Polymeric hrp-nanobody conjugates, methods for their preparation and use

Abstract

This invention discloses a poly-HRP-nanobody conjugate, its preparation method, and its application. The aim is to provide a method that utilizes periodate oxidation, through systematic optimization of key parameters such as reaction temperature, oxidation time, and reactant molar ratio, to generate multiple active aldehyde groups on the surface of horseradish peroxidase. Subsequently, the activated HRP reacts with the primary amine groups on the nanobody to form a high-molecular-weight functional polymer conjugate. This method leverages the small molecular weight of nanobodies, overcoming the limitation of traditional IgG antibodies in forming high-valence functional polymers due to steric hindrance during conjugation. The prepared conjugate significantly improves the detection sensitivity of immunoassays, exhibits high reaction yield, and its crude product can be directly applied to detection without purification, greatly simplifying the preparation process and reducing application costs. It provides a simple, universal, and efficient solution for developing novel high-sensitivity immunoassay reagents and belongs to the fields of biotechnology and in vitro diagnostics.

Description

Technical Field

[0001] This invention belongs to the fields of biotechnology and in vitro diagnostics, specifically relating to a polyHRP-nano antibody conjugate, its preparation method, and its application. Background Technology

[0002] Enzyme-linked immunosorbent assay (ELISA) has become the "gold standard" technology for biomolecular detection in medical diagnostics, life science research, and public safety monitoring due to its high specificity, high sensitivity, ease of operation, and stable and reliable results. With the continuous expansion of application scenarios, the requirements for detection sensitivity are becoming increasingly stringent. Therefore, developing efficient signal amplification strategies has become a core research direction for improving ELISA performance. Conjugating HRP to antibodies is the most direct strategy. The periodate oxidation method is a landmark technology in this field and remains the basis for the production of most commercial HRP-labeled reagents. This method generates aldehyde groups by oxidizing the sugar chains on the surface of HRP, enabling them to react with the amino groups on the antibody to form a conjugate.

[0003] However, this classic method faces a core bottleneck when applied to signal amplification: the molecular weight of traditional immunoglobulin G (IgG) is enormous (approximately 150 kDa), resulting in significant steric hindrance. When attempting to increase the coupling ratio of HRP to IgG to form polymers, it easily leads to a decrease in antibody activity and the formation of non-functional precipitates or aggregates, which in turn reduces the performance of the detection reagent.

[0004] Therefore, it is particularly necessary to develop high molecular weight polyHRP-nanobody conjugates to label multiple HRP molecules on a single nanobody binding site and significantly amplify the signal, and to develop a simple, efficient and cost-controllable construction method to overcome the limitations of traditional methods. Summary of the Invention

[0005] The purpose of this invention is to overcome the shortcomings of the prior art. The first objective of this invention is to provide a high molecular weight polyHRP-nanobody conjugate that can label multiple HRP molecules on a single nanobody binding site, thereby significantly amplifying the signal.

[0006] A second objective of this invention is to provide a method for preparing high molecular weight polyHRP-nanobody conjugates by efficiently polymerizing horseradish peroxidase (HRP) into nanobody conjugates using nanobodies as functional scaffolds and bridges. This method effectively activates HRP while maximizing the preservation of its enzymatic activity and promoting the formation of a stable and efficient polymer structure with the nanobody.

[0007] A third objective of this invention is to provide applications of the poly-HRP-nanobody conjugate.

[0008] Therefore, the first technical solution provided by this invention is as follows:

[0009] A method for preparing a poly-HRP-nanobody conjugate includes the following steps:

[0010] 1) Add periodate to HRP and carry out an oxidation reaction in an acidic buffer to obtain activated HRP;

[0011] 2) The HRP activated in step 1) is coupled with nanobodies in an alkaline buffer to form Schiff base-bonded HRP-nanobodies conjugates.

[0012] 3) Add a reducing agent to the Schiff base-bonded HRP-nanobody conjugate prepared in step 2) to obtain a single-bonded polyHRP-nanobody conjugate.

[0013] 4) Add glycine to the polyHRP-nanobody conjugate prepared in step 3) to block the unreacted aldehyde sites, and obtain the target product polyHRP-nanobody conjugate.

[0014] Furthermore, in the above-mentioned method for preparing polyHRP-nanobody conjugates, the periodate in step 1) is sodium periodate; the acidic buffer is an acetate buffer with pH 5.0-6.0; and the alkaline buffer in step 2) is a carbonate buffer with pH 9.0-9.5.

