Single-domain antibodies against llama igg2b fc tag and uses thereof

Abstract

The present application relates to a kind of anti-lama IgG2b Fc label single-domain antibody, the single-domain antibody has the following complement determining region: CDR1, CDR2 and CDR3;Wherein, the amino acid sequence of CDR1 is as any one of SEQ ID NO.31-60 shows, the amino acid sequence of CDR2 is as any one of SEQ ID NO.61-90 shows, the amino acid sequence of CDR3 is as any one of SEQ ID NO.91-120 shows.The single-domain antibody of anti-lama IgG2b Fc label of the present application has short development cycle, high antibody quality, has outstanding advantages in antibody affinity and targeting specificity, small molecule size, stability and reworkability, production cost and the like, and the advantage of antigen recognition specificity is outstanding.The single-domain antibody also has strong tissue penetration, can more easily penetrate tissue and cell barrier, in-depth to the diseased tissue inside play a role, can also tolerate wide range of temperature, pH value and chemical environment change, so it has advantages in a variety of application scenarios.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to single-domain antibodies with an anti-alpaca IgG2b Fc tag and their applications. Background Technology

[0002] Alpacas have garnered significant attention in immunoglobulin research due to the unique properties and diverse functions of their IgG antibodies. Traditionally, serum antibodies are classified into four subtypes: IgG1, IgG2, IgG3, and IgG4. Alpaca serum antibodies primarily consist of two types: IgG1 and IgG2. IgG1 has a structure similar to traditional antibodies, containing two heavy chains and two light chains; IgG2 lacks the CH1 and light chain domains, hence it is also known as a heavy chain antibody (hcAb). IgG1 plays a crucial role in the early stages of the immune response, rapidly recognizing and binding antigens, activating the complement system, promoting phagocytosis and clearance of pathogens by phagocytes, and exhibiting high affinity for various antigens, making a significant contribution to resisting primary infection. IgG2 is produced in large quantities, especially during secondary immune responses, and can maintain a prolonged state of immune protection. IgG2a excels in activating the complement system, efficiently triggering complement cascade reactions to kill pathogens, exhibiting strong affinity for complex antigens, and demonstrating excellent precise binding ability. IgG2b focuses on immune regulation, binding to Fc receptors on the surface of immune cells to regulate their activity, such as stimulating B cell proliferation and differentiation and promoting antibody production. It is indispensable in maintaining immune homeostasis and also has high affinity for specific antigens, enabling it to synergistically enhance immune defense with other subtypes. These subtypes of alpaca IgG antibodies cooperate and complement each other, jointly constructing a sophisticated and efficient immune defense system. This not only protects the health of alpacas themselves but also provides rich resources and inspiration for biomedical research, demonstrating potential application value in disease diagnosis, immunotherapy, and vaccine development, and contributing to further development and innovation in related fields.

[0003] Alpaca IgG2b antibodies contain an antigen-binding fragment and an Fc fragment. The Fc fragment, serving as a tag for recombinant protein antigens, has been widely used in animal immunization experiments and in vivo studies. It can promote recombinant protein dimerization, thereby enhancing immunogenicity (while the tag itself does not possess strong immunogenicity). This facilitates the expression and purification of recombinant proteins, resulting in good activity, long half-life, and good stability, contributing to better antigen-specific immune responses. Furthermore, proteins fused with the alpaca IgG2b Fc tag are easier to detect (via the binding of the Fc tag to the secondary antibody) and purified (using the Fc tag to affinity chromatography columns for protein A or protein G). Therefore, developing antibodies against alpaca IgG2b Fc has multiple important applications. In terms of detection and quantitative analysis, it can be used as a primary antibody in Western blotting and ELISA experiments, or to capture recombinant proteins containing this tag, determining the expression status and quantifying their concentration, thus aiding in the optimization of expression conditions and quality control. For purifying recombinant proteins, this technology can immobilize them on the chromatography column matrix, enabling efficient separation of recombinant proteins from mixtures through specific binding, thereby improving protein purity to meet subsequent research needs. In studying immune response mechanisms, it helps to elucidate the intrinsic principles behind the tag's enhancement of the immunogenicity of recombinant proteins, explore the interactions between antibodies, tags, and recombinant proteins, and provide a basis for developing immunotherapy strategies. In the development of alpaca nanobodies, it allows for the evaluation of the binding status of nanobodies to the IgG2b Fc tag, optimization of design and screening, and monitoring of the stability and distribution of nanobodily-recombinant protein complexes in vivo, providing crucial references for the in vivo application of nanobodies and promoting in-depth development of related research and applications.

[0004] Since Hamers et al. discovered naturally occurring heavy chain antibodies lacking light chains in camel blood in 1993, single-domain antibodies (sdAbs) have gradually replaced other small antibodies and become a hot topic in the development of novel antibody drugs. Single-domain antibodies, also known as nanobodies, are typically only about 15 kDa, approximately one-tenth the size of traditional antibodies. They contain disulfide bonds internally and have numerous hydrophilic residues on their surface, exhibiting strong resistance to heat and pH. The absence of Fc fragments and light chains in sdAbs allows them to recognize cryptic or small epitopes that traditional antibodies cannot, while avoiding complement reactions. Furthermore, single-domain antibodies possess numerous advantages, including high stability, low toxicity, high solubility, ease of target screening, ease of direct expression in prokaryotic microorganisms, and good cost-effectiveness. Sequence homology analysis showed that the VHH germline gene sequence of camel sdAb is highly homologous to that of human VH3, but CDR1 and CDR3 are slightly longer than those in humans. CDR3 protrudes outward in the tertiary structure, suggesting higher antigen binding specificity and affinity. Developing single-domain antibodies against the alpaca IgG2b Fc tag based on its excellent properties would better leverage its important functions, further enhancing its effectiveness.

[0005] With the continuous development of the biopharmaceutical industry, the demand for novel, high-performance antibodies is increasing. However, the production process also faces many significant challenges, such as high costs and long preparation cycles. The relatively limited number of alpacas in livestock farming, coupled with the high technical and cost investment required for antibody production and preparation, results in a relatively high price for alpaca IgG2b Fc-tagged antibodies, which to some extent limits their widespread adoption in large-scale applications. Therefore, efficiently screening for high-affinity anti-alpaca IgG2b Fc-tagged single-domain antibodies has broad and far-reaching significance for serving the biopharmaceutical diagnostic and therapeutic field. Summary of the Invention

[0006] In view of this, a single-domain antibody against alpaca IgG2b Fc tag and its application are provided. It has a short research and development cycle, high antibody quality, and outstanding advantages in antibody affinity and target specificity, small molecular size, stability and modifiability, and production cost. It also has strong tissue penetration and stability, which can more easily penetrate tissue and cell barriers and penetrate deep into the diseased tissue to exert its effects. It can also tolerate a wide range of temperature, pH and chemical environment changes.

