Polymerized antigen construct for rapid immunodetection

Abstract

The invention relates to an antigen construct for rapid immune detection, in particular to an antigen molecule for realizing dimerization or trimerization through foldon or a variant thereof. The antigen construct can enhance the conformation stability and improve the antibody binding efficiency, so that the sensitivity and shelf life stability in colloidal gold, fluorescent microspheres, ELISA (enzyme-linked immuno sorbent assay) and other in-vitro diagnosis methods in immunochromatography are remarkably improved. The construct is suitable for antibody detection of rabies, influenza, coronavirus, Brucella, tuberculosis and the like in zoonosis and HIV, HBV, HCV, syphilis and the like in human infectious diseases, and can be widely applied to clinical screening, animal quarantine, family self-test and public health emergency monitoring.

Description

Technical Field

[0001] This invention relates to the fields of biotechnology and in vitro diagnostics, and more specifically, to a multimeric antigen construct stabilized by an oligomerization domain, and its application in rapid immunoassay. Background Technology

[0002] Serological testing is one of the most common and effective methods in infectious disease prevention and control and clinical diagnosis. Immunochromatographic techniques, especially colloidal gold immunochromatography and fluorescent microsphere immunochromatography, are widely used in primary healthcare, animal disease prevention, and home testing due to their rapid detection, ease of operation, and lack of the need for large instruments. In these detection methods, the quality of the antigen as the detection element directly determines the sensitivity and specificity. Compared with traditional detection methods that rely on monoclonal or polyclonal antibodies, the use of recombinant antigens has significant advantages. On the one hand, antigens are derived from the pathogen itself and usually contain conserved immunodominant epitopes across species, making them suitable for cross-species detection in humans and animals, avoiding inconsistencies caused by host differences. On the other hand, the acquisition of recombinant antigens does not rely on complex processes such as animal immunization and hybridoma screening, allowing for rapid construction and application to the detection of new pathogens in a short period, significantly shortening the product development cycle. Therefore, antigen-based immunoassay is considered one of the most flexible and widely applicable strategies in infectious disease diagnosis.

[0003] However, currently used monomeric recombinant antigens have significant drawbacks. Many viral envelope proteins (such as rabies G protein and influenza HA protein) are natively oligomers (dimers or trimers), and their key antibody epitopes depend on the correct spatial conformation. When expressed in monomeric form, conformational epitopes are easily lost, leading to insufficient detection sensitivity in low-titer sera, high false-negative rates, and monomeric antigens are prone to inactivation or aggregation at room temperature or under thermal stress, making it difficult to meet the 18-24 month shelf-life requirement of commercial diagnostic reagents. Foldon (derived from phage T4 fibritin) and GCN4-pII (artificial dimer zipper) are known oligomerization tags, mainly used in vaccine design and structural biology research. In diagnostic applications, the forced trimerization of wild-type foldon is unsuitable for natural dimer antigens. Furthermore, their thermal stability and long-term storage stability under diagnostic conditions are insufficient, and there is a lack of systematic modification and validation for different antigen conformation requirements. Therefore, I propose a multimeric antigen construct for rapid immunoassay. Summary of the Invention

[0004] The purpose of this invention is to overcome the shortcomings of the prior art and provide a multimeric antigen construct for rapid immunoassay, which can significantly improve the detection sensitivity of antibodies (especially low-titer antibodies) and has excellent storage stability.

[0005] To achieve the above objectives, the present invention provides the following technical solution: an antigen construct for rapid immunoassay, comprising a target antigen fragment and an oligomerization domain, wherein the oligomerization domain is operatively connected to the target antigen fragment, such that the target antigen forms a dimer or trimer conformation; preferably, the oligomerization domain is selected from: foldon of bacteriophage T4 fibritin or its functional variants, or GCN4-pII dimer zipper;

[0006] More preferably, the foldon functional variant is a variant that has been truncated or site-directedly mutated to form a dimer conformation, or a variant that has undergone amino acid substitution at a hydrophobic core residue (such as Ile→Leu) to improve thermal stability.

