concentration of PEP-35 ranges from 17.21 μM to 3442.3 μM, the B / B0 value decreases significantly, from 0.987 to 0.232. This indicates that with the increase in the concentration of PEP-35, the inhibition rate of anti-SUs mAb shows a significant concentration-dependent increase, and the enhancing effect of PEP-35 on the inhibitory activity of the antibody has an obvious dose-dependence.

[0102] The above results show that PEP-35 enhances the activity of the antibody to recognize the antigen through positive allosteric regulation and improves the affinity of the antibody.

[0103] 2. Effect of the allosteric regulatory molecule (PEP-35) on the sensitivity of the anti-sulfonylurea antibody

[0104] Antibody sensitivity is a core parameter in immunoassay analysis, directly reflecting the ability of the antibody to recognize and bind to the antigen. By detecting changes in antibody sensitivity, not only can it be revealed whether the allosteric regulatory molecule can enhance or weaken its affinity for the antigen by changing the antibody conformation, but it also provides an important reference for the development of highly sensitive immunoassay methods.

[0105] In this example, the ic-ELISA method was used to detect the change in the sensitivity of anti-SUs mAb in the presence of the allosteric regulatory molecule (PEP-35). The specific operation steps are as follows:

[0106] (1) Coating: The same as step (1) in "1. Effect of Allosteric Regulatory Molecule on Inhibition Rate of Anti-Sulfonylurea Antibody" of this example.

[0107] (2) Blocking: The same as step (2) in "1. Effect of Allosteric Regulatory Molecule on Inhibition Rate of Anti-Sulfonylurea Antibody" of this example.

[0108] (3) Preparation of Allosteric Regulatory Molecule-Antibody Complex: Basically the same as step (3) in "1. Effect of Allosteric Regulatory Molecule on Inhibition Rate of Anti-Sulfonylurea Antibody" of this example. The molar concentrations of PEP-35 in the obtained allosteric regulatory molecule-antibody complexes are 0.7 mM and 1.5 mM respectively.

[0109] (4) Sample addition: Chlorpropamide was diluted with PBST to obtain drug standard dilutions with different concentrations, where the concentrations of chlorpropamide were 0.01 ng / mL, 0.1 ng / mL, 1 ng / mL, 10 ng / mL, 100 ng / mL, 1000 ng / mL, and 10000 ng / mL respectively. 50 μL / well of the drug standard dilutions with different concentrations were added to the even-numbered columns of a 96-well enzyme-linked immunosorbent assay (ELISA) plate, and 50 μL / well of PBST was added to the odd-numbered columns. Then, 100 μL / well of the allosteric regulatory molecule-antibody complex was added to each column. The addition of 100 μL / well of the antibody dilution was used as a negative control. After incubation at 37 °C for 40 min, the plate was washed 5 times with the washing solution and patted dry.

[0110] (5) Addition of secondary antibody: The same as step (5) in "1. Effect of Allosteric Regulatory Molecule on Inhibition Rate of Anti-Sulfonylurea Antibody" of this example.

[0111] (6) Color development: The same as step (6) in "1. Effect of Allosteric Regulatory Molecule on Inhibition Rate of Anti-Sulfonylurea Antibody" of this example.

[0112] (7) Termination: The same as step (7) in "1. Effect of Allosteric Regulatory Molecule on Inhibition Rate of Anti-Sulfonylurea Antibody" of this example.

[0113] (8) Reading: The same as step (8) in "1. Effect of Allosteric Regulatory Molecule on Inhibition Rate of Anti-Sulfonylurea Antibody" of this example.

[0114] (9) Standard curve establishment: The standard curve was plotted using the four-parameter fitting module of Origin 9.0 software. The logarithm of the chlorpropamide concentration was used as the abscissa, and the B / B0 value was used as the ordinate, where B is the A of each well with added chlorpropamide 450 , and B0 is the A of each well with a chlorpropamide concentration of 0 450 . The equation of the fitting function is: y = (A - D) / [1 + (x / C) B + D; where A is the A when the chlorpropamide concentration is the lowest 450, D is A when the concentration of chlorpropamide is the highest 450 , C is the half-inhibitory concentration IC 50 , generally located at the inflection point of the standard curve, B is the steepness of the curve and is called the slope factor.