[0007] A single-domain antibody against an alpaca IgG2b Fc tag, the single-domain antibody having the following complementarity-determining regions: CDR1, CDR2, and CDR3;

[0008] The amino acid sequence of CDR1 is shown in any one of SEQ ID NO.31-60, the amino acid sequence of CDR2 is shown in any one of SEQ ID NO.61-90, and the amino acid sequence of CDR3 is shown in any one of SEQ ID NO.91-120.

[0009] Preferably, the complementarity-determining region of the single-domain antibody of the anti-alpaca IgG2b Fc tag is shown in any one of (1) - (30) below:

[0010] (1) CDR1 is shown as SEQ ID NO.31, CDR2 is shown as SEQ ID NO.61, and CDR3 is shown as SEQ ID NO.91;

[0011] (2) CDR1 is shown in SEQ ID NO.32, CDR2 is shown in SEQ ID NO.62, and CDR3 is shown in SEQ ID NO.92;

[0012] (3) CDR1 is shown in SEQ ID NO.33, CDR2 is shown in SEQ ID NO.63, and CDR3 is shown in SEQ ID NO.93;

[0013] (4) CDR1 is shown in SEQ ID NO.34, CDR2 is shown in SEQ ID NO.64, and CDR3 is shown in SEQ ID NO.94;

[0014] (5) CDR1 is shown in SEQ ID NO.35, CDR2 is shown in SEQ ID NO.65, and CDR3 is shown in SEQ ID NO.95;

[0015] (6) CDR1 is shown in SEQ ID NO.36, CDR2 is shown in SEQ ID NO.66, and CDR3 is shown in SEQ ID NO.96;

[0016] (7) CDR1 is shown in SEQ ID NO.37, CDR2 is shown in SEQ ID NO.67, and CDR3 is shown in SEQ ID NO.97;

[0017] (8) CDR1 is shown in SEQ ID NO.38, CDR2 is shown in SEQ ID NO.68, and CDR3 is shown in SEQ ID NO.98;

[0018] (9) CDR1 is shown in SEQ ID NO.39, CDR2 is shown in SEQ ID NO.69, and CDR3 is shown in SEQ ID NO.99;

[0019] (10) CDR1 is shown in SEQ ID NO.40, CDR2 is shown in SEQ ID NO.70, and CDR3 is shown in SEQ ID NO.100;

[0020] (11) CDR1 is shown as SEQ ID NO.41, CDR2 is shown as SEQ ID NO.71, and CDR3 is shown as SEQ ID NO.101;

[0021] (12) CDR1 is shown as SEQ ID NO.42, CDR2 is shown as SEQ ID NO.72, and CDR3 is shown as SEQ ID NO.102;

[0022] (13) CDR1 is shown as SEQ ID NO.43, CDR2 is shown as SEQ ID NO.73, and CDR3 is shown as SEQ ID NO.103;

[0023] (14) CDR1 is shown as SEQ ID NO.44, CDR2 is shown as SEQ ID NO.74, and CDR3 is shown as SEQ ID NO.104;

[0024] (15) CDR1 is shown in SEQ ID NO.45, CDR2 is shown in SEQ ID NO.75, and CDR3 is shown in SEQ ID NO.105;

[0025] (16) CDR1 is shown in SEQ ID NO.46, CDR2 is shown in SEQ ID NO.76, and CDR3 is shown in SEQ ID NO.106;

[0026] (17) CDR1 is shown in SEQ ID NO.47, CDR2 is shown in SEQ ID NO.77, and CDR3 is shown in SEQ ID NO.107;

[0027] (18) CDR1 is shown in SEQ ID NO.48, CDR2 is shown in SEQ ID NO.78, and CDR3 is shown in SEQ ID NO.108;

[0028] (19) CDR1 is shown in SEQ ID NO.49, CDR2 is shown in SEQ ID NO.79, and CDR3 is shown in SEQ ID NO.109;

[0029] (20) CDR1 is shown in SEQ ID NO.50, CDR2 is shown in SEQ ID NO.80, and CDR3 is shown in SEQ ID NO.110;

[0030] (21) CDR1 is shown in SEQ ID NO.51, CDR2 is shown in SEQ ID NO.81, and CDR3 is shown in SEQ ID NO.111;

[0031] (22) CDR1 is shown in SEQ ID NO.52, CDR2 is shown in SEQ ID NO.82, and CDR3 is shown in SEQ ID NO.112;

[0032] (23) CDR1 is shown in SEQ ID NO.53, CDR2 is shown in SEQ ID NO.83, and CDR3 is shown in SEQ ID NO.113;

[0033] (24) CDR1 is shown in SEQ ID NO.54, CDR2 is shown in SEQ ID NO.84, and CDR3 is shown in SEQ ID NO.114;

[0034] (25) CDR1 is shown in SEQ ID NO.55, CDR2 is shown in SEQ ID NO.85, and CDR3 is shown in SEQ ID NO.115;

[0035] (26) CDR1 is shown in SEQ ID NO.56, CDR2 is shown in SEQ ID NO.86, and CDR3 is shown in SEQ ID NO.116;

[0036] (27) CDR1 is shown in SEQ ID NO.57, CDR2 is shown in SEQ ID NO.87, and CDR3 is shown in SEQ ID NO.117;

[0037] (28) CDR1 is shown in SEQ ID NO.58, CDR2 is shown in SEQ ID NO.88, and CDR3 is shown in SEQ ID NO.118;

[0038] (29) CDR1 is shown in SEQ ID NO.59, CDR2 is shown in SEQ ID NO.89, and CDR3 is shown in SEQ ID NO.119;

[0039] (30) CDR1 is shown as SEQ ID NO.60, CDR2 is shown as SEQ ID NO.90, and CDR3 is shown as SEQ ID NO.120.

[0040] Preferably, the single-domain antibody of the anti-alpaca IgG2b Fc tag has the following frame regions: FR1, FR2, FR3, and FR4; wherein the amino acid sequence of FR1 is shown in any one of SEQ ID NO. 121-150; the amino acid sequence of FR2 is shown in any one of SEQ ID NO. 151-180; the amino acid sequence of FR3 is shown in any one of SEQ ID NO. 181-210; and the amino acid sequence of FR4 is shown in any one of SEQ ID NO. 211-240.

