Nanometer antibody 29A1 of anti-vitamin B12 antibody complex and application of nanometer antibody 29A1

Abstract

The invention discloses a nano antibody 29A1 of an anti-VB12 antibody compound. The sequence of a variable region of the nano antibody 29A1 is shown as SEQ ID NO: 1. The antibody sequence of the nano antibody 29A1 is as shown in SEQ ID NO: 2. The nano antibody can specifically recognize a compound formed by VB12 and an anti-VB12 monoclonal antibody 34C1, can be paired with a monoclonal antibody 39A1, and is developed into an immunodetection reagent based on a sandwich method principle.

Description

Technical Field

[0001] The present invention relates to a nano antibody 29A1 of an anti-vitamin B12 antibody complex and application thereof, belonging to the technical field of antibodies. Background Art

[0002] Vitamin B12 (VB12), also known as cobalamin, is an essential micronutrient for maintaining normal human metabolism and function. As a cofactor for methyltransferases, it participates in the synthesis of substances such as methionine and thymine, as well as the transfer and storage of folate within cells. It is a crucial coenzyme in DNA synthesis. Vitamin B12 deficiency impairs folate utilization, which is associated with early miscarriage and recurrent miscarriage. It can also lead to megaloblastic anemia, neuropathy (such as peripheral nerve damage, cognitive impairment, and depression), coronary artery disease, and metabolic abnormalities.

[0003] Because VB12 requires intrinsic factor for absorption in the ileum, malabsorption (such as atrophic gastritis, intestinal diseases), drug interference (such as proton pump inhibitors), or insufficient dietary intake (vegetarians) can all lead to VB12 deficiency. Testing VB12 levels is crucial for early diagnosis and intervention, so VB12 testing is of great clinical significance.

[0004] Chemiluminescence and immunofluorescence assays, based on the principle of immune binding, offer advantages in terms of labor and instrument costs, sensitivity, and detection throughput. Currently, chemiluminescence immunoassays and immunofluorescence assays are primarily used for the detection of VB12 using competitive methods. During the test, VB12 antigens are coated on the sample. Upon addition of the sample, the antigen in the sample competes with the antigen coupled to the magnetic beads or microspheres for binding to the labeled VB12 detection antibody or VB12-specific binding protein.

[0005] Due to the diversity of VB12 active forms in organisms, there are differences in their affinity with antibodies or specific binding proteins, resulting in the competitive method still having problems such as low sensitivity, poor specificity, poor reproducibility, and narrow detection range.

[0006] The sandwich method theoretically boasts higher detection sensitivity than the competition method. While the sensitivity of the competition method primarily depends on the binding capacity (affinity) of the antibody / specific binding protein, the sensitivity of the sandwich method is less affected by affinity and primarily depends on the signal gain in the presence of the analyte and the measurement error in its absence. Signal response curves reveal different locations for the hook effect in the sandwich and competition methods: the hook effect in the sandwich method occurs at high antibody / specific binding protein doses, while in the competition method, it occurs at low antibody / specific binding protein doses. Measuring a strong signal against a low background is generally much easier than measuring the difference between two weak signals. Furthermore, kinetically, using excess capture and tracer antibodies / specific binding proteins facilitates the formation of the "antibody / specific binding protein-antigen-antibody / specific binding protein" sandwich complex, thereby improving sensitivity.

[0007] Therefore, the development of a sandwich-based immunoassay for VB12 would help improve the sensitivity of VB12 detection in biological samples. However, the existing technology lacks antibodies with high affinity and specificity for the complex formed by VB12 and monoclonal antibodies. Summary of the Invention The present invention provides a nanobody 29A1 of an anti-vitamin B12 antibody complex and its application, which can effectively solve the above problems.

[0008] A nanobody 29A1 of an anti-vitamin B12 antibody complex, whose variable region sequence is shown in SEQ ID NO: 1. In some embodiments, the antibody sequence of Nanobody 29A1 of the anti-vitamin B12 antibody complex is shown in SEQ ID NO:2.

[0009] A vitamin B12 detection reagent comprises the nanobody 29A1 of the anti-vitamin B12 antibody complex.

[0010] A vitamin B12 detection kit comprises the nanobody 29A1 of the anti-vitamin B12 antibody complex.

[0011] A method for detecting vitamin B12 uses the nanobody 29A1 of the anti-vitamin B12 antibody complex to detect vitamin B12.

[0012] In some embodiments, the detection method is a sandwich method, using the anti-VB12 monoclonal antibody 34C1 as the capture antibody and the nanobody 29A1 as the detection antibody; the heavy chain of the anti-VB12 monoclonal antibody 34C1 is shown in SEQ ID NO: 3, and the light chain is shown in SEQ ID NO: 4.

