Nanobody-based lateral flow immunoassay for rapid antigen detection

Abstract

A nanobody-based point-of-care lateral flow immunoassay (LFA) for the rapid, cost-effective detection of SARS-CoV-2 and MERS-CoV proteins in biological samples is disclosed. The assay described herein uses nanobody-based binding agents that selectively capture and detect viral antigens, such as spike (S) proteins and receptor-binding domains (RBDs), with high sensitivity and specificity. The LFA utilizes a colorimetric readout visible to the naked eye, eliminating the need for specialized equipment. The assay supports single and multiplex detection formats, enabling simultaneous analysis of multiple viral analytes. The LFAs are stable under standard storage conditions and provide a practical solution for decentralized and scalable testing.

Description

[0001] ATTORNEY DOCKET NO. KAUST 2025-014-02 PCT

[0002] NANOBODY-BASED LATERAL FLOW IMMUNOASSAY

[0003] FOR RAPID ANTIGEN DETECTION CROSS -REFERNCE TO RELATED APPLICATIONS This application claims priority to and the benefit of U. S. Serial No. 63 / 736,983, filed December 20, 2024, the entire content of which is hereby incorporated by reference for all purposes in its entirety.

[0004] FIELD OF THE INVENTION

[0005] The invention is generally related to an assay method for detecting the presence of an analyte in a sample and devices and kits for performing the same.

[0006] BACKGROUND OF THE INVENTION

[0007] Coronaviruses are a large family of viruses that can cause respiratory diseases in humans, with symptoms ranging from the mild common cold to severe and potentially lethal conditions.1To date, three coronaviruses have caused significant outbreaks in humans: SARS coronavirus (SARS-CoV), which led to the SARS outbreak from 2002 to 2004; MERS coronavirus (MERS-CoV), which caused the MERS outbreak in 2012; and SARS-CoV-2, which emerged in 2019 and is responsible for the COVID-19 pandemic.2MERS-CoV was first identified in 2012 in Saudi Arabia and has since spread to several countries.3Despite causing a relatively small epidemic, MERS-CoV has proven to be highly lethal, with a mortality rate of approximately 35% among diagnosed cases. MERS-CoV is a zoonotic virus, with dromedary camels serving as the primary reservoir host.4Although camels infected with MERS-CoV typically exhibit only mild symptoms, they still pose a high risk of transmission.5Individuals who have close contact with camels are at a heightened risk of MERS-CoV infection.6Notably, there have been four reported cases of MERS-CoV between December 2021 and October 2022, with three of these cases having direct exposure to camels. In the wake of its outbreak in 2002, SARS-CoV infected over 8,000 individuals across 30 countries, leading to a minimum of 774 fatalities globally. The primary phase of the outbreak spanned approximately eight months, with the World Health Organization declaring SARS contained on July 5, 2003.

[0008] SARS-CoV-2, first identified in 2019, has caused a global pandemic.7,8This pandemic has resulted in a significant loss of life and has overwhelmed healthcare systems worldwide.9,10It led to unprecedented disruptions in the global economy, causing job losses and business closures.11Additionally, it has had profound effects on mental health,12 13and disrupted the education sector forcing to shift institutions to remote learning.14The virus primarily targets the upper respiratory tract (URT), subsequently spreading to the lower respiratory tract and causing 45808996.1 1 ATTORNEY DOCKET NO. KAUST 2025-014-02 PCT

[0009] typical symptoms such as fever, cough, and shortness of breath.7, 15Infected individuals transmit the virus from their URT to their surroundings, thereby facilitating the spread of the infection. Consequently, most country’ s responses to the pandemic included strict public health measures such as lockdowns, travel restrictions, and mass testing campaigns.16The COVID-19 pandemic has spurred the rapid development of diagnostic assays to enable mass screening and testing, with the aim of alerting individuals and preventing exposure of high-risk groups to the potentially lethal virus.17 19Currently, the most reliable and robust diagnostic test is reverse transcription polymerase chain reaction (RT-PCR), which is utilized in numerous certified private and governmental laboratories and hospitals worldwide. Despite significant efforts to introduce alternative antibody-based tests, RT-PCR remains the ‘gold standard’ due to its unmatched accuracy in identifying infected individuals.17, 19, 20The primary obstacles to expanding RT-PCR capacities are a shortage of specific chemical supplies and kits, the need for highly expensive and specialized instruments that typically take hours to yield results, and their availability primarily only in centralized service laboratories.17, 21, 22These factors significantly limit the number of tests that can be conducted, especially in countries without local production capabilities.17