[0041] Preferably, the frame region of the single-domain antibody is as shown in any one of (31)-(60) below:

[0042] (31) FR1 is shown in SEQ ID NO.121, FR2 is shown in SEQ ID NO.151, FR3 is shown in SEQ ID NO.181; FR4 is shown in SEQ ID NO.211;

[0043] (32) FR1 is shown in SEQ ID NO.122, FR2 is shown in SEQ ID NO.152, FR3 is shown in SEQ ID NO.182; FR4 is shown in SEQ ID NO.212;

[0044] (33) FR1 is shown in SEQ ID NO.123, FR2 is shown in SEQ ID NO.153, FR3 is shown in SEQ ID NO.183; FR4 is shown in SEQ ID NO.213;

[0045] (34) FR1 is shown in SEQ ID NO.124, FR2 is shown in SEQ ID NO.154, FR3 is shown in SEQ ID NO.184; FR4 is shown in SEQ ID NO.214;

[0046] (35) FR1 is shown in SEQ ID NO.125, FR2 is shown in SEQ ID NO.155, FR3 is shown in SEQ ID NO.185; FR4 is shown in SEQ ID NO.215;

[0047] (36) FR1 is shown in SEQ ID NO.126, FR2 is shown in SEQ ID NO.156, FR3 is shown in SEQ ID NO.186; FR4 is shown in SEQ ID NO.216;

[0048] (37) FR1 is shown in SEQ ID NO.127, FR2 is shown in SEQ ID NO.157, FR3 is shown in SEQ ID NO.187; FR4 is shown in SEQ ID NO.217;

[0049] (38) FR1 is shown in SEQ ID NO.128, FR2 is shown in SEQ ID NO.158, FR3 is shown in SEQ ID NO.188; FR4 is shown in SEQ ID NO.218;

[0050] (39) FR1 is shown in SEQ ID NO.129, FR2 is shown in SEQ ID NO.159, FR3 is shown in SEQ ID NO.189; FR4 is shown in SEQ ID NO.219;

[0051] (40) FR1 is shown in SEQ ID NO.130, FR2 is shown in SEQ ID NO.160, FR3 is shown in SEQ ID NO.190; FR4 is shown in SEQ ID NO.220;

[0052] (41) FR1 is shown in SEQ ID NO.131, FR2 is shown in SEQ ID NO.161, FR3 is shown in SEQ ID NO.191; FR4 is shown in SEQ ID NO.221;

[0053] (42) FR1 is shown in SEQ ID NO.132, FR2 is shown in SEQ ID NO.162, FR3 is shown in SEQ ID NO.192; FR4 is shown in SEQ ID NO.222;

[0054] (43) FR1 is shown in SEQ ID NO.133, FR2 is shown in SEQ ID NO.163, FR3 is shown in SEQ ID NO.193; FR4 is shown in SEQ ID NO.223;

[0055] (44) FR1 is shown in SEQ ID NO.134, FR2 is shown in SEQ ID NO.164, FR3 is shown in SEQ ID NO.194; FR4 is shown in SEQ ID NO.224;

[0056] (45) FR1 is shown in SEQ ID NO.135, FR2 is shown in SEQ ID NO.165, FR3 is shown in SEQ ID NO.195; FR4 is shown in SEQ ID NO.225;

[0057] (46) FR1 is shown in SEQ ID NO.136, FR2 is shown in SEQ ID NO.166, FR3 is shown in SEQ ID NO.196; FR4 is shown in SEQ ID NO.226;

[0058] (47) FR1 is shown in SEQ ID NO.137, FR2 is shown in SEQ ID NO.167, FR3 is shown in SEQ ID NO.197; FR4 is shown in SEQ ID NO.227;

[0059] (48) FR1 is shown in SEQ ID NO.138, FR2 is shown in SEQ ID NO.168, FR3 is shown in SEQ ID NO.198; FR4 is shown in SEQ ID NO.228;

[0060] (49) FR1 is shown in SEQ ID NO.139, FR2 is shown in SEQ ID NO.169, FR3 is shown in SEQ ID NO.199; FR4 is shown in SEQ ID NO.229;

[0061] (50) FR1 is shown in SEQ ID NO.140, FR2 is shown in SEQ ID NO.170, FR3 is shown in SEQ ID NO.200; FR4 is shown in SEQ ID NO.230;

[0062] (51) FR1 is shown in SEQ ID NO.141, FR2 is shown in SEQ ID NO.171, FR3 is shown in SEQ ID NO.201; FR4 is shown in SEQ ID NO.231;

[0063] (52) FR1 is shown in SEQ ID NO.142, FR2 is shown in SEQ ID NO.172, FR3 is shown in SEQ ID NO.202; FR4 is shown in SEQ ID NO.232;

[0064] (53) FR1 is shown in SEQ ID NO.143, FR2 is shown in SEQ ID NO.173, FR3 is shown in SEQ ID NO.203; FR4 is shown in SEQ ID NO.233;

[0065] (54) FR1 is shown in SEQ ID NO.144, FR2 is shown in SEQ ID NO.174, FR3 is shown in SEQ ID NO.204; FR4 is shown in SEQ ID NO.234;

[0066] (55) FR1 is shown in SEQ ID NO.145, FR2 is shown in SEQ ID NO.175, FR3 is shown in SEQ ID NO.205; FR4 is shown in SEQ ID NO.235;

[0067] (56) FR1 is shown in SEQ ID NO.146, FR2 is shown in SEQ ID NO.176, FR3 is shown in SEQ ID NO.206; FR4 is shown in SEQ ID NO.236;

[0068] (57) FR1 is shown in SEQ ID NO.147, FR2 is shown in SEQ ID NO.177, FR3 is shown in SEQ ID NO.207; FR4 is shown in SEQ ID NO.237;

[0069] (58) FR1 is shown in SEQ ID NO.148, FR2 is shown in SEQ ID NO.178, FR3 is shown in SEQ ID NO.208; FR4 is shown in SEQ ID NO.238;

[0070] (59) FR1 is shown in SEQ ID NO.149, FR2 is shown in SEQ ID NO.179, FR3 is shown in SEQ ID NO.209; FR4 is shown in SEQ ID NO.239;

[0071] (60) FR1 is shown in SEQ ID NO.150, FR2 is shown in SEQ ID NO.180, FR3 is shown in SEQ ID NO.210; FR4 is shown in SEQ ID NO.240.

[0072] Preferably, the amino acid sequence of the single-domain antibody is shown in any one of SEQ ID NO.1-30.

[0073] Preferably, the framework region is a heavy chain framework region, and at least a portion of the heavy chain framework region is independently derived from alpaca-derived antibodies.

