Anti-idiotypic nano antibody 28E1 of vitamin B12 and application of anti-idiotypic nano antibody 28E1

Abstract

The invention provides an anti-idiotypic nano antibody 28E1 of vitamin B12. A variable region sequence of the anti-idiotypic nano antibody 28E1 is shown as SEQ ID NO: 1. The antibody sequence of the anti-idiotype nano antibody 28E1 of the vitamin B12 is as shown in SEQ ID NO: 2. The antibody can specifically recognize the anti-VB12 monoclonal antibody 34C1 and inhibit the combination of the anti-VB12 monoclonal antibody 34C1 and VB12, and can be used for developing an immunodetection reagent based on a competition law principle.

Description

Technical Field

[0001] The present invention relates to an anti-idiotypic nano antibody 28E1 of vitamin B12 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 folic acid within cells. It is also a crucial coenzyme in DNA synthesis.

[0003] If the human body is deficient in vitamin B12, it will affect the utilization of folic acid, which is related to conditions such as early pregnancy miscarriage and recurrent miscarriage. It may also cause megaloblastic anemia, neuropathy (such as peripheral nerve damage, cognitive impairment, depression), coronary heart disease, and metabolic abnormalities. Because VB12 requires the help of intrinsic factor to be absorbed in the ileum, absorption disorders (such as atrophic gastritis, intestinal diseases), drug interference (such as proton pump inhibitors), or insufficient dietary intake (such as vegetarians) may all lead to VB12 deficiency in the human body. Testing VB12 levels is extremely critical for early diagnosis and intervention. Therefore, VB12 testing is of great clinical significance.

[0004] Chemiluminescence and immunofluorescence methods, based on the principle of immune binding, have certain advantages in terms of labor and instrument costs, sensitivity, and detection throughput. However, the existing technology lacks vitamin B12 antibodies with high detection sensitivity and strong specificity. Summary of the Invention The present invention provides an anti-idiotypic nanoantibody 28E1 of vitamin B12 and its application, which can effectively solve the above problems.

[0005] A vitamin B12 anti-idiotypic nanobody 28E1, whose variable region sequence is shown in SEQ ID NO: 1. In some embodiments, the anti-idiotypic nanobody 28E1 against vitamin B12 has an antibody sequence as shown in SEQ ID NO: 2.

[0006] A vitamin B12 detection reagent comprises the anti-vitamin B12 anti-idiotypic nanoantibody 28E1.

[0007] A vitamin B12 detection kit comprises the anti-vitamin B12 anti-idiotypic nanoantibody 28E1.

[0008] A method for detecting vitamin B12, characterized in that vitamin B12 is detected using the anti-vitamin B12 anti-idiotypic nanoantibody 28E1.

[0009] In some embodiments, the detection method is a competitive method, with the anti-VB12 monoclonal antibody 34C1 as the coating antibody and the nanobody 28E1 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.

[0010] The beneficial effects of the present invention are: This invention provides a nanobody targeting vitamin B12 (VB12), designated 28E1. This nanobody exhibits high specificity, accurately recognizing and targeting the anti-VB12 monoclonal antibody 34C1. Through this specific recognition, 28E1 effectively inhibits the binding of 34C1 to VB12. Based on this property, 28E1 exhibits significant potential for the development of competitive immunoassays. By leveraging the competitive binding mechanism between 28E1 and 34C1, highly sensitive and specific immunoassays can be designed, providing powerful tools and support for the diagnosis and treatment of VB12-related diseases. DETAILED DESCRIPTION

[0011] Example 1 1. Preparation of Nanobody 28E1 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.

[0012] 29A1 was screened from a phage library.

[0013] Alpaca nanobody phage library and library screening were constructed according to "Phage Display: A General Protocol" (Tim Clackson, Henry B. Lowman).

[0014] 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 mAb 34C1 but not the VB12 / mAb 34C1 complex. Clone 28E1 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.

[0015] The variable region sequence of the Nanobody 28E1 is: QVKLEESGGGSVQAEGSLRLSCAASGYTYRNYYMAWFRQAPGKEREAVAGISSSGTWTTYADSVKGRFTISRDNAKKTVYLQMNSLRPEDTAMYYCAARHRASRPARNWWALPYFRRYEYNYWGQGTQVTVSS (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.