[0015] Furthermore, in the above-mentioned method for preparing polyHRP-nanobody conjugates, the molar ratio of periodate to HRP in step 1) is 50:1 to 400:1; and the molar ratio of HRP to nanobody in step 2) is 1:1.

[0016] Furthermore, in the above-mentioned method for preparing polyHRP-nano antibody conjugates, the oxidation reaction temperature is 1.2℃~6℃; the reaction time is 30~60min.

[0017] Furthermore, in the above-mentioned method for preparing poly-HRP-nanobody conjugates, the coupling reaction time is 2-8 hours and the temperature is room temperature.

[0018] Furthermore, in the above-mentioned method for preparing polyHRP-nanobody conjugates, the reducing agent is sodium cyanoborohydride.

[0019] The second technical solution provided by the present invention is a polyHRP-nanobody conjugate, which is prepared by the method described in the first technical solution.

[0020] Furthermore, in the above-mentioned polyHRP-nanobody conjugate, the molecular weight of the polyHRP-nanobody conjugate is greater than 250 kDa, and the molar ratio of nanobody to HRP is greater than 2:1.

[0021] This invention also provides the application of the above-mentioned poly-HRP-nanobody conjugate in the preparation of immunoassay diagnostic reagents.

[0022] Compared with the prior art, the present invention has the following significant advantages:

[0023] 1. The conjugate prepared by this invention is a high molecular weight polymer with a molecular weight far exceeding 250kDa, which enables the labeling of multiple HRP molecules on multiple nanobody binding sites, resulting in a significant signal amplification effect.

[0024] 2. The technical solution provided by this invention uses periodate to oxidize HRP under mild acidic conditions, so that the sugar chains on the surface of HRP are moderately oxidized to generate multiple active aldehyde groups, providing sufficient reaction sites for subsequent polymerization reactions, while avoiding excessive oxidation that could lead to enzyme inactivation. The oxidized and activated HRP is then mixed with nanobodies in an alkaline buffer system, allowing the active aldehyde groups on the HRP to undergo nucleophilic addition reactions with the primary amine groups on the surface of the nanobodies, forming unstable Schiff base intermediates. Because nanobodies are small (approximately 15 kDa) and have low steric hindrance, they can act as efficient "connectors," crosslinking multiple HRP molecules together to form high-molecular-weight network or chain polymers. A mild reducing agent is then added to the reaction system to selectively reduce the unstable Schiff base to stable secondary amine bonds, thereby forming structurally stable poly-HRP-nanobody conjugates. Subsequently, a blocking agent can be added to react with the remaining unreacted aldehyde groups in the system, terminating the coupling process and increasing the stability of the product.

[0025] 3. In the technical solution provided by this invention, the performance of the crude reaction product mixture can reach 89% of the purified polymer components. Therefore, this conjugate can be directly used for immunoassay without undergoing complex chromatographic separation and purification steps, greatly simplifying the production process. Attached Figure Description

[0026] Figure 1 These are electrophoretic images of the reaction products at different temperatures;

[0027] Figure 2 This is a comparison of the ELISA performance of reaction products at different temperatures;

[0028] Figure 3 These are the size exclusion chromatography elution results of the reaction products at different temperatures;

[0029] Figure 4This is a comparison of the ELISA performance of different elution components before optimization;

[0030] Figure 5 The results are from the SEC elution of the optimized product;

[0031] Figure 6 It is based on the detection performance of sandwich ELISA using polyHRP-nanobody conjugates;

[0032] Figure 7 This is a comparison chart of the ELISA performance of different elution components after optimization. Detailed Implementation

[0033] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments. It should be understood that the embodiments described herein are only for explaining the present invention and are not intended to limit the scope of the present invention.

[0034] The 3.5 mg / mL anti-GSIV nanobody HC2 solution described in the following examples was obtained by dissolving the nanobody targeting the giant salamander iridovirus disclosed in CN119331084A in PBS buffer (pH 7.4).

[0035] Example 1

[0036] This embodiment provides a polyHRP-nanobody conjugate, which is prepared by the following steps:

[0037] 1) HRP Oxidation Activation: Take 30 μL of horseradish peroxidase (HRP) solution with a concentration of 20 mg / mL, add 1.44 μL of 3M sodium acetate buffer (pH 5.2), and then add 12 μL of 227 mM sodium periodate (NaIO4) aqueous solution to make the final molar ratio of NaIO4 to HRP 200:1 and the final pH of the reaction system 5.2. React the mixture at 1.2℃ in the dark for 60 min to generate active aldehyde groups on the sugar chains of HRP. After the reaction, desalt the activated HRP using a G-25 desalting column to remove excess sodium periodate.