[0074] Preferably, the single-domain antibody against the alpaca IgG2b Fc tag is obtained by immunizing camels with alpaca IgG2b Fc recombinant protein antigen, collecting peripheral blood cells from the immunized camels, isolating alpaca IgG2b Fc affinity lymphocytes, extracting total RNA and reverse transcribing it into cDNA, cloning the V region of the camel heavy chain antibody using Nest-PCR technology, inserting it into the phage plasmid pMES4, constructing a phage expression library, and then performing multiple rounds of screening on the alpaca IgG2b Fc antigen using phage display technology. The binding affinity of the obtained single-domain antibodies is verified by enzyme-linked immunosorbent assay, thereby screening out single-domain antibodies against the alpaca IgG2b Fc tag with high affinity activity.

[0075] Preferably, the DNA sequence of the single-domain antibody with the anti-alpaca IgG2b Fc tag is shown in any one of SEQ ID NO.241-270.

[0076] Another aspect of this application provides a fusion protein containing a single-domain antibody with an anti-alpaca IgG2b Fc tag as described above.

[0077] Furthermore, this application also provides a product containing a single-domain antibody with an anti-alpaca IgG2b Fc tag as described above or a fusion protein as described above, wherein the product is a diagnostic reagent, an in vitro diagnostic reagent, an antitumor drug, an immune cell for cell immunotherapy, or a carrier for delivering drugs at a site expressing alpaca IgG2b Fc.

[0078] This application utilizes the alpaca IgG2b Fc recombinant protein antigen to immunize camels, successfully screening for alpaca IgG2b Fc single-domain antibodies with high affinity activity. The heavy chain variable region of the single-domain antibody has three CDR regions. CDR1 and CDR3 are slightly longer than those in humans, and CDR3 protrudes outward in the tertiary structure. Therefore, the single-domain antibody exhibits higher antigen-binding specificity and affinity than traditional antibodies. Furthermore, the anti-alpaca IgG2b Fc-tagged single-domain antibody and its applications described in this application have a short development cycle, high antibody quality, and significant advantages in antibody affinity and targeting specificity, small molecule size, stability and modifiability, and production cost. In addition, the alpaca IgG2b Fc single-domain antibody also possesses strong tissue penetration and stability, enabling it to more easily penetrate tissue and cell barriers, deeply penetrate diseased tissues to exert its effects, and tolerate a wide range of temperature, pH, and chemical environmental changes. It maintains its antigen-binding activity in complex in vivo and in vitro environments, making it advantageous in various application scenarios (such as in vivo therapy and in vitro diagnostic reagent development). It has advantages in the early diagnosis of diseases (especially in cases requiring rapid and highly sensitive detection), targeted imaging and treatment of tumors (due to its good tissue penetration), and some research and application scenarios that require precise identification of subtle differences in antigens, such as the development of highly specific diagnostic reagents and novel targeted therapeutic drugs.

[0079] Furthermore, this invention provides a preparation method for the supernatant of the above-mentioned alpaca IgG2b Fc single-domain antibody, which expresses the alpaca IgG2b Fc single-domain antibody in a small amount, effectively reducing the development and production cost of alpaca IgG2b Fc antibody. The single-domain antibody has been verified by the ELISA system to have high specificity and high affinity for alpaca IgG2b Fc, indicating that the alpaca IgG2b Fc single-domain antibody obtained by this invention has further development value. Attached Figure Description

[0080] Figure 1 This is a schematic diagram of the camel immune serum titer test results for single-domain antibodies against the anti-alpaca IgG2b Fc tag.

[0081] Figure 2 This is a schematic diagram of the results of detecting PCR products using agarose gel electrophoresis.

[0082] Figure 3 This is a graph showing the results of panning the alpaca IgG2b Fc single-domain antibody phage library.

[0083] Figure 4 This is a graph of ELISA test results evaluating the enrichment level of specific antibodies.

[0084] Figure 5This is a screenshot of the screening results for single-domain antibody-positive clones that identify alpaca IgG2b Fc specificity, showing the ELISA-1-44 results.

[0085] Figure 6 This is a screenshot of the screening results for single-domain antibody-positive clones that identify alpaca IgG2b Fc specificity, showing the ELISA-45-136 results.

[0086] Figure 7 This is a screenshot of the screening results for single-domain antibody-positive clones that identify alpaca IgG2b Fc specificity, showing the ELISA-137-228 results.

[0087] Figure 8 This is a graph showing the screening results of single-domain antibody-positive clones that identify alpaca IgG2b Fc specificity, displaying the ELISA-229-312 results.

[0088] Figure 9 This is a screenshot of the screening results for single-domain antibody-positive clones that identify alpaca IgG2b Fc specificity, showing the results of ELISA-313-395.

[0089] Figure 10 This is a screenshot of the screening results for single-domain antibody-positive clones that identify alpaca IgG2b Fc specificity, showing the results of ELISA-396-475.

[0090] Figure 11 This is a graph showing the screening results of single-domain antibody-positive clones that identify alpaca IgG2b Fc specificity, displaying the results of ELISA-476-486.

[0091] Figure 12 This is a screenshot of the screening results for single-domain antibody-positive clones that identify alpaca IgG2b Fc specificity, showing the results of ELISA-487-528.

[0092] Figure 13 This is a screenshot of the screening results for single-domain antibody-positive clones that identify alpaca IgG2b Fc specificity, showing the results of ELISA-529-572.

[0093] Figure 14 This is a screenshot of the screening results for single-domain antibody-positive clones that identify alpaca IgG2b Fc specificity, showing the results of ELISA-573-582.

[0094] Figure 15 The results of the unique sequence analysis of the positive clone are shown.

[0095] Figure 16 This displays the ELISA validation results based on phage supernatant.

[0096] Figure 17This shows the ELISA validation results based on the periplasmic space expression product (VHH nanobody). Detailed Implementation

[0097] The present invention will now be described in detail with reference to specific embodiments and accompanying drawings.

[0098] This invention provides a single-domain antibody with an anti-alpaca IgG2b Fc tag, characterized in that the single-domain antibody has the following complementarity-determining regions: CDR1, CDR2, and CDR3; wherein the amino acid sequence of CDR1 is shown in any one of SEQ ID NO. 31-60, the amino acid sequence of CDR2 is shown in any one of SEQ ID NO. 61-90, and the amino acid sequence of CDR3 is shown in any one of SEQ ID NO. 91-120. Preferably, the complementarity-determining regions of CDR1, CDR2, and CDR3 of the anti-alpaca IgG2b Fc tag single-domain antibody are matched as shown in the sequence listing. Preferably, the single-domain antibody of the anti-alpaca IgG2bFc tag has the following heavy chain framework regions, namely FR1, FR2, FR3, and FR4; wherein the amino acid sequence of FR1 is as shown in any one of SEQ ID NO. 121-150; the amino acid sequence of FR2 is as shown in any one of SEQ ID NO. 151-180; the amino acid sequence of FR3 is as shown in any one of SEQ ID NO. 181-210; and the amino acid sequence of FR4 is as shown in any one of SEQ ID NO. 211-240. Preferably, the amino acid sequence of the single-domain antibody is as shown in any one of SEQ ID NO. 1-30. Preferably, the framework region is a heavy chain framework region, and at least a portion of the heavy chain framework region is independently derived from an alpaca-derived antibody. Preferably, the DNA sequence of the single-domain antibody of the anti-alpaca IgG2bFc tag is as shown in any one of SEQ ID NO. 241-270.