[0010] Lateral Flow Assays (LFAs), also known as lateral flow immunochromatographic assays, have proven to be such a complementary assay to RT-PCR. Most coronaviruses targeting LFAs targeting the spike (S) protein and nucleoprotein (N) due to their high immunogenicity.23For the detection of SARS-CoV-2, numerous lateral flow immunoassays have already received emergency use authorization from the United States Food and Drug Administration (FDA).24In contrast, the detection of MERS-CoV still relies primarily on reverse transcription polymerase chain reaction (RT-PCR) or enzyme-linked immunosorbent assay (ELISA), with only a few lateral flow tests reported.25, 26This highlights the need for further development and approval of rapid, point-of-care diagnostic tests for MERS-CoV to enhance outbreak management and control.

[0011] Although traditional commercial LFAs generally exhibit lower sensitivity and specificity compared to standard laboratory tests such as PCR, their ease of use and cost-effectiveness have made them integral parts of population-scale disease testing and prevention. Moreover, considerable efforts have been made to improve LFAs. Recent advancements have concentrated on bolstering sensitivity and robustness through the optimization of assay kinetics and signal amplification.27, 28

[0012] One elegant approach to enhancing the performance of LFAs involves the use of nanobodies (Nbs), also known as VHHs, which are single-domain antibodies.29Nanobodies are 45808996.1 2 ATTORNEY DOCKET NO. KAUST 2025-014-02 PCT

[0013] the antigen-recognizing domains derived from a unique class of single-chain antibodies found in camelids, with llamas and camels being the most common sources.29They are small in size (~15 kDa, 4 nm long and 2.5 nm wide), highly soluble, stable under a wide range of conditions, and can bind their targets with high affinity and specificity.30Unlike regular antibodies, nanobodies can be produced more quickly, inexpensively, and in larger quantities from Escherichia coli (E. coli) bacterial cultures.31

[0014] Despite their potential to enhance LFAs, nanobodies are still rarely used in these tests. The small size of nanobodies allows for a high density of receptor units, which can potentially increase sensitivity and reduce background noise, thereby significantly improving the performance of LFAs. Recently, an LFA capable of detecting the SARS-CoV-2 spike protein using a combination of the angiotensin-converting enzyme 2 (ACE2) receptor and a nanobody has been reported.32the ACE2 receptor to capture the antigen, while detection was achieved using a gold-conjugated nanobody.32Approaches which use a nanobody to capture the antigen and a second nanobody for detection are still needed.

[0015] It is an object of the invention to provide a LFA using nanobodies.

[0016] It is an object of the invention to provide a point-of-care assay with higher sensitivity and specificity for rapid detection of viral antigens such as SARS-CoV-2 AND MERS-CoV antigens in a biological sample from a subject at the place of collection to provide immediate results.

[0017] It is also an object of the present invention to provide kits for a point-of-care assay for detecting SARS-CoV-2 and MERS-CoV antigens in a biological sample.

[0018] BRIEF SUMMARY OF THE INVENTION

[0019] Lateral flow assay (LFA) devices and methods of use thereof are disclosed. The devices are used to determine the presence of a SARS-CoV-2 antigen, MERS-CoV antigen, RSV antigen or Zika virus antigen (each referred to herein as analyte) in a biological sample.