[0007] Preferably, the target antigen fragment is selected from viral antigens (such as rabies virus G protein, influenza virus HA protein, coronavirus S protein, HIV gp41 / gp120, HBsAg, HCV core / non-structural protein, syphilis TpN15 / TpN17 / TpN47), bacterial antigens (such as Brucella outer membrane protein, Mycobacterium tuberculosis ESAT-6 / CFP-10) or parasitic antigens (such as Toxoplasma gondii SAG1).

[0008] Preferably, the oligomerizing domain is linked to the target antigen fragment by a linker peptide of 15-35 amino acids in length (such as a (G4S)n repeat sequence or an EAAAK repeat sequence). The linker region may further include a protease-cleavable site (such as a TEV or HRV-3C protease recognition sequence).

[0009] Preferably, the antigen construct is expressed and purified using a mammalian cell system (such as HEK293 or CHO cells).

[0010] Compared with existing technologies, this antigen construct for rapid immune detection has the following advantages:

[0011] I. This invention significantly improves the binding affinity to antibodies (especially low-titer antibodies) by restoring or mimicking the natural oligomeric conformation of antigens, thereby greatly enhancing detection sensitivity and reducing the false negative rate.

[0012] Second, the optimized oligomerization domains (such as heat-resistant mutant foldon) and linker peptide design of this invention enable the antigen to remain stable under thermal stress (such as 37°C) and long-term storage conditions, thereby extending the shelf life of diagnostic products.

[0013] Third, by selecting wild-type foldon (trimerization), dimerized foldon variants, or GCN4-pII (dimerization), this invention can flexibly adapt to the natural conformation requirements of different antigens, avoid conformational distortion, and has a wide range of applications.

[0014] Fourth, the constructs of this invention are small in size, easy to express and purify efficiently in mammalian cells, suitable for large-scale production, and have good commercial prospects.

[0015] Other advantages, objectives and features of the invention will be set forth in part in the description which follows, and in part will be apparent to those skilled in the art from the following examination or study, or may be learned from the practice of the invention. Detailed Implementation

[0016] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0017] In a first aspect, the present invention provides an antigen construct for rapid immunoassay, comprising a target antigen fragment linked to an oligomerizing domain. The target antigen fragment is selected from: viral antigens, including but not limited to rabies virus G protein, influenza virus HA protein, coronavirus S protein, human immunodeficiency virus (HIV) gp41 or gp120, hepatitis B virus surface antigen (HBsAg), hepatitis C virus core or non-structural protein, Treponema pallidum TpN15, TpN17, and TpN47; bacterial antigens, including but not limited to Brucella outer membrane protein, Mycobacterium tuberculosis ESAT-6 or CFP-10; and parasitic antigens, including but not limited to Toxoplasma gondii. The oligomerizing domain is a foldon derived from bacteriophage T4 fibritin or a functional variant thereof, or a GCN4-pII dimerizing zipper, thereby enabling the target antigen to form a dimer or trimer conformation to enhance stability and improve the detection sensitivity to the target antibody. The oligomerizing domain is further selected from immunoglobulin Fc fragments, COMP pentamerizing domains, and IgG CH3. The dimerizing domain or Ferritin nanoparticle display domain is truncated or site-directed mutated to form a dimer conformation. The oligomerizing domain is linked to the target antigen fragment by a 15–35 amino acid linker peptide. The linker region contains cleavable sites selected from TEV protease recognition sequences or HRV-3C protease recognition sequences. The linker peptide includes EAAAK or (G4S) repeat sequences. The foldon domain contains amino acid substitutions of hydrophobic core residues (e.g., Ile→Leu) to improve thermal stability and long-term storage stability.

[0018] Secondly, antigen constructs, expressed and purified through mammalian cell systems (HEK293 or CHO), exhibit higher thermal stability and long-term storage stability than monomeric antigens. In immunochromatographic test strips or ELISA systems, antigen constructs serve as detection lines, capture antigens, or markers. Rapid immunoassay products, containing antigen constructs and immunochromatographic carriers, are suitable for detecting antibodies against human or animal infectious diseases in serum, plasma, or whole blood samples.