[0099] Compared to traditional alpaca IgG2b Fc-tagged antibodies, the functional characteristics of alpaca IgG2b Fc-tagged single-domain antibodies are mainly manifested in the following aspects. First, they exhibit a significant advantage in antigen recognition specificity. The VHH domain of alpaca IgG2b Fc-tagged single-domain antibodies possesses highly specific antigen recognition capabilities. It can recognize and bind to specific epitopes on antigens. This binding is based on the complementary amino acid sequence and spatial structure of the VHH domain, forming a tight non-covalent bond with the antigen. Due to the simpler structure of single-domain antibodies, the epitopes they bind to may be difficult for traditional antibodies to access, such as some epitopes hidden within the antigen molecule. This provides possibilities for the discovery and application of novel antigen targets. Second, they can bind with high affinity to antigens. Although there is only one antigen-binding site, the VHH domain of single-domain antibodies can bind to antigens with high affinity. Its affinity constant (Kd) can typically reach 10⁻⁻⁶. 7 - 10⁻ 9 At M and even lower levels, effective antigen binding can be achieved at lower antibody concentrations, which is significant in disease diagnosis and treatment, for example, for developing highly sensitive diagnostic reagents or highly effective therapeutic antibodies. Although the affinity of a single binding site may be relatively low, it has advantages in recognizing certain hidden antigenic epitopes that are difficult for traditional antibodies to access. Moreover, its binding specificity is high, enabling more precise identification of subtle differences on antigens, such as distinguishing different isoforms or mutants of antigens. Third, it has strong tissue penetration and stability. Due to its small molecular weight, alpaca IgG2b Fc-tagged single-domain antibodies have good tissue penetration, making it easier to penetrate tissue and cell barriers and penetrate deep into diseased tissues to exert their effects. Fourth, it also has high stability, tolerating a wide range of temperature, pH, and chemical environmental changes, and maintaining its antigen-binding activity in complex in vivo and in vitro environments, which makes it advantageous in various application scenarios (such as in vivo therapy and in vitro diagnostic reagent development). It has advantages in the early diagnosis of diseases (especially in cases requiring rapid and highly sensitive detection), targeted imaging and treatment of tumors (due to its good tissue penetration), and some research and application scenarios that require precise identification of subtle differences in antigens, such as the development of highly specific diagnostic reagents and novel targeted therapeutic drugs.

[0100] Preferably, the single-domain antibody against the alpaca IgG2b Fc tag is obtained by immunizing camels with alpaca IgG2b Fc recombinant protein antigen, collecting peripheral blood cells from the immunized camels, isolating alpaca IgG2b Fc affinity lymphocytes, extracting total RNA and reverse transcribing it into cDNA, cloning the V region of the camel heavy chain antibody using Nest-PCR technology, inserting it into the phage plasmid pMES4, constructing a phage expression library, and then performing multiple rounds of screening on the alpaca IgG2b Fc antigen using phage display technology. The binding affinity of the obtained single-domain antibodies is verified by enzyme-linked immunosorbent assay, thereby screening out single-domain antibodies against the alpaca IgG2b Fc tag with high affinity activity.

[0101] Therefore, the screening of single-domain antibodies against the anti-alpaca IgG2b Fc tag in this application is divided into the following stages: ① camel immunization and serum titer determination; ② construction of camel single-domain antibody phage display library; ③ amplification and rescue of camel single-domain antibody phage display library; ④ panning for single-domain antibodies that specifically bind to alpaca IgG2b Fc using phage display technology; ⑤ identification of positive clones of single-domain antibodies specific to alpaca IgG2b Fc; ⑥ sequence analysis of positive clones; ⑦ verification of unique clones that specifically bind to alpaca IgG2b Fc.

[0102] The following specific examples illustrate each of the above stages.

[0103] ① Camel immunization and serum titer determination

[0104] (1) Immune camels with alpaca IgG2b Fc

[0105] 1 mg of alpaca IgG2b Fc was mixed with an equal volume of Freund's adjuvant and injected subcutaneously into the camel's neck at 3-5 points. Blood was collected from the marginal ear vein before immunization. Immunization was repeated every two weeks for a total of 5 injections; 5 mL of peripheral blood was collected from the camel at each immunization. During blood collection, the camel's head was fixed to one side. The skin at the blood collection site was shaved, disinfected with 75% alcohol, and allowed to dry before blood collection. The jugular vein groove was pressed with a finger until the vein engorged. After disinfection, the needle was inserted to collect 5 mL of blood, which was then allowed to stand for serum preparation for titer evaluation. Seven days after the final immunization, 50 mL of blood was collected in an EDTA anticoagulant tube, immediately and continuously and slowly shaken to mix thoroughly, placed on ice, and transported back to the laboratory.

[0106] (2) Serum titer detection

[0107] 100 ng of alpaca IgG2b Fc antigen was coated overnight at 4°C on a 96-well high-adsorption ELISA plate. The coated antigen was washed three times with PBST, and then blocked with 200 μL of 2% BSA at room temperature for 2 hours. Serum samples collected before and after each immunization were diluted to different concentration gradients. The blocking solution was discarded, and the ELISA plate was washed 3-5 times with PBST. 100 μL of serum samples of different dilutions were added to each well and incubated at room temperature for 2 hours. The serum was then discarded, and the plate was washed 5 times with PBST. Camelid IgG-HRP was diluted with 1% BSA, and 100 μL was added to each well. The plate was incubated at room temperature in the dark for 1.5 hours. The plate was then washed 5 times, and 100 μL of TMB chromogenic solution was added to each well. The plate was incubated at room temperature for 10-30 minutes. OD450 was measured after adding stop solution. Serum titer results are shown in the appendix. Figure 1 .

[0108] (3) Isolation of blood lymphocyte samples

[0109] Lymphocytes were isolated from blood samples collected after the last immunization, using the following method:

[0110] i. Add 7 mL of Ficoll lymphocyte separation medium to each 15 mL centrifuge tube;

[0111] ii. Add an equal volume of PBS (1×) or physiological saline to fresh whole blood that has been treated with an anticoagulant (EDTA) and mix thoroughly.