[0020] The LFA uses a lateral flow test strip which includes overlapping membranes (solid substrate) that are mounted on a backing card for better stability and handling. The test trip includes a solid substrate, such as a membrane, having: (i) a sample application point, (ii) a conjugate zone in which is immobilized a first plurality of nanobodies specific for analyte, where the analyte is a SARS-CoV-2 antigen, MERS-CoV antigen, RSV antigen and / or Zika virus antigen, (iii) a capture zone which includes a test line on which is immobilized a second plurality of nanobodies specific for a SARS-CoV-2 antigen, MERS-CoV antigen, RSV antigen and / or Zika virus antigen, and (iv) a control zone which includes a control line on which is immobilized a plurality of binding agents (such as another protein, e.g. nanobody or antibody) specific for the conjugate nanobody. FIG. 1.

[0021] 45808996.1 3 ATTORNEY DOCKET NO. KAUST 2025-014-02 PCT

[0022] Exemplary nanobodies specific for SARS-CoV-2 antigen, MERS-CoV antigen, RSV antigen and / or Zika virus antigen are presented in SEQ ID Nos: 2-9 and 13-22 (include nanobody sequences and additional tags / modulators / flexible linkers). Thus the disclosed devices include nanobody pairs specific for SARS-CoV-2 antigen, MERS-CoV antigen, RSV antigen and / or Zika virus, which are present in SEQ ID Nos: 2-9 and 13-22, conjugated via a linker to a modulator domain, and to a tether particle via the modulator domain.

[0023] The first set of nanobodies specific for an analyte are immobilized in the conjugate zone (herein, conjugate nanobodies), each nanobody conjugated to a tether particle and including a detectable label, for example, a fluorescent label or a colored label, via modulator domain. Preferably, the nanobody is conjugated to the modulator via a flexible linker.

[0024] In some forms, the tether particle is a gold nanoparticle. In some forms, the modulator domain is strep tag or GST (Glutathione- S. transferase)-tag. In some forms, the modulator domain is a lysine-rich protein tag, such as spycatcher preferably including at least 7 lysine residues. In some forms, the flexible linker is SGGGS (SEQ ID NO:25).

[0025] The second set of nanobodies specific for the analyte are immobilized on a capture line in the capture zone ((herein, capture nanobodies).

[0026] The conjugate nanobodies and the capture nanobodies bind to different epitopes on the analyte.

[0027] A first optical signal from the detectable label can be detected at the test line.

[0028] A second optical signal from the detectable label can be detected at the control line. In some forms, the device includes, in (ii), a conjugate zone (or conjugate pad) in which is immobilized (a) a plurality of a first conjugate nanobody specific for a first analyte, which is conjugated to a first detectable label and (b) a plurality of a second conjugate nanobody specific for a second analyte, which is conjugated to a second detectable label, in (iii) a first test line on which is immobilized a plurality of a first capture nanobody specific for the first analyte, and a second test line on which is immobilized a plurality of a first capture nanobody specific for the second analyte, and in (iv) a test line, on which is immobilized a binding partner for the first or second conjugate nanobody. FIG. 13. The first and second detectable labels can be different from each other, or they can be the same.

[0029] In some forms, the device includes two membrane strips each having (i) an application point (or sample pad), (ii) a conjugate zone (or conjugate pad) which contains one or more conjugate nanobodies, (iii) a capture zone (including test line) for conjugate nanobody immobilization, one which is immobilized one or more capture nanobodies, and (iv) a control zone (including a control line). FIG. 13C. The conjugate and capture nanobodies in the first 45808996.1 4 ATTORNEY DOCKET NO. KAUST 2025-014-02 PCT

[0030] membrane strip are specific for a first analyte and the conjugate and capture nanobodies in the second membrane strip are specific for a second and different analyte.

[0031] An overview of various design configurations are shown in FIGs. 13A-E.

[0032] In some forms, LFA includes an anti-SARS-CoV-2 nanobody combination, having a first nanobody that specifically binds SARS-CoV-2 analyte, as the binding agent for the SARS-CoV-2 analyte to be detected and also uses a second nobody that specifically binds the same SARS-CoV-2 analyte at the capture agent to capture the first nanobody-analyte complex.

[0033] In some forms, LFA includes an anti-MERS-CoV nanobody combination, having a first nanobody that specifically binds MERS-CoV 2 analyte, as the binding agent for the MERS-CoV analyte to be detected and also uses a second nobody that specifically binds the same MERS-CoV analyte at the capture agent to capture the first nanobody-analyte complex.