[0115] Such as Figure 6 As shown, when PEP-35 was not added (i.e., mAb + 0 mM PEP-35), the IC 50 of anti-SUs mAb was 161.73 ng / mL; after adding 0.7 mM PEP-35 (i.e., mAb + 0.7 mM PEP-35), the IC 50 of anti-SUs mAb decreased to 89.40 ng / mL, and the sensitivity increased nearly 2-fold; after adding 1.5 mM PEP-35 (i.e., mAb + 1.5 mM PEP-35), the IC 50 of anti-SUs mAb decreased to 27.45 ng / mL, and the sensitivity increased up to nearly 6-fold at most.

[0116] The above results indicate that PEP-35 remotely and positively allosterically regulates the antigen recognition ability of the Fab variable region of the antibody by binding to the Fc region of the antibody, thereby improving the detection sensitivity of the antibody.

[0117] 3. Effect of the allosteric regulator molecule (PEP-35) on the thermal stability of anti-sulfonylurea antibody

[0118] Differential scanning fluorimetry (DSF) was used to determine the effect of the allosteric regulator molecule (PEP-35) on the thermal stability of the antibody. DSF is an efficient and sensitive technique that can rapidly evaluate the thermal stability of proteins by monitoring the change in the binding of a fluorescent dye to hydrophobic regions during the heating process. The specific experimental steps are as follows:

[0119] (1) Sample preparation: Dilute anti-SUs mAb and the dry powder of the allosteric regulator molecule (PEP-35) to 1 mg / mL with PBST respectively, and dilute 5000× SYPRO Orange fluorescent dye to 50× for subsequent use.

[0120] (2) Preparation of the reaction system: Prepare a mixed system according to the molar ratio of anti-SUs mAb to PEP-35 of 1:50. Add 6 μL of 50× SYPRO Orange fluorescent dye to the system, and make up to 60 μL with PBST buffer to make the final concentration of the fluorescent dye 5×. After gently mixing, incubate at room temperature in the dark for 5 min to allow the dye to fully bind to the hydrophobic region of the antibody. Replace the allosteric regulator molecule with an equal volume of PBST buffer as the control group.

[0121] (3) Sample addition: Transfer the prepared reaction system to a 96-well PCR plate, add 20 μL of the reaction system to each well, and set up 3 replicates.

[0122] (4) DSF determination: Use a real-time fluorescence quantitative PCR instrument for determination. Set the temperature range from 25 °C to 95 °C, and the heating rate is 1 °C / min. Monitor the fluorescence signal during the heating process in real time. The excitation wavelength is 490 nm, and the emission wavelength is 575 nm.

[0123] (5) Data analysis: After the experiment, use Protein Thermal Shift software to plot the melting curve of fluorescence intensity versus temperature. Determine the melting temperature (Tm value) by fitting the curve, that is, the temperature when the fluorescence intensity reaches half of the maximum value. Compare and analyze with the Tm value of the control group to evaluate the change in the Tm value of the antibody caused by the allosteric regulatory molecule, and evaluate its effect on the thermal stability of the antibody. Use GraphPad Prism software for chart drawing.

[0124] As Figure 7 shown, compared with the control group without PEP-35 (i.e., Anti-SUs mAb), when PEP-35 is added and the molar concentration of PEP-35 to anti-SUs mAb is 50:1 (i.e., Anti-SUs mAb+PEP-35), the Tm value of anti-SUs mAb increases by 1.20 °C.

[0125] The above results indicate that the allosteric regulatory molecule (PEP-35) binds to the Fc segment of the antibody, induces conformational changes, and then improves the thermal stability of the antibody.

[0126] Example 3 Effect of allosteric regulatory molecule (PEP-35) on the performance of anti-phenolphthalein antibody and anti-bis(4-hydroxyphenyl)butyric acid antibody

[0127] 1. Effect of allosteric regulatory molecule (PEP-35) on the sensitivity of anti-phenolphthalein antibody

[0128] According to the operation steps of "2. Effect of allosteric regulatory molecule (PEP-35) on the sensitivity of anti-sulfonylurea antibody" in Example 2, detect the change in the sensitivity of anti-phenolphthalein antibody (i.e., the monoclonal antibody prepared with PT-6C-BSA as the immunogen in the prior art "CN117126123A") in the presence of the allosteric regulatory molecule (PEP-35). The difference is that the coating antigen is PT-4C-OVA in the prior art "CN117126123A", the molar concentration of PEP-35 in the allosteric regulatory molecule-antibody complex is 1.5 mM, and the antigen drug is phenolphthalein (CAS#: 77-09-8).