[0038] 2) Polymerization and Coupling Reaction: Add 58 μL of a 3.5 mg / mL anti-GSIV nanobody HC2 solution (HRP to nanobody molar ratio approximately 1:1) to the desalted activated HRP solution, and immediately add 10 μL of 0.2 M sodium carbonate buffer (pH 9.2) to adjust the reaction system to alkaline. Shake the mixture at room temperature for 0.5 h to promote the formation of Schiff bases between the aldehyde groups on the HRP and the amino groups on the nanobody, thereby crosslinking into a polymer.

[0039] 3) Reduction and Blocking: Add 1.1 μL of sodium cyanoborohydride (NaCNBH3, dissolved in 1NNaOH) solution to the above reaction solution, and continue the reaction at room temperature for 2 h to reduce the unstable Schiff base to a stable secondary amine bond. After the reaction is complete, add 11 μL of 2M glycine solution and incubate for another 30 min to block all unreacted aldehyde sites in the system, thus terminating the reaction and obtaining the polyHRP-nanobody conjugate.

[0040] Example 2

[0041] This embodiment provides a polyHRP-nanobody conjugate, the preparation method and parameters of which are basically the same as those in the previous embodiment, the only difference being that the mixture is reacted at 6°C in the dark for 60 min.

[0042] The conjugates obtained in Examples 1 and 2 were analyzed by electrophoresis. The results are as follows: Figure 1 As shown, a high molecular weight polymer (A9) was formed in the product, and the content of the polymer was higher under oxidation conditions at 6℃, resulting in a darker protein band. ELISA performance comparison: Figure 2 As shown, the slope is higher under oxidation conditions at 6℃, corresponding to higher sensitivity. The product is separated by SEC, as shown... Figure 3 As shown, the polymer peaks are more pronounced under oxidation conditions at 6℃. The elution products were then tested using ELISA. Figure 4 As shown, the ELISA signal response of polymers is significantly better than that of oligomers (corresponding to A14).

[0043] Example 3

[0044] This embodiment provides a polyHRP-nanobody conjugate, which is prepared by the following steps:

[0045] 1) HRP Oxidation Activation: Take 30 μL of horseradish peroxidase (HRP) solution with a concentration of 20 mg / mL, add 1.44 μL of 3M sodium acetate buffer (pH 5.2), and then add 12 μL of 227 mM sodium periodate (NaIO4) aqueous solution, so that the final molar ratio of NaIO4 to HRP is 200:1 and the final pH of the reaction system is 5.2. React the mixture at 6℃ in the dark for 60 min to generate active aldehyde groups on the sugar chains of HRP. After the reaction, desalt the activated HRP using a G-25 desalting column to remove excess sodium periodate.

[0046] 2) Polymerization and Coupling Reaction: Add 58 μL of 3.5 mg / mL anti-GSIV nanobody HC2 solution (HRP to nanobody molar ratio approximately 1:1) to the desalted activated HRP solution, and immediately add 10 μL of 0.2 M sodium carbonate buffer (pH 9.2) to adjust the reaction system to alkaline. Shake the mixture at room temperature for 4 h to promote the formation of Schiff bases between the aldehyde groups on HRP and the amino groups on the nanobody, thereby cross-linking into a polymer.

[0047] 3) Reduction and Blocking: Add 1.1 μL of sodium cyanoborohydride (NaCNBH3, dissolved in 1NNaOH) solution to the above reaction solution, and continue the reaction at room temperature for 4 h to reduce the unstable Schiff base to a stable secondary amine bond. After the reaction is complete, add 11 μL of 2M glycine solution and incubate for another 30 min to block all unreacted aldehyde sites in the system, thus terminating the reaction and obtaining the polyHRP-nanobody conjugate.

[0048] Analysis of the product prepared in Example 3: A small amount of the crude conjugate obtained above (hereinafter referred to as "purification-free conjugate") was analyzed by size exclusion chromatography (SEC). Results Figure 5 As shown, a large number of high molecular weight polymers (mainly A9 component) were formed in the product, with a molecular weight much greater than 250 kDa, proving that this method can efficiently prepare polymer-type couplings.

[0049] To verify the performance of the poly-HRP-nanobody conjugate prepared in this application, the following experiments and results of the establishment and performance verification of the GSIV sandwich ELISA are presented.

[0050] 1. ELISA Operation Procedure:

[0051] Coating: Coat a 96-well microplate with capture antibody (anti-GSIV polyclonal antibody, pAb) at a concentration of 2 µg / mL and incubate overnight at 4°C.