[0034] In some forms, LFA includes an anti-RSV nanobody combination, having a first nanobody that specifically binds RSV analyte, as the binding agent for the RSV analyte to be detected and also uses a second nobody that specifically binds the same RSV analyte at the capture agent to capture the first nanobody- analyte complex.

[0035] In some forms, LFA includes an anti-zika virus nanobody combination, having a first nanobody that specifically binds zika virus analyte, as the binding agent for the zika virus analyte to be detected and also uses a second nobody that specifically binds the same zika virus analyte at the capture agent to capture the first nanobody-analyte complex.

[0036] The disclosed LFA can be used to detect the presence of an analyte in a sample, such as viral antigens.

[0037] A method for detecting the presence of a SARS-CoV-2 antigen, MERS-CoV antigen, RSV antigen and / or Zika virus antigen in a biological sample, includes:

[0038] b) applying a biological sample suspected of containing the antigen(s) to the application point of the LFA device.

[0039] b) allowing the biological sample to flow from the application point through: (i) the conjugation zone, (ii) to the capture zone(s) and (iii) to the test zone.

[0040] c) detecting a first signal from the detectable label present at the test line and a second signal from the detectable label present at the test line

[0041] In some forms, the method includes detecting a second signal such as an optical or fluorescent signal from the second detectable label present at the second capture region and test line. This method thereby detects the presence of the first and second analytes in the biological sample.

[0042] 45808996.1 5 ATTORNEY DOCKET NO. KAUST 2025-014-02 PCT

[0043] While preferred embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention.

[0044] While preferred embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention.

[0045] BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1A shows principle of nanobody-based heterologous lateral flow assay. When the antigen is added (Step 1-2), it initially forms a complex with the nanobody and gold nanoparticles (AuNPs) (Step 3). This antigen-nanobody-AuNP complex is then immobilized on the test line by a capture nanobody (Step 4). Any remaining nanobody- AuNP complexes are subsequently immobilized on the control line by an anti-VHH antibody (Step 5). The LFA test is considered positive when both the test and control line are visible to the naked eye. It is negative when only the control line is visible. The assay is invalid if no control line is detected by the naked eye or if only the test line is visible (Step 6). FIG. IB. Detailed lateral flow strip scheme. Distances of test line and control line from conjugate pad are as indicated. FIG. 1C. Scheme of nanobody-based sandwich lateral flow. When the ZIKV NS1 protein antigen is added, it initially forms a complex with the AuNP-conjugated ZIKV_NbD6. This antigen-nanobody-AuNP complex is then immobilized on the test line by the capture nanobody ZIKV_Nb32. Any remaining nanobody- AuNP complexes are subsequently immobilized on the control line by an anti-VHH antibody. The LFA is considered positive when both the test and control lines are visible to the naked eye. It is negative when only the control line is visible. The assay is invalid if the control line is not visible.

[0046] FIGS. 2A-2B show nanobody pairing test using LFA (no control line). (FIG.2A) anti-SARS-CoV-2 nanobody combinations. Sample: running buffer at pH 8.0, 940 nM SARS-CoV-2 RBD and 327 nM SARS-Cov-2 SI protein. (FIG. 2B) anti-MERS-CoV nanobody combinations. Sample: running buffer at pH 8.0 and 175 nM MERS-CoV S protein.

[0047] 45808996.1 6 ATTORNEY DOCKET NO. KAUST 2025-014-02 PCT

[0048] FIGS.3A-3D show optimization of LFA blocking buffer and running buffer. Sample: only running buffer. Strip 1: 0% BSA. Strip 2: 0.25% BSA. Strip 3: 0.6% 377 BSA. Strip 4: 1%BSA. AB. AuNP-NM1230 with printed anti-VHH antibody and printed NM1226 in running buffer (FIG.3A) pH 7.0 and (FIG.3B) pH 8.0. CD. AuNP-378 VHH83 with printed anti-VHH antibody and printed VHH84 in running buffer (FIG.3C) pH 7.0 and (FIG.3D) pH 8.0.