[0112] iii. Take a 15 mL centrifuge tube containing lymphocyte separation medium and carefully and slowly transfer it into another 15 mL centrifuge tube containing lymphocyte separation medium, ensuring that the mixture is above the surface of the lymphocyte separation medium (i.e., do not mix the two liquids and maintain a clear interface). Centrifuge at 3,000 g for 20 min.

[0113] iv. Carefully transfer the supernatant to a 1.5 mL cell cryopreservation tube using a 1 mL pipette, label it with the animal number and the word "plasma," place it in a small cloth bag with a drawstring, and store it in a liquid nitrogen container. Carefully separate the leukocyte layer into a 15 mL centrifuge tube using a 1 mL pipette; fill the tube with PBS (1×) to 15 mL; wash the leukocytes with PBS (1×), centrifuge (3,000 g, 20 min), carefully discard the supernatant, without disturbing the cell clumps at the bottom of the tube, and recover the leukocytes in the remaining 0.1-0.2 mL of PBS.

[0114] vi. Add 5 times the volume of RNA later, gently mix the cell clumps, divide into 2 portions and place into 1.5 mL cell cryopreservation tubes, then store in liquid nitrogen.

[0115] ② Construction of camel single-domain antibody phage display library

[0116] (1) Total RNA extraction

[0117] Take a sample of frozen lymphocytes, add 1 mL of Trizol, let stand at room temperature for 10 min, then add 0.2 mL of chloroform, shake vigorously, let stand at room temperature until the solution separates into layers (about 10 min), centrifuge at 12,000 rpm, collect the upper aqueous phase, add an equal volume of isopropanol, mix well, let stand at room temperature for 15 min until nucleic acid precipitation, centrifuge at high speed to remove the supernatant, add 1 mL of 75% ethanol (prepared with DEPC water) to the RNA precipitate for washing, centrifuge at high speed to remove the supernatant, drain the water, dissolve the RNA in nuclease-free water, and take 1 μL for concentration and purity determination.

[0118] (2) cDNA synthesis

[0119] Take 1 μg of RNA and synthesize cDNA using the cDNA first-strand synthesis kit (Super Script™ III First - StrandSynthesis SuperMix (Invitrogen)). Use Oligo dT as the reverse transcription primer. Store the synthesized cDNA at -20°C.

[0120] (3) Phage display library construction and PCR amplification

[0121] Using the synthesized cDNA as a template, the V region (VHH) of the camel heavy chain antibody was amplified by Nest-PCR. The table below shows the names and sequences of the Nest-PCR primers:

[0122]

[0123] a. First-round PCR reaction system: cDNA 1 uL; Mix 12.5 uL; CALL001 0.5 uL; CALL002 0.5 uL; water to a final volume of 25 uL. First-round PCR reaction conditions: 95 ℃ for 5 min; 94 ℃ for 1 min, 57 ℃ for 1 min, 72 ℃ for 1 min, 35 cycles; 72 ℃ for 5 min.

[0124] b. Second round PCR reaction system: 40 ng of first round PCR product; 25 uL of Mix; 1 uL of VHH-Back; 1 uL of VHH-For; water to a final volume of 50 uL. Second round PCR reaction conditions: 95 ℃ for 5 min; 94 ℃ for 45 s, 60 ℃ for 45 s, 72 ℃ for 45 s, 15 cycles; 72 ℃ for 5 min.

[0125] c. After the PCR reaction, the PCR products were detected by 1.5% agarose gel electrophoresis. In the first round of PCR, the target gene fragment was located at 700 bp. The gel was excised, and the target band was recovered using the QIAEX II Gel Extraction Kit. In the second round of PCR, the target gene fragment was located at 500 bp. The target band, i.e., the VHH fragment, was then excised and recovered. The electrophoresis results are attached. Figure 2 .

[0126] (4) Enzyme digestion of phage vector and ligation of target fragment

[0127] The VHH fragment and pMES4 vector were double-digested with restriction endonucleases Eco91I and PstI, respectively. The reaction system is as follows:

[0128] a. Vector digestion system: pMES4 vector 20 μg; PstI 10 μL; Eco91I 20 μL; Cutsmart buffer 50 μL; add H2O to 500 μL.

[0129] b. Fragment digestion system: VHH fragment 5 μg; PstI 7 μL; Eco91I 14 μL; Cutsmart buffer 50 μL; add H2O to 500 μL. Digest overnight at 37 ℃. After agarose gel electrophoresis, excise and recover the digested product. Mix the digested products of the vector and VHH fragment, and ligate overnight at 16 ℃ using T4 DNA Ligase ligase.

[0130] ③ Camel single-domain antibody phage display library amplification and rescue

[0131] (1) Construction of phage display library

[0132] After purification using a PCR Purification Kit, 1 μL of the ligation product was transformed into *E. coli* TG1 competent cells. The cells were incubated at 37 °C for 2 h and serially diluted to 10¹, 10², and 10³. 300 μL of each solution was plated and incubated overnight at 37 °C. The clone count was calculated to be approximately 10⁵ clones per plate. This transformation was repeated in large-scale trials using the same method until the clone count of the library reached over 10⁸. All clones were eluted with LB elution solution, centrifuged at 5,000 g for 5 min, and the precipitate was resuspended in 2 mL of LB elution solution. An equal volume of 30% glycerol was added, and the cells were stored at -80 °C.

[0133] (2) Library diversity detection

[0134] Eighty clones from (1) were randomly selected for sequencing to detect recombination rate and assess library quality. The recombination rate of the constructed alpaca IgG2b Fc single-domain antibody library was 92.5%. The diversity of the alpaca IgG2b Fc single-domain antibody library was analyzed. Sequencing results showed that 64 single clones had 54 amino acid sequences, indicating that the constructed library had good diversity.

[0135] (3) Phage amplification and rescue

[0136] A phage library of alpaca IgG2b Fc single-domain antibody was amplified and rescued using helper phages. The preserved monoclonal library was inoculated into 100 mL of medium and cultured to the logarithmic growth phase. Helper phages with an MOI of 20 were added, and the culture was incubated at room temperature for 30 min. After low-speed centrifugation, the precipitate was resuspended in medium and inoculated into 300 mL of medium, and cultured overnight. The next day, the phage was centrifuged at 3,000 g for 30 min, the supernatant was collected, PEG was added to precipitate the phage, and the culture was incubated on ice for 30 min. It was then centrifuged at 3,000 g for 30 min, yielding the alpaca IgG2b Fc single-domain antibody phage library. After resuspending the precipitate in PBS, its titer was determined to be 2 x 10¹³ pfu / mL.