[0019] In the specific construction process, the target antigen fragment and the polymerized domain are usually linked by a flexible or rigid linker peptide of 15–35 amino acids to ensure spatial independence and avoid epitope occlusion. For antigens that are naturally trimeric, wild-type foldon can be used directly to maintain their trimeric state; for antigens that are naturally dimer, truncated or site-directed mutant forms of foldon or GCN4-pII dimer zippers can be used to induce dimerization; if enhanced thermal stability and shelf-life performance are required, amino acid substitutions can be made to the core residues of foldon to improve hydrophobic packaging ability. In practical applications, these fusion antigens can be efficiently secreted and expressed in mammalian cells (such as HEK293 or CHO). After purification by affinity chromatography and molecular sieve chromatography, they can be directly applied to rapid detection platforms such as colloidal gold immunochromatography, fluorescent microsphere detection, or ELISA.

[0020] Although this invention is applicable to a variety of pathogen antigens, to more intuitively demonstrate its effects, the following examples select only three representative antigens as demonstrations: rabies virus G protein (to illustrate the maintenance of the trimeric conformation), syphilis TpN47 protein (to illustrate the adaptation of the dimer conformation, including a comparison between the foldon dimer mutant and the GCN4-pII dimer zipper), and influenza HA protein (to illustrate the effect of stability optimization). These three types of antigens can cover different natural conformation requirements and technical modification methods, thus fully demonstrating the universality and practical feasibility of this invention.

[0021] The following examples will describe the gene construction, expression purification, and preservation methods of these three types of antigens, as well as their performance comparison in immunochromatography and ELISA detection. Related data show that the antigen constructs of this invention are superior to monomeric antigens in terms of sensitivity, stability, and applicability, validating the broad application potential of this strategy in rapid immunoassays.

[0022] Example 1: Construction and application of foldon trimer antigen of rabies virus G protein:

[0023] The tPA signal peptide (SEQ ID NO:1) was linked to the external domain of the rabies virus G protein (ECD, SEQ ID NO:2), and then a flexible linker peptide (G4S)4 (SEQ ID NO:3), a T4 fibritin foldon (SEQ ID NO:4), and a 6×His tag (SEQ ID NO:5) were sequentially linked at the C-terminus to obtain the complete fusion construct (SEQ ID NO:6). In the control group construction, only the tPA signal peptide (SEQ ID NO:1) was linked to the rabies G protein ECD (SEQ ID NO:2), and a 6×His tag (SEQ ID NO:5) was directly attached to the C-terminus. The resulting monomeric construct is shown in SEQ ID NO:7. All amino acid sequences were designed according to the codon usage preference principle of the human expression system and cloned into the pcDNA3.1 expression vector. The above constructs were transfected into HEK293F suspension cells. After 5 days of culture, the supernatant was collected, and the target protein was purified by Ni-NTA affinity chromatography. Impurities and aggregates were removed by molecular sieve chromatography. Under SDS-PAGE (denaturing conditions) analysis, the SEQ ID NO:6 protein showed a monomeric band of approximately 66 kDa, and the SEQ ID NO:7 protein showed approximately 64 kDa. The purity of both was greater than 95%. In blue in situ gel and molecular sieve chromatography analysis, SEQ ID NO:6 protein mainly existed in trimer form, with a molecular weight of approximately 200 kDa, accounting for over 80%; while SEQ ID NO:7 protein only showed a monomer peak. SEQ ID NO:6 and SEQ ID NO:7 proteins were coated onto the detection lines of colloidal gold immunochromatographic test strips, respectively. Serum samples from dogs vaccinated against rabies (n=40) were selected. All sera were confirmed as antibody-positive by neutralization experiments or commercial ELISA, but the titers varied considerably (1:20–1:640). Serum was uniformly diluted 1:10, and 100 μL was added. After incubation at room temperature for 15 minutes, color development was read. To accelerate stability testing, it was shown that after storage at 37°C for 30 days, the test strip prepared with SEQ ID NO:6 protein retained approximately 85% of the signal, while the test strip prepared with SEQ ID NO:7 protein retained only approximately 60%.

[0024] The detection performance of Foldon trimer antigen (SEQ ID NO:6) and monomer control (SEQ ID NO:7) is compared in the table below.