[0049] FIGS.4A-4C shows SAS-CoV-2 lateral flow LOD and specificity with different BSA blocking concentration. Strip 1-5: Serial dilution of SARS-CoV-2 SI 401 protein from 327 nM to 0.0327 nM. Strip 6-10: Serial dilution of SARS-CoV-2 RBD from 940 nM to 0.094 nM (FIG.

[0050] 4A) 0.25% BSA. (FIG.4B) 0.6% BSA. (FIG.4C) 1% 402 BSA.

[0051] FIG.5 shows inactivated SARS-CoV-2 virus test. Wildtype heat-inactivated virus was directly added, 10x and 100x diluted in running buffer at pH 8.0.

[0052] FIGS.6A-6C. MERS-CoV lateral flow LOD and specificity under different BSA blocking concentration. Strip 1-5: Serial dilution of MERS-CoV S 419 protein from 175 nM to 0.0175 nM. Strip 6-10: Serial dilution of MERS-CoV RBD from 900 nM to 0.009 nM. (FIG. 6A) 0.25% BSA. (FIG.6B) 0.6% BSA. (FIG.6C) 1% 420 BSA.

[0053] FIG.7 shows inactivated MERS-CoV virus test. Wildtype heat-inactivated virus was directly added as sample.

[0054] FIGS.8A-8D show Multiplex LFA for SARS-CoV-2 and MERS-CoV test. Sample 1: 32.7 nM SARS-CoV-2 SI. Sample 2:17.5 nM MERS-CoV S. Sample 3: Mixture of 32.7 nM SARS-CoV-2 SI and 17.5 nM MERS-CoV S. (FIG.8A) 0.25% BSA. (FIG.8B) 0.6% BSA. (FIG.8C) 1% BSA. (FIG.8D) Inactivated virus test with undiluted heat-inactivated virus using 0.6% BSA blocked LFA strips.

[0055] FIGS.9A-9B show distinction of SARS-CoV-2 and MERS-CoV. (FIG.9A) Separate detection by single-analyte LFAs. Sample 1: 32.7 nM SARS-CoV-2 SI. Sample 2: 17.5 nM MERS-CoV S using 0.6% BSA blocked LFA strips. (FIG.9B) Single multiplex LFA detection. Mixture of 32.7 nM SARS-CoV-2 SI and 17.5 nM MERS-CoV S using 0.6% BSA blocked LFA strip.

[0056] FIGs. 10A-10B show stability of SARS-CoV-2 and MERS-CoV LFA at room temperature and 4°C. (FIG. 10A) SARS-CoV LFA. Left sample: running buffer at pH 8.0, right sample: 32.7 nM SARS-CoV-2 SI using 0.6% BSA blocked LFA strips. (FIG. 10B) MERS-CoV LFA. Left sample: running buffer at pH 8.0, right sample: 17.5 nM MERS-CoV S using 0.6% BSA blocked LFA strips. RT: room temperature.

[0057] FIGs. 11A-11E show SDS-PAGE and western blot. (FIG. 11A) SDS-PAGE of mNeonGreen and its nanobodies. Line 1: mNeonGreen (26 kDa); Line 2: anti-mNeonGreen 45808996.1 7 ATTORNEY DOCKET NO. KAUST 2025-014-02 PCT