[0013] The beneficial effects of the present invention are: The nanobody 29A1 provided by the present invention possesses highly specific recognition capabilities, accurately identifying specific complexes formed by VB12 and the anti-VB12 monoclonal antibody 34C1. Furthermore, the nanobody 29A1 can effectively pair with the monoclonal antibody 39A1. Based on this property, it can be further developed into a sandwich-based immunoassay reagent. This reagent utilizes the synergistic effect of the nanobody 29A1 and the monoclonal antibody 39A1 to achieve sensitive detection of target substances, thus playing an important role in clinical diagnosis, disease monitoring, and other fields. DETAILED DESCRIPTION

[0014] In order to make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention. Therefore, the detailed description of the embodiments of the present invention provided below is not intended to limit the scope of the invention claimed, but merely represents selected embodiments of the present invention.

[0015] Example 1 1. Preparation of Nanobody 29A1 Nanobodies are derived from the variable heavy-chain region (VHH) of camelids (such as alpacas) and sharks. They naturally lack light chains and consist solely of the variable heavy-chain region (VHH), which is composed of four framework regions (FR) and three complementarity-determining regions (CDRs). The CDR3 region is long and has a convex loop structure, enabling it to recognize epitopes difficult for traditional antibodies to bind (such as cryptic sites). With a molecular weight of only 12-15 kDa, it is the smallest known antigen-binding unit. Nanobodies possess unique structural and performance advantages and are widely used in the biopharmaceutical field.

[0016] 29A1 was screened from a phage library.

[0017] Alpaca nanobody phage library and library screening were constructed according to "Phage Display: A General Protocol" (Tim Clackson, Henry B. Lowman).

[0018] Anti-VB12 monoclonal antibody 34C1 was biotinylated using the ImmunoPure Sulfo-NHS-LC-Biotin Kit (ThermoFisher). 200 μl of the nanobody phage library was pre-incubated with 10 μl of streptavidin-coated magnetic beads (Yisheng Bio) and 0.5 μg of biotinylated mAb 34C1 overnight in BSA / PBS. Unbound phage were separated from the magnetic beads, and 100 μl of the beads were incubated with 100 ng of VB12, 500 ng of biotinylated mAb 34C1, and 5 μl of streptavidin-coated magnetic beads at room temperature on a shaker for 1 hour. The beads were washed five times with 0.5 ml of PBS, and the bound phage were eluted with 100 μl of HCl (pH 2.2) for 30 minutes. The eluted phage were neutralized with 1 M Tris and infected with E. coli XL1-Blue cells were used. Phage was purified. Five rounds of panning were performed. Phage ELISA was used to select clones that specifically recognized the VB12 / mAb 34C1 complex but not mAb 34C1. Clone 29A1 was screened and sequenced at Shanghai Bioengineering. The above procedures are well known to those skilled in the art; other methods known in the art can also be used to construct phage libraries and perform screening.

[0019] The variable region sequence of the Nanobody is: QVKLEESGGGSVQAGGSLRLSCAASGYTYSSRYMGWFRQAPGKEREWVASISRGGGTTYYADSVKGRFTISRDNARNTLYLQMNILKPEDTAMYYCVARRGSYWVPGIGFSYWGQGTQVTVSS (SEQ ID NO: 1) The nanobody can be fused with an Fc sequence from any species to construct a complete antibody, preferably mouse IgG1 Fc.

[0020] The antibody sequence is: QVKLEESGGGSVQAGGSLRLSCAASGYTYSSRYMGWFRQAPGKEREWVASISRGGGTTYYADSVKGRFTISRDNARNTLYLQMNILKPEDTAMYYCVARRGSYWVPGIGFSYWGQGTQVTVSSVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMNTNGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK (SEQ ID NO:2) The anti-VB12 monoclonal antibody 34C1 was prepared by a conventional method. Its heavy chain sequence is as follows: QEQLKESGGRLVTPGTPLTLTCTVSGFSLSSYTMGWVRQAPGKGLEYIGIIYASGSTYYAAWARGRFTISKTSTTVDLKMTSLTTEDTATYFCARGGYGIYGYGTYFNLWGQGTLVTVSSPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSPRPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMNTNGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK (SEQ ID NO:3) The light chain sequence is: QFVLTQPQSVSGSLGQTVSISCNRDSGNIEDYYVHWYQQHPGKAPTTVIYNDDQRPSGVPDRFSGSIDSTSNSASLTITGLLAEDEADYYCLSSDSSANPV FGGGTQLTVTSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO:4).

[0021] The heavy chain variable region sequence is: QEQLKESGGRLVTPGTPLTLTCTVSGFSLSSYTMGWVRQAPGKGLEYIGIIYASGSTYYAAWARGRFTISKTSTTVDLKMTSLTTEDTATYFCARGGYGIYGYGTYFNLWGQGTLVTVSS (SEQ ID NO: 5).