[0025] serum titer range Sample size Monomeric antigen detection count Foldon trimeric antigen detection count Detection rate increase factor 1:20–1:80 20 10 18 1.8× ≥1:160 20 20 20 —

[0026] This embodiment demonstrates that the foldon fusion antigen (SEQ ID NO:6) has a higher detection rate in low-titer serum of immunized dogs and exhibits superior storage stability compared to the monomer control (SEQ ID NO:7). Under SDS-PAGE, the two constructed monomers have similar molecular weights, but the foldon fusion antigen can form a stable trimer under native conditions, proving that the multimerization strategy achieved through the foldon domain in this invention can significantly improve the sensitivity and reliability of rapid immunoassay.

[0027] Example 2: Construction and application of Treponema pallidum TpN47 protein dimer antigen:

[0028] The TpN47 outer domain (SEQ ID NO:8) was used as the core antigen. The N-terminus was linked to the tPA signal peptide (SEQ ID NO:1), and the C-terminus was sequentially linked to a (G4S)4 linker (SEQ ID NO:3), different polymerization domains, and a His tag (SEQ ID NO:5). The structure is as follows:

[0029] SEQ ID NO:9: Monomer control (SP + TpN47 ECD + Linker + His),

[0030] SEQ ID NO:10: Trimeric foldon fusion (SP + TpN47 ECD + Linker + WT foldon + His),

[0031] SEQ ID NO:11: Dimeric foldon^Di-SS fusion (SP + TpN47 ECD + Linker + foldon^Di-SS + His),

[0032] SEQ ID NO:12: Dimeric foldon^Di-core fusion (SP + TpN47 ECD + Linker + foldon^Di-core + His),

[0033] SEQ ID NO:13: GCN4-pII dimer fusion (SP + TpN47 ECD + Linker + GCN4-pII + His),

[0034] All sequences were codon-optimized and cloned into the pcDNA3.1 expression vector. The coding sequences of SEQ ID NO:9 to SEQ ID NO:13 were cloned into the pcDNA3.1 vector, with each vector containing only one construct. HEK293F suspension cells were then transfected and cultured at 37 °C and 5% CO2 for 5 days. Cell supernatants were collected, purified by Ni-NTA affinity chromatography, and further polished using molecular sieve chromatography (Superdex 200 Increase) to obtain high-purity recombinant proteins. Under SDS-PAGE (denaturing conditions), each protein showed the expected monomeric bands, with a molecular weight of approximately 49–50 kDa and a purity greater than 90%.

[0035] SEQ ID NO:9 (monomer control) only shows monomer peaks.

[0036] SEQ ID NO:10 (trimester foldon fusion) mainly exists in trimer form.

[0037] SEQ ID NO:11 and SEQ ID NO:12 (dimeric foldon mutants) are predominantly dimer, with a significantly reduced proportion of trimers.

[0038] SEQ ID NO:13 (GCN4-pII fusion) stably forms a dimer, with a proportion exceeding 80%.

[0039] Under non-reducing SDS-PAGE, SEQ ID NO:11 and SEQ ID NO:12 showed molecular weight shifted bands, which were restored to monomer bands after adding a reducing agent, verifying the existence of cross-chain disulfide bonds. Different forms of TpN47 fusion antigen were coated onto the detection lines of the immunochromatographic test strips, and colloidal gold immunochromatographic strips were prepared using a unified process.

[0040] Parallel testing was performed using 60 clinical serum samples (30 confirmed positive cases and 30 negative cases). Serum samples were uniformly diluted 1:10, with 100 μL added and incubated at room temperature for 15 minutes before reading the results. The monomeric control showed the lowest sensitivity, while the trimer fusion slightly improved the detection rate but resulted in false positives. The dimeric foldon mutants (11, 12) and the GCN4-pII fusion (13) significantly improved the detection rate while maintaining high specificity, with GCN4-pII showing the best performance. A comparison of the detection performance of different polymerization strategies for TpN47 is shown in the table below:

[0041] Construction Form Positive results detected (30 cases) False positives (30 cases) SEQ ID NO:9 Monomer Control 22 0 SEQ ID NO:10 Trimeric Foldon 25 1 SEQ ID NO:11 Di-saturated foldon^Di-SS 28 0 SEQ ID NO:12 Di-core foldon^Di-core 27 0 SEQ ID NO:13 GCN4-pII dimer 29 0