[0058] nanobody VHH0025 (commercial); Line 3: anti-mNeonGreen nanobody VHH0054 (commercial). (FIG. 11B) SDS-PAGE of GFP and its nanobody. Line 1: GFP (27.7 kDa); Line 2: anti-GFP nanobody (13.9 kDa). (FIG. 11C) SDS-PAGE of SARS-CoV-2 Spike / RBD and four anti- SARS-CoV-2 nanobodies. Line 1: anti-SARS-CoV-2 nanobody NM1226 (16 kDa); Line 2: anti-SARS-CoV-2 nanobody NM1230 (15.6 kDa); Line 3: anti-SARS-CoV-2 nanobody C5 (14 kDa); Line 4: anti-SARS-CoV-2 nanobody F2 (15 kDa); Line 5: SARS-CoV-2 spike SI protein (76.5 kDa); Line 6: SARS-CoV-2 RBD (26.5 kDa); (FIG. 11D) SDS-PAGE of MERS-CoV Spike / RBD and four anti-MERS-CoV nanobodies. Line 1: MERS-CoV spike protein (142.5 kDa); Line 2: MERS-CoV RBD (27.7 kDa); Line 3: anti-MERS-CoV VHH1 (14.1 kDa); Line 4: anti-MERS-CoV VHH55 (14.9 kDa); Line 5: anti-MERS-CoV VHH83 (14.2 kDa); Line 6: anti-MERS-CoV VHH84 (14.1 kDa). (FIG. 11E) Western blot of all nanobodies blotted anti-6x His antibody. Line 1: anti-mNeonGreen nanobody VHH0025; Line 2: anti-mNeonGreen nanobody VHH0054; Line 3: anti-GFP nanobody; Line 4: anti-SARS-CoV-2 nanobody NM1226; Line 5: anti-SARS-CoV-2 nanobody NM1230; Line 6: anti-SARS-CoV-2 nanobody C5; Line 7: anti-SARS-CoV-2 nanobody F2; Line 8: anti-MERS-CoV VHH1; Line 9: anti-MERS-CoV VHH55; Line 10: anti-MERS-CoV VHH83; Line 11: anti-MERS-CoV VHH84.

[0059] FIGS. 12A-12B show Lateral flow detecting mNeonGreen. (FIG. 12A) Proof of concept. Control line: anti-VHH antibody. Test line: anti-mNeonGreen VHH54. Conjugate pad: anti-mNeonGreen VHH25-AuNP. (FIG. 12B) Control line: anti-GFP nanobody. Test line: anti-mNeonGreen VHH54. Conjugate pad: anti-mNeonGreen VHH25-AuNP and GFP- AuNP. Strip 1: 1.9 pM mNeonGreen; Strip 2: 0.2 pM mNeonGreen; Strip 3: 19.2 nM mNeonGreen; Strip 4: 1.9 nM mNeonGreen; Strip 5: 0.2 nM mNeonGreen.

[0060] FIGs. 13A-13I show a structural overview and internal design of test housings. FIG. 13A shows Deca test housing, external view. FIG. 13B shows single test housing, external view.

[0061] FIG. 13C shows dual test housing, external view. FIG. 13D shows triple test housing, external view. FIG. 13E shows internal layout of the Deca test housing, showing organized test strips. FIG. 13F shows internal layout of the Dual test housing, demonstrating identical strip placement. FIG. 13G shows 3D model of the textured grip structure designed to enhance handling stability. FIG. 13H shows single test housing featuring an integrated hinge for one-piece printing. FIG. 131 shows single test housing immediately after printing, showcasing its one-piece fabrication as it emerges from the printer.

[0062] FIG. 14 shows Nanobody pairing results.

[0063] FIG. 15A-15F shows Benchmark results using commercial RSV test kit.

[0064] FIG. 16 shows detection of preF and postF protein.

[0065] 45808996.1 8 ATTORNEY DOCKET NO. KAUST 2025-014-02 PCT

[0066] FIG. 17 shows detection of antigens spiked in nasopharyngeal fluid (NF).

[0067] FIG. 18A-18B. SDS-PAGE of expressed nanobodies. Stained SDS-PAGE of anti-FIG.

[0068] 18A. RSV nanobodies; M: Marker; 1. His-VHH-4; 2. His-VHH-L66; 3. His-VHH-C1184; 4. His-ALX0171; 5. Strep-VHH-4; 6. Strep- VHH-L66; 7. Strep-VHH-C1184; 8. Strep-ALX0171. FIG.

[0069] 18B. (B) Western blot of anti-RSV nanobodies, blotted with 6xHisTag antibody-HRP; M:

[0070] Marker; 1. His-VHH-4; 2. His-VHH-L66; 3. His-VHH-C1184

[0071] FIG. 19. SDS-PAGE of purified nanobodies. 1: GST-NbD6 (39.7 kDa); 2: GST-Nb32 (39.7 kDa); 3: NbD6-Strep (14.9 kDa); 4: Nb32-Strep (15.1 kDa).