[0022] The light chain variable region sequence is: QFVLTQPQSVSGSLGQTVSISCNRDSGNIEDYYVHWYQQHPGKAPTTVIYNDDQRPSGVPDRFSGSIDSTSNSASLTITGLLAEDEADYYCLSSDSSANPVFGGGTQLTVT (SEQ ID NO: 6).

[0023] Sequence synthesis and expression Shanghai Sangon Biotechnology (Shanghai) Co., Ltd. was commissioned to synthesize the nanobody 29A1 sequence and insert it into the PTT5 vector. Following the Thermo Fisher FreeStyle™293 Expression System User Manual, the vector containing the recombinant protein sequence was transfected into HEK293-F cells, which were then cultured and the cell culture supernatant collected.

[0024] The above operations are well known to those skilled in the art, and other plasmid construction, cell transfection and culture methods known in the art may also be used to obtain the above single-chain antibody.

[0025] separation and purification Supernatant from HEK293-F cells was loaded onto a Protein A affinity chromatography column equilibrated with 20mM phosphate buffer, pH 7.4, at a linear flow rate of 200-400 cm / h to enrich the target protein. After loading, the column was eluted with 20mM acetate buffer, pH 3.5, and the eluted fractions were collected.

[0026] The eluted fractions from the avidin affinity chromatography were concentrated by ultrafiltration using a 30 kD ultrafiltration tube and buffer exchanged. The recombinant protein was stored in 20 mM phosphate buffer containing 100 mM sodium chloride, pH 7.4.

[0027] The above operations are well known to those skilled in the art, and other purification and separation methods known in the art may also be used to obtain the above recombinant protein. 2. Sandwich ELISA to detect the specificity and affinity of nanoantibody 29A1 Sandwich ELISA assays were performed using the anti-VB12 monoclonal antibody 34C1 as the coating antibody and the HRP-labeled nanobody 29A1 as the primary antibody. The small molecule VB12 was diluted 5-fold in PBS and added to the assay wells for incubation with the monoclonal antibody 34C1 for 1 hour. The assay was then incubated with the HRP-labeled nanobody 29A1 for colorimetric reading. The results are shown in Table 1.

[0028] Table 1 ELISA detection of nanobody 29A1 activity

[0029] From the detailed experimental data shown in Table 1, it can be clearly observed that the detection wells without the addition of the small molecule VB12 showed a negative reaction, which clearly indicates that there is no nonspecific binding between the nanobody 29A1 and the anti-VB12 monoclonal antibody 34C1. This important discovery provides a solid and reliable theoretical and practical foundation for the subsequent development of sandwich-based immunoassay reagents using this combination of antibodies.

[0030] As the amount of small molecule VB12 added gradually increased, the OD450 readings obtained by ELISA testing also continued to increase. This trend of change fully demonstrates that the nanobody 29A1 has the ability to specifically recognize the small molecule antibody complex formed by the binding of small molecule VB12 and anti-VB12 monoclonal antibody 34C1. More importantly, this specific recognition process is not interfered with by the monoclonal antibody 34C1 that is not bound to the small molecule VB12. In addition, the test results showed that there was a good correlation between the OD450 reading and the concentration of small molecule VB12, which means that the antibody combination can accurately and sensitively reflect the actual concentration of small molecule VB12 in the test sample. Based on these significant experimental results, it can be confirmed that the antibody combination has broad application prospects and high practical value in the development of immunoassay reagents based on the sandwich principle.

[0031] The foregoing description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Those skilled in the art will readily appreciate that various modifications and variations are possible. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention are intended to be within the scope of protection of the present invention.

Claims

1. A nanobody 29A1 of an anti-vitamin B12 antibody complex, characterized in that: The variable region sequence is shown in SEQ ID NO:

1.

2. The Nanobody 29A1 of the anti-vitamin B12 antibody complex according to claim 1, characterized in that The antibody sequence is shown in SEQ ID NO:

2.

3. A detection reagent for vitamin B12, characterized in that Nanobody 29A1 comprising the anti-vitamin B12 antibody complex of claim 1 or 2.

4. A detection kit for vitamin B12, characterized in that: Nanobody 29A1 comprising the anti-vitamin B12 antibody complex of claim 1 or 2.

5. A method for detecting vitamin B12, characterized in that: Vitamin B12 is detected using the nanobody 29A1 of the anti-vitamin B12 antibody complex according to claim 1 or 2.

6. The method for detecting vitamin B12 according to claim 5, wherein The detection method is a sandwich method, using the anti-VB12 monoclonal antibody 34C1 as the capture antibody and the nanobody 29A1 as the detection antibody; the heavy chain of the anti-VB12 monoclonal antibody 34C1 is shown in SEQ ID NO: 3, and the light chain is shown in SEQ ID NO: 4.

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