[0129] As Figure 8As shown in A in [reference], when no PEP-35 was added (i.e., mAb + 0 mM PEP-35), the IC of the anti-phenolphthalein antibody 50 was 3.03 ng / mL; after adding 1.5 mM PEP-35 (i.e., mAb + 1.5 mM PEP-35), the IC of the anti-phenolphthalein antibody 50 decreased to 1.07 ng / mL, and the sensitivity was increased by nearly 3 times at most.

[0130] 2. Effect of allosteric regulatory molecule (PEP-35) on the sensitivity of anti-bis(4-hydroxyphenyl)propane antibody

[0131] According to the operation steps of "2. Effect of allosteric regulatory molecule (PEP-35) on the sensitivity of anti-sulfonylurea antibody" in Example 2, the change in the sensitivity of anti-bis(4-hydroxyphenyl)propane antibody (i.e., the monoclonal antibody prepared with OPH-1-BSA as the immunogen in the prior art "CN117447380A") was detected in the presence of allosteric regulatory molecule (PEP-35). The difference was that the coating antigen was OPH-2-OVA in the prior art "CN117447380A", the molar concentration of PEP-35 in the allosteric regulatory molecule-antibody complex was 1.5 mM, and the antigen drug was bis(4-hydroxyphenyl)propane (CAS#: 2943075-86-1).

[0132] As Figure 8 shown in B in [reference], when no PEP-35 was added (i.e., mAb + 0 mM PEP-35), the IC of the anti-bis(4-hydroxyphenyl)propane antibody 50 was 24.11 ng / mL; after adding 1.5 mM PEP-35 (i.e., mAb + 1.5 mM PEP-35), the IC of the anti-bis(4-hydroxyphenyl)propane antibody 50 decreased to 10.98 ng / mL, and the sensitivity was increased by nearly 2.2 times at most.

[0133] The above results show that PEP-35 enhances the antibody recognition activity of various small molecule haptens through positive allosteric effects and has excellent universality.

[0134] Based on the above results, it is shown that PEP-35 binds to the allosteric site of the antibody Fc, causing conformational conduction from Fc to Fab and remotely allosterically regulating the activity of Fab inside the antibody. PEP-35 is an allosteric regulatory molecule that has a positive allosteric regulatory effect on antibodies.

[0135] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit the protection scope of the present invention. For those of ordinary skill in the art, other different forms of changes or modifications can be made based on the above description and ideas. It is not necessary and impossible to list all the implementation manners here. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the claims of the present invention.

Claims

1. Use of a compound with a structural formula as shown in formula (I) in improving antibody performance, 2. The application according to claim 1, wherein wherein the antibody performance includes at least one of antibody affinity, antibody detection sensitivity, or antibody stability.

3. Use of a compound with a structural formula as shown in formula (I) in preparing a positive allosteric modulator of an antibody, 4. Use of a compound with a structural formula as shown in formula (I) in combination with an antibody in preparing a detection product, 5. The application according to any one of claims 1 to 4, characterized in that wherein the compound uses the Fc segment of the antibody as an allosteric site.

6. The application according to any one of claims 1 to 4, characterized in that, The antibody is an antibody against a small molecule compound.

7. The application according to claim 6, wherein The antibody includes any one or several of an anti-sulfonylurea antibody, an anti-phenolphthalein antibody, or an anti-bis(4-hydroxyphenyl)butane antibody.

8. A positive allosteric modulator of an antibody, characterized in that, including a compound with a structural formula as shown in formula (I), 9. Use of the positive allosteric modulator according to claim 8 in combination with an antibody in preparing a detection product.

10. A composition, characterized in that, including a compound with a structural formula as shown in formula (I) or the positive allosteric modulator according to claim 8, and an antibody;

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