[0052] Sealing: After washing the plate, add 300 μL of 3% skim milk powder solution to each well and seal for 1 hour.

[0053] Sample loading: After washing the plate, add 100 μL / well of serially diluted GSIV virus sample and incubate for 1 h.

[0054] Detection: After washing the plate, add the "purification-free conjugate" prepared in the above steps diluted with 3% BSA (the working concentration is defined as 1 µg / mL according to the nanobody components) and incubate at 37°C for 1 h.

[0055] Color development and reading: After washing the plate, TMB substrate solution was added for color development, and then the reaction was terminated with 2MH2SO4. The absorbance (OD value) was measured at a wavelength of 450 nm.

[0056] 2. Performance Verification:

[0057] Sensitivity and linear range: Based on the optimized ELISA conditions described above, a standard curve for GSIV detection was established (e.g., Figure 6 (As shown). The results show that the method is effective in the range of 0~1×10⁻⁶. 7 The method exhibits good linearity (R² > 0.99) across the PFU / mL concentration range. The limit of detection (LOD) calculated using the three-standard-deviation method is 3.7 × 10⁻⁶. 4 The PFU / mL concentration demonstrates extremely high sensitivity.

[0058] The effectiveness of "purification-free" application: A comparison was made between the "purification-free conjugate" and the SEC-purified high molecular weight polymer component (A9) and monomer component (A14) (e.g.) Figure 7 (As shown). The results show that polymer component A9 has the highest detection sensitivity. Crucially, the detection sensitivity (slope) of the "purification-free conjugate" reaches 89% of that of the purified A9 component, demonstrating the high efficiency of the preparation method of this invention. Its crude product can be directly used for high-performance detection without purification, making it highly practical.

[0059] In summary, the method provided in this embodiment successfully prepared high-performance polyHRP-nanobody conjugates and demonstrated their significant advantages in constructing high-sensitivity ELISAs, especially their great potential for "purification-free" applications, providing a simple, efficient, and low-cost technical solution for developing novel immunodiagnostic reagents.

Claims

1. A method for preparing a polyHRP-nanobody conjugate, characterized in that, Includes the following steps: 1) Add periodate to HRP and carry out an oxidation reaction in an acidic buffer to obtain activated HRP; 2) The HRP activated in step 1) is coupled with nanobodies in an alkaline buffer to form Schiff base-bonded HRP-nanobodies conjugates. 3) Add a reducing agent to the Schiff base-bonded HRP-nanobody conjugate prepared in step 2) to obtain a single-bonded polyHRP-nanobody conjugate. 4) Add glycine to the polyHRP-nanobody conjugate prepared in step 3) to block the unreacted aldehyde sites, and obtain the target product polyHRP-nanobody conjugate. The oxidation temperature described in step 1) is 6°C; In step 1), the molar ratio of periodate to HRP is 200:1; The reaction time for the coupling reaction described in step 2) is 4 hours.

2. The method for preparing the poly-HRP-nanobody conjugate according to claim 1, characterized in that, The nanobody described is a nanobody targeting the iridovirus of the giant salamander.

3. The method for preparing the poly-HRP-nanobody conjugate according to claim 1, characterized in that, In step 1), the periodate is sodium periodate; the acidic buffer is an acetate buffer with a pH of 5.0 to 6.0; and in step 2), the alkaline buffer is a carbonate buffer with a pH of 9.0 to 9.

5.

4. The method for preparing the poly-HRP-nanobody conjugate according to claim 1, characterized in that, Step 2) The molar ratio of HRP to nanobody is 1:

1.

5. The method for preparing the poly-HRP-nanobody conjugate according to claim 1, characterized in that, The reaction time for the oxidation reaction is 30-60 minutes.

6. The method for preparing the poly-HRP-nanobody conjugate according to claim 1, characterized in that, The coupling reaction was performed at room temperature.

7. The method for preparing the poly-HRP-nanobody conjugate according to claim 1, characterized in that, The reducing agent is sodium cyanoborohydride.

8. A poly-HRP-nanobody conjugate, characterized in that, It is prepared by the method described in any one of claims 1-6.

9. The poly-HRP-nanobody conjugate according to claim 8, characterized in that, The polymeric HRP-nanobody conjugate has a molecular weight greater than 250 kDa, and the molar ratio of nanobody to HRP is greater than 2:

1.

10. The use of the polyHRP-nanobody conjugate according to claim 8 in the preparation of immunoassay diagnostic reagents.

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