[0016] The sequence of the Nanobody 28E1 is: QVKLEESGGGSVQAEGSLRLSCAASGYTYRNYYMAWFRQAPGKEREAVAGISSSGTWTTYADSVKGRFTISRDNAKKTVYLQMNSLRPEDTAMYYCAARHRASRPARNWWALPYFRRYEYNYWGQGTQVTVSSVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMNTNGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK (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).

[0017] The heavy chain variable region sequence is: QEQLKESGGRLVTPGTPLTLTCTVSGFSLSSYTMGWVRQAPGKGLEYIGIIYASGSTYYAAWARGRFTISKTSTTVDLKMTSLTTEDTATYFCARGGYGIYGYGTYFNLWGQGTLVTVSS (SEQ ID NO: 5).

[0018] The light chain variable region sequence is: QFVLTQPQSVSGSLGQTVSISCNRDSGNIEDYYVHWYQQHPGKAPTTVIYNDDQRPSGVPDRFSGSIDSTSNSASLTITGLLAEDEADYYCLSSDSSANPVFGGGTQLTVT (SEQ ID NO: 6).

[0019] Sequence synthesis and expression Shanghai Sangon Biotechnology (Shanghai) Co., Ltd. was commissioned to synthesize the nanobody 28E1 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. The cells were cultured and the cell culture supernatant was collected.

[0020] 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.

[0021] separation and purification The supernatant of HEK293-F cells expressing the target protein 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.

[0022] The eluted fractions from the avidin affinity chromatography were concentrated by ultrafiltration using a 30 kD ultrafiltration tube and subjected to buffer exchange. The recombinant protein was stored in 20 mM phosphate buffer at pH 7.4 containing 100 mM sodium chloride.

[0023] 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. Competitive ELISA to detect the specificity and affinity of nanobody 28E1 Competitive ELISA was performed using the anti-VB12 monoclonal antibody 34C1 as the coating antibody and the HRP-labeled nanobody 28E1 as the primary antibody. The small molecule VB12 was diluted 5-fold in PBS and added to the test wells for incubation with the monoclonal antibody 34C1 for 1 hour. The colorimetric reading was then incubated with the HRP-labeled nanobody 28E1. The test results are shown in the following table: Table 1 ELISA detection of nanobody 28E1 activity

[0024] The detailed experimental data presented in Table 1 clearly demonstrate that, in the absence of small molecule VB12, the assay wells exhibited a significantly strong positive reaction, demonstrating the ability of nanobody 28E1 to specifically bind to the anti-VB12 monoclonal antibody 34C1. As the amount of small molecule VB12 added increased, the OD450 readings obtained by ELISA assay showed a continuous downward trend. This change indicates that after the anti-VB12 monoclonal antibody 34C1 specifically binds to the added small molecule VB12, the site where nanobody 28E1 originally bound to monoclonal antibody 34C1 is effectively blocked, preventing stable binding. This suggests that nanobody 28E1 and small molecule VB12 compete for the VB12 binding site on monoclonal antibody 34C1, further confirming the anti-idiotypic nature of nanobody 28E1. Furthermore, the assay results in Table 1 show a good correlation with the concentration of small molecule VB12, demonstrating that this antibody combination accurately reflects the actual concentration of small molecule VB12 in the test sample. Based on this characteristic, this antibody combination is expected to be used in the development of immunoassay reagents based on the principle of competition, providing a powerful tool for detection work in related fields.

[0025] 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 of the present invention are possible. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention shall be included within the scope of protection of the present invention.

Claims

1. An anti-idiotypic nanobody 28E1 against vitamin B12, characterized in that: The variable region sequence is shown in SEQ ID NO:

1.

2. The anti-idiotypic nanobody 28E1 of vitamin B12 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 The invention comprises the anti-vitamin B12 anti-idiotypic nanobody 28E1 according to claim 1 or 2.

4. A detection kit for vitamin B12, characterized in that: The invention comprises the anti-vitamin B12 anti-idiotypic nanobody 28E1 according to claim 1 or 2.

5. A method for detecting vitamin B12, characterized in that: Vitamin B12 is detected using the anti-vitamin B12 anti-idiotypic nanoantibody 28E1 described in claim 1 or 2.

6. The method for detecting vitamin B12 according to claim 5, wherein The detection method is a competitive method, with the anti-VB12 monoclonal antibody 34C1 as the coating antibody and the nanobody 28E1 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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