[0137] ④ Use phage display technology to screen for single-domain antibodies that specifically bind to alpaca IgG2b Fc.

[0138] (1) Washing of alpaca IgG2b Fc single-domain antibody phage library

[0139] Four rounds of panning were performed. Alpaca IgG2b Fc antigen was used to coat ELISA plates, which were incubated overnight at 4°C. The next day, rescued alpaca IgG2b Fc single-domain antibody phages were added and incubated at room temperature for 2 hours. The wells were washed 10 times with PBST, and 100 μL of triethylamine was added. The plates were incubated at room temperature for 30 minutes. The collected phages constituted the alpaca IgG2b Fc single-domain antibody phage library obtained through affinity panning. 10 μL of the phages were used to infect TG1 cells and plated for determining the number of clones after screening. The remaining phages were used for amplification. Results of each round of panning are attached. Figure 3 .

[0140] (2) Amplification and rescue of phages after screening

[0141] The amplification and rescue methods are the same as above. The obtained PBS suspension is the phage after the first round of screening. It is stored at 4°C and used for the next round of screening. The same screening steps are followed, and the amount of antigen is gradually reduced for 3-4 rounds of screening.

[0142] (3) ELISA to evaluate the enrichment of specific antibodies

[0143] ELISA plates were coated with 100 ng of alpaca IgG2b Fc antigen and incubated overnight at 4 °C. The next day, 2% BSA was added for blocking at room temperature for 1 h. The experimental groups were added with phage amplified after each wash, while the control group was added with an equal amount of wild-type phage. Incubation was performed at room temperature for 2 h. The plates were washed 10 times with PBST to remove unbound phage. HRP-labeled anti-M13 antibody was added, and incubation was performed at room temperature for 1 h. Chromogenic buffer was added, and the plates were reacted in the dark for 60 min. Absorbance was measured; the absorbance gradually increased with each wash, indicating that specific antibodies were enriched. ELISA results are attached. Figure 4 .

[0144] ⑤ Identification of positive single-domain antibody clones specific to alpaca IgG2b Fc

[0145] The experimental group ELISA plates were coated with 100 ng of alpaca IgG2b Fc antigen, while the control group ELISA plates were coated with 100 μL PBS and incubated overnight at 4 ℃. From the phage plates obtained in the third and final rounds of screening, 1056 single clones were randomly selected and placed in 1 mL of culture medium. The plates were incubated at 37 ℃ until the logarithmic growth phase, then M13 was added for 30 min at room temperature, followed by centrifugation to replace the medium with fresh medium. The next day, the supernatant was collected by centrifugation. Simultaneously, ELISA plates were blocked with 2% BSA at room temperature for 2 h. Supernatant was added to each well in both the experimental and control groups, and the plates were incubated at room temperature for 2 h. The plates were washed 5 times with PBST, and M13 antibody was added, followed by incubation at room temperature for 1 h. The plates were then washed 3-5 times with PBST. TMB substrate was added, and the reaction was carried out for 10 min. The absorbance was read on a microplate reader. A positive clone was identified when the absorbance ratio of the positive clone to the control well was greater than 2. ELISA verification results showed 582 positive clones. The positive clone screening results are attached. Figure 5 .

[0146] ⑥ Positive clone sequence analysis

[0147] Sequencing was performed on the 582 positive clones obtained. The sequencing results yielded 30 nucleotide sequences. Analysis of their amino acid sequences revealed that all 30 sequences exhibited typical single-domain antibody structures, consisting of a framework region (FR1, FR2, FR3, and FR4) and a complementarity-determining region (CDR1, CDR2, and CDR3). Unique sequence analysis of the positive clones is attached. Figure 6 .

[0148] ⑦ Validating a unique clone specific to alpaca IgG2b Fc

[0149] The experimental group ELISA plates were coated with 100 ng of alpaca IgG2b Fc antigen, and the control group ELISA plates were coated with 100 uL PBS. Incubation was performed overnight at 4 ℃. Two aliquots of 30 unique monoclonal antibodies were picked from each well and placed in 1 mL of culture medium. The plates were incubated at 37 ℃ until the logarithmic growth phase. One aliquot was inoculated with M13 at room temperature for 30 min, then centrifuged to replace the medium with fresh medium. The next day, the supernatant was collected by centrifugation. Simultaneously, the ELISA plates were blocked with 2% BSA at room temperature for 2 h. For both the experimental and control groups, supernatant was added to each well and incubated at room temperature for 2 h. The plates were washed 5 times with PBST, and M13-HRP secondary antibody was added, incubated at room temperature for 1 h. The other aliquot was induced overnight with 1 mM IPTG. The next day, the bacterial sediment was collected by centrifugation, lysed, and centrifuged at 5,000 g for 15 min. The supernatant was collected. Simultaneously, the ELISA plates were blocked with 2% BSA at room temperature for 2 h. For both the experimental and control groups, the monoclonal antibody supernatant was added to each well and incubated at room temperature for 2 h. h; wash 5 times with PBST, add VHH-HRP secondary antibody, incubate at room temperature for 1 h; after secondary antibody incubation, wash 3-5 times with PBST; add TMB substrate, react for 10 min, and read the absorbance on a microplate reader; when the ratio of absorbance to control well is greater than 2, it is considered a positive clone; ELISA verification results showed that all 30 unique clones were positive in secondary verification. This indicates that all 30 positive sequences can specifically bind to alpaca IgG2b Fc antigen. The unique clone verification results are attached. Figure 7 and attached Figure 8 .

[0150] This invention successfully screened a single-domain antibody sequence that specifically binds to alpaca IgG2b Fc using phage display technology, and analyzed the binding affinity of the alpaca IgG2b Fc single-domain antibody using ELISA.

[0151] Example 1: Polyclonal Phage ELISA Detection of Alpaca IgG2b Fc Specific Antibody Enrichment

[0152] The enrichment of alpaca IgG2bFc antibodies in the original library and the libraries rescued after rounds 1, 2, 3, and 4 enrichment was detected by polyclonal phage ELISA. The experimental group was coated with 100 ng of alpaca IgG2b Fc onto an ELISA plate, while the blank group was coated with PBS without antigen. The plates were incubated overnight at 4°C. The next day, 2% BSA was added for blocking at room temperature for 1 h. Phage supernatant collected from each round of alpaca IgG2b Fc panning and the original library was used as primary antibody for incubation. Antibodies were added to the control and experimental groups, respectively, and incubated at room temperature for 2 h. After washing 10 times with PBST, secondary antibody was added and incubated at room temperature for 1 h. TMB substrate was added, and the reaction was allowed to proceed for 10-20 min. The OD 450 absorbance was read on an ELISA reader. The results showed that after four rounds of panning, antibodies specifically binding to alpaca IgG2b Fc were effectively enriched (see attached image). Figure 4 ).