[0042] This embodiment illustrates that for antigens like TpN47, which naturally tend towards a dimer conformation, dimerization is significantly superior to monomerization and trimerization. Dimerized antigens significantly improve the detection rate of low-titer sera while maintaining specificity and avoiding the non-specific binding problems commonly encountered with Fc fusions. The dimerization strategy of this invention has the advantages of small molecular weight, high expression efficiency, and good batch-to-batch consistency, making it particularly suitable for the development of rapid immunodiagnostic products.

[0043] Example 3: Construction and application of influenza virus HA protein stabilized trimer antigen:

[0044] Following standard gene optimization and mammalian cell expression strategies, the HA outer domain (SEQ ID NO:14) was linked to the EAAAK×5 rigid linker peptide (SEQ ID NO:15) and the modified heat-resistant mutant foldon (SEQ ID NO:16), with a His tag added to the end for purification. The corresponding wild-type foldon was also constructed simultaneously as a control. All sequences were codon-optimized and cloned into the HEK293 expression vector, followed by transient transfection to obtain the secreted protein. The protein was then purified by Ni-NTA affinity chromatography and molecular sieve chromatography, with product purity exceeding 95%. The constructed plasmid was transiently transfected into HEK293F cells, and the supernatant was collected after 5–7 days of culture. The target protein was purified by Ni-NTA affinity chromatography and Superdex200 molecular sieve chromatography. SDS-PAGE showed a major band of approximately 75 kDa. SEC-MALS analysis indicated that both constructs were predominantly trimers, but construct B showed a more concentrated peak with no obvious aggregation. All constructs were used on standard colloidal gold immunochromatographic strips, using proteins as the detection line antigen. The test samples included 30 H1N1 positive sera, 30 negative sera, and 10 sera each of H3N2, influenza B, RSV, adenovirus, rhinovirus, and four seasonal coronaviruses. The interpretation criteria were: color development of the detection line indicated a positive result, and no color development indicated a negative result. Overall specificity was calculated. Under untreated conditions, both wild-type foldon and heat-resistant mutant foldon constructs performed well, but the construct without the EAAAK linker showed a significantly lower detection rate in low-titer sera, only 90%. The addition of EAAAK increased the detection rate to 100%, indicating that the rigid linker peptides indeed improved epitope exposure and conformational rationality. After high-temperature pretreatment, the performance of the wild-type foldon construction decreased, with the detection rate of H1N1 positive serum dropping to 83.3%, and a small number of false positives appearing in the cross-reactivity panel, resulting in an overall specificity reduction to 95%. In contrast, the heat-resistant mutant foldon construction maintained its integrity after the same heat treatment, with a 100% positive serum detection rate, no increase in cross-reactivity, and an overall specificity of 99% (95% CI: 96.3–100%).

[0045] The experimental results are summarized in the table below:

[0046] Build type Linker Foldon type Handling method H1N1 positive detection rate (n=30) False positive rate (n=30) Cross-reactivity rate (n=60) Overall specificity (%) HA–ECD–foldon–His none WT foldon Unprocessed 90% 0% 1.70% 97.5 HA–ECD–foldon–His none WT foldon 52 °C × 45 min 83.30% 0% 3.30% 95 HA–ECD–EAAAK–WTfoldon–His have WT foldon Unprocessed 100% 0% 0% 100 HA–ECD–EAAAK–WTfoldon–His have WT foldon 52 °C × 45 min 90% 0% 3.30% 96.5 HA–ECD–EAAAK–foldon^Stab–His have Heat-resistant mutation foldon Unprocessed 100% 0% 0% 100 HA–ECD–EAAAK–foldon^Stab–His have Heat-resistant mutation foldon 52 °C × 45 min 100% 0% 0% 100

[0047] In this embodiment, an EAAAK×5 rigid linker peptide was specifically introduced between the external domain and the C-terminal polymerized domain of the H1N1 HA protein. This design was chosen because influenza virus HA, in its natural state, is fixed to the viral membrane surface by its C-terminal transmembrane region. However, in recombinant construction, after removing the transmembrane region, direct fusion with the foldon often leads to heterogeneous trimer formation due to spatial folding interference, and may even affect epitope exposure. While flexible linker peptides (such as (G4S)3) can provide some degree of freedom, excessive flexibility in a large, conformation-dependent antigen like HA often results in folding instability and aggregation risks. In contrast, the EAAAK rigid linker peptide can form a stable α-helix arrangement, providing a fixed spatial interval between HAECD and the foldon, avoiding epitope obstruction and reducing unnecessary folding interference.