[0072] FIG. 20A-20C Binding analysis of ZIKV_Nb32 and ZIKV_NbD6 to the Zika NS1 antigen using BLI. Data were fit with a 1:1 binding model. (FIG. 20A) GST-tagged nanobodies immobilized on anti-GST sensors were exposed to a two-fold dilution series of Zika NS1 starting at 100 nM. (FIG. 20B) NS1 target immobilized on Ni-NTA sensors exposed to the same GST-tagged nanobodies provided in the solution. The two-fold dilution series started from 100 nM (Nb32) or 12.5 nM (NbD6). (FIG. 20C) Ni-NTA-immobilized NS1 exposed to StrepTag-II versions of both nanobodies using the same dilutions as in B. The 13 nM trace was excluded as an outlier from the analysis of Nb32 (not shown). Structures of nanobodies are based on AlphaFold3 models.46The NS1 tetramer and GST structures were taken from PDB (8WBG and 1GNW, respectively).

[0073] FIG. 21A-21B. Characterization of Zika NS 1 by mass photometry. (FIG.21A) The contrast distribution of Zika NS1 single molecule collisions at varying concentrations (25, 50, 100, and 250 nM) indicated the presence of oligomeric species at higher concentrations. (FIG.

[0074] 21B) Apparent molecular weights (± 1 SD) deduced from contrast distribution peaks in A are plotted against NS1 (monomer) concentration (Mrof monomer = 43.5 kDa). The expected Mrfor different oligomers (monomer Mr= 43.5 kDa) are shown as broken lines with shading indicating the average standard deviation (expected accuracy) from contrast distribution peaks in that size range.

[0075] FIG. 22A-22C. Competitive BLI assay for (FIG. 22A) binding of NbD6 alone and (FIG.

[0076] 22B) binding of NbD6 after sensor saturation with Nb32. Results were fit to a 1:1 binding model. Zika NS1 was immobilized on Ni-NTA sensors as before. NbD6 was applied in varying concentrations (two-fold serial dilution starting from 100 nM). (FIG. 22C) representative sensor trace (50 nM NbD6) showing all the steps in the competitive assay. Structures of nanobodies are based on AlphaFold3 models.46The NS1 tetramer and GST structures were taken from PDB (8WBG and 1GNW, respectively).

[0077] 45808996.1 9 ATTORNEY DOCKET NO. KAUST 2025-014-02 PCT

[0078] FIG. 23A-23C. Validation of LFA using different nanobody constructs. NC membrane blocked with 0.6% BSA. (FIG. 23A) Capture Nb: GST-Nb32, detection Nb: GST-NbD6. (FIG.

[0079] 23B) Capture Nb: Nb32-Strep, detection Nb: NbD6-Strep. (FIG.23C) Capture Nb: GST-Nb32, detection Nb: NbD6-Strep.

[0080] FIG. 24A-24B. LOD determination of ZIKV LFA in running buffer. Lane 1-7: Dilution series of ZIKV NS1 protein. Lane 1: 2.5 pg / ml, lane 2: 250 ng / ml, lane 3: 25 ng / ml, lane 4: 2.5 ng / ml, lane 5: 0.25 ng / ml. (FIG.24A) Capture Nb: Nb32-Strep, detection Nb: NbD6-Strep. NC membrane blocked with 0.6% BSA. (FIG. 24B) Capture Nb: GST-Nb32, detection Nb: NbD6-Strep. NC membrane blocked with 0.6% BSA.

[0081] FIG. 25A-25B. Validation of ZIKV LFA using Strep-tagged nanobodies in human serum (FIG. 25 A) and bovine urine (FIG. 25B). Capture Nb: Nb32-Strep, detection Nb: NbD6-Strep. NC membrane blocked with 0.6% BSA. Lanel-8: Dilution series of ZIKV NS1 protein. Lane 1: 2.5 pg / ml, lane 2: 0.5 pg / ml, lane 3: 0.1 pg / ml, lane 4: 20 ng / ml, lane 5: 4 ng / ml, lane 6: 2 ng / ml, lane 7: 1 ng / ml, lane 8: 0.5 ng / ml.