[0153] Example 2: ELISA analysis of the binding affinity of alpaca IgG2b Fc single-domain antibody

[0154] The experimental group was coated with 100 ng of alpaca IgG2b Fc protein onto an ELISA plate, while the blank group was coated with PBS. Both were incubated overnight at 4 °C. The next day, 2% BSA was added for blocking at room temperature for 1 h. The supernatant of the M13 monoclonal antibody of alpaca IgG2b Fc was used as the primary antibody for incubation. Antibodies were added to both the control and experimental groups, and incubated at room temperature for 2 h. After washing 10 times with PBST, secondary antibody was added, and incubation was continued at room temperature for 1 h. Substrate was added, and the reaction was allowed to proceed for 10-20 min. Absorbance values ​​were then read on an ELISA reader. ELISA results showed that the alpaca IgG2b Fc single-domain antibody exhibited excellent specificity for alpaca IgG2b Fc antigen, with signal values ​​in the experimental group significantly exceeding those in the blank group (see attached image). Figure 7 ).

[0155] Example 3: ELISA analysis of periplasmic expression products to determine the binding affinity of alpaca IgG2b Fc single-domain antibody.

[0156] The experimental group was coated with 100 ng of alpaca IgG2b Fc protein onto an ELISA plate, while the blank group was coated with PBS. Both were incubated overnight at 4 °C. The next day, 2% BSA was added for blocking at room temperature for 1 h. The periplasmic expression product of alpaca IgG2b Fc induced by IPTG was used as the primary antibody for incubation. Antibodies were added to both the control and experimental groups, and incubated at room temperature for 2 h. After washing 10 times with PBST, secondary antibody was added, and incubation was continued at room temperature for 1 h. Substrate was added, and the reaction was allowed to proceed for 10-20 min. Absorbance values ​​were then read on an ELISA reader. ELISA results showed that the alpaca IgG2b Fc single-domain antibody exhibited excellent specificity for alpaca IgG2b Fc antigen, with signal values ​​in the experimental group significantly exceeding those in the blank group (see attached image). Figure 8 ).

[0157] Another aspect of this application provides a fusion protein containing a single-domain antibody with an anti-alpaca IgG2b Fc tag as described above.

[0158] Furthermore, this application also provides a product containing a single-domain antibody with an anti-alpaca IgG2b Fc tag as described above or a fusion protein as described above, wherein the product is an anti-tumor drug, an immune cell for cell immunotherapy, or a carrier for delivering a drug to a site expressing alpaca IgG2b Fc.

[0159] This application utilizes the recombinant alpaca IgG2b Fc protein antigen to immunize camels, successfully screening for alpaca IgG2b Fc single-domain antibodies with high affinity. The heavy chain variable region of the single-domain antibody has three CDR regions. Among them, CDR1 and CDR3 are slightly longer than those in humans, and CDR3 protrudes outward in the tertiary structure. Therefore, the single-domain antibody exhibits higher antigen-binding specificity and affinity than traditional antibodies. Furthermore, this application addresses the problem that "existing technologies for the development of alpaca IgG2b Fc antibodies focus on traditional monoclonal antibodies. Traditional monoclonal antibody screening methods are time-consuming and labor-intensive, traditional antibodies cannot be expressed in prokaryotic systems, have large molecular weights and complex structures, poor tissue permeability, long development cycles, high production costs, and large batch-to-batch variations, which severely limit the development of alpaca IgG2b Fc antibody drugs in my country and cannot meet the diagnostic and treatment needs of Chinese patients." This application provides a single-domain antibody based on the anti-alpaca IgG2b Fc tag and its application. This antibody has a short development cycle, high quality, and significant advantages in antibody affinity and target specificity, small molecule size, stability and modifiability, and production cost.

[0160] It should be noted that the present invention is not limited to the above-described embodiments. Based on the inventive spirit of the present invention, those skilled in the art can make other changes, and these changes made in accordance with the inventive spirit of the present invention should be included within the scope of protection claimed by the present invention.

Claims

1. A single-domain antibody against an alpaca IgG2b Fc tag, characterized in that, The single-domain antibody has the following complementarity-determining regions: CDR1, CDR2, and CDR3; the complementarity-determining regions of the single-domain antibody with the anti-alpaca IgG2b Fc tag are shown below: (1) CDR1 is shown as SEQ ID NO.31, CDR2 is shown as SEQ ID NO.61, and CDR3 is shown as SEQ ID NO.

91.

2. The single-domain antibody against alpaca IgG2b Fc tag according to claim 1, characterized in that, The single-domain antibody of the anti-alpaca IgG2b Fc tag has the following frame regions: FR1, FR2, FR3, FR4; wherein, the frame regions of the single-domain antibody are as follows: (31) FR1 is shown in SEQ ID NO.121, FR2 is shown in SEQ ID NO.151, FR3 is shown in SEQ ID NO.181; FR4 is shown in SEQ ID NO.

211.

3. The single-domain antibody against alpaca IgG2b Fc tag according to claim 1, characterized in that, The amino acid sequence of the single-domain antibody is shown in SEQ ID NO.

1.

4. The single-domain antibody against alpaca IgG2b Fc tag according to claim 2, characterized in that, The framework region is a heavy chain framework region, and at least a portion of the heavy chain framework region is independently derived from camel-derived antibodies.

5. The single-domain antibody against alpaca IgG2b Fc tag according to claim 1, characterized in that, The single-domain antibody against alpaca IgG2b Fc tag was obtained by immunizing camels with alpaca IgG2b Fc recombinant protein antigen, collecting peripheral blood cells from immunized camels, isolating alpaca IgG2b Fc affinity lymphocytes, extracting total RNA and reverse transcribing it into cDNA, cloning the V region of camel heavy chain antibody using Nest-PCR technology, inserting it into phage plasmid pMES4, constructing a phage expression library, and then screening alpaca IgG2b Fc antigen through multiple rounds of phage display technology. The binding affinity of the obtained single-domain antibodies was verified by enzyme-linked immunosorbent assay, thus screening out single-domain antibodies against alpaca IgG2b Fc tag with high affinity activity.

6. The single-domain antibody against alpaca IgG2b Fc tag according to claim 1, characterized in that, The DNA sequence of the single-domain antibody with the anti-alpaca IgG2b Fc tag is shown in SEQ ID NO.241.

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