[0048] On the other hand, this embodiment introduces heat-resistant mutations, such as E19L and T25V, into the foldon domain. These sites are located in the hydrophobic core region of the triple α-helix, determining the tightness of the helical bundle. By replacing more isomeric amino acids with residues with more regular side chains and higher hydrophobicity, the cohesion of the triple helix is ​​enhanced, thereby improving the overall melting temperature and stability under thermal stress. Experimental results show that under 52 °C heat pretreatment, the wild-type foldon construction exhibits a decrease in detection rate and slight cross-reactivity. However, after 52 °C pretreatment, HA–ECD–EAAAK–foldon^Stab–His showed a 100% positive detection rate (30 / 30) and an overall specificity of 100% (0 / 90 false positives), consistent with the untreated group, and no increase in cross-reactivity was observed.

[0049] Further experiments showed that the HA construct incorporating the EAAAK rigid linker exhibited more uniform structural stability in molecular sieve analysis and maintained a high signal intensity in accelerated storage at 45 °C, while the heat-resistant mutant foldon significantly reduced batch-to-batch variability caused by depolymerization or aggregation. The combination of these two factors resulted in a construct that not only structurally resembled the natural trimer but also demonstrated excellent stability under long-term storage and industrial transportation conditions.

[0050] In summary, this embodiment verifies that the combined design of "EAAAK rigid linker + heat-resistant mutant foldon" has a significant effect on improving the performance of HA fusion antigen in rapid immunoassay. This strategy not only overcomes the limitations of monomer and ordinary polymer construction, but also exhibits reliable stability under process and storage conditions, further demonstrating the application value and promotion prospects of this invention in the industrialization of in vitro diagnostic products.

[0051] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A multimeric antigen construct for rapid immunoassay, characterized in that, comprises a target antigen fragment and an oligomerization domain operatively linked to the target antigen fragment, the oligomerization domain is selected from a foldon or a functional variant thereof, a GCN4-pII dimerization zipper, the antigen construct is capable of allowing the target antigen fragment to form a dimeric or trimeric conformation.

2. The antigen construct of claim 1, wherein: the functional variant of the foldon is a variant modified to form a dimeric conformation; the foldon or the functional variant thereof comprises one or more amino acid substitutions for improved stability; the amino acid substitutions are located at the hydrophobic core residues of the foldon.

3. The antigen construct of claim 1, wherein, the target antigen fragment is selected from a viral antigen, a bacterial antigen, or a parasitic antigen.

4. An antigenic construct according to claim 3, characterised in that, the antigen comprises a viral antigen, a bacterial antigen, or a parasitic antigen.

5. The antigen construct of claim 1, wherein: the oligomerization domain is connected to the target antigen fragment via a linker peptide; the linker peptide is 15-35 amino acids in length and is selected from a (G4S)n repeat sequence or an EAAAK repeat sequence; the linker peptide comprises a protease cleavable site; the cleavable site is selected from a TEV protease recognition sequence, a HRV-3C protease recognition sequence.

6. An immunochromatographic test reagent, characterized by comprises the antigen construct of any one of claims 1-5.

7. The immunochromatographic test reagent according to claim 6, characterized by the reagent is an immunochromatographic test strip or an ELISA kit.

8. An immunochromatographic test method, characterized by: the antigen construct of any one of claims 1-5 is used as a detection line, a capture antigen, or a label in an immunochromatographic test strip, an ELISA system. the antigen construct of any one of claims 1-5 is used as a detection line, a capture antigen, or a label in an immunochromatographic test strip, an ELISA system.