[0082] FIG. 26. Dengue NS1 test in our LFA using Strep-tagged nanobodies. Capture Nb: Nb32-Strep, detection Nb: NbD6-Strep. NC membrane blocked with 0.6% BSA. Sample 1: 8.5 pg / ml Dengue serotype 1 NS1 protein, sample 2: 26.7 pg / ml Dengue serotype 2 NS1 protein, sample 3: 15.6 pg / ml Dengue serotype 3 NS1 protein, sample 4: 11.0 pg / ml Dengue serotype 4 NS1 protein.

[0083] FIG. 27. Stability test of our LFA using Strep-tagged nanobodies at room temperature or 4°C. Capture Nb: Nb32-Strep, detection Nb: NbD6-Strep. NC membrane blocked with 0.6% BSA.

[0084] FIG. 28. Stability test. Stability tests were conducted every 7 days. Test strips were stored at either 4°C or room temperature.

[0085] DETAILED DESCRIPTION OF THE INVENTION

[0086] Disclosed herein are devices, systems, methods and kits for performing immunoassay tests on a sample. The immunoassay devices may be used in conjunction with diagnostic reader systems for obtaining a sensitive read-out of the immunoassay results. The immunoassay devices may be especially suited for the detection of at least a first analyte and a second analyte in a sample. The immunoassay devices and methods may utilize a competitive binding-like assay and a sandwich binding assay to detect analytes in a sample.

[0087] A. Definitions

[0088] The term “assay” refers to an in vitro procedure for analyzing a sample to determine the presence, absence, or quantity of one or more analytes of interest.

[0089] 45808996.1 10 ATTORNEY DOCKET NO. KAUST 2025-014-02 PCT

[0090] The terms “control” and “calibration” as used in connection with analytes, are used interchangeably to refer to analytes used as internal standards.

[0091] The term “analyte” refers to a chemical substance of interest that is a potential constituent of a biological sample and is to be analyzed by an assay.

[0092] The term “small analyte” refers to an analyte that is too small to be specifically bound by two antibodies that are specific for the analyte. For example, a small analyte may have a molecular weight of less than 2,000 Daltons, more preferably less than 1,500 Daltons, most preferably less than 1,000 Daltons. The small molecule can be a hydrophilic, hydrophobic, or amphiphilic compound.

[0093] A “lateral flow” assay device is a device intended to detect the presence (or absence) of a target analyte in sample in which the test sample flows along a solid substrate via capillary action.

[0094] The term “membrane” as used herein refers to a solid substrate with sufficient porosity to allow movement of binding agent bound to analyte by capillary action along its surface and through its interior.

[0095] The term “membrane strip” or “test strip” refers to a length and width of membrane sufficient to allow separation and detection of analyte.

[0096] The term “application point” is the position on the membrane where a fluid can be applied.

[0097] The term “binding agent” refers to a compound that specifically binds to an analyte. The term “capture agent” refers to an immobilized compound that selectively binds analyte complexed with binding agent (capture complex) or free binding agent (as a control). The capture agent may be conjugated to an immobilized capture particle. Binding agents and capture agents may be linked (directly or indirectly) to a detectable label. A binding agent is indirectly linked to a detectable label if it is bound to a particle that is directly linked to the detectable label.

[0098] The term “capture complex” refers to a complex formed by the specific binding of a binding agent to an analyte. The capture complex is immobilized for detection when captured by an immobilized capture agent.

[0099] The term “sandwich complex” refers to a complex formed by the specific binding of an immobilized capture agent to a binding agent and an analyte.

[0100] The term “immobilized” refers to chemical or physical fixation of an agent or particle to a location on or in a substrate, such as a membrane. For example, capture agents may be

[0101] 45808996.1 11 ATTORNEY DOCKET NO. KAUST 2025-014-02 PCT

[0102] chemically conjugated to a membrane, and particles coated with capture agents may be physically trapped within a membrane.

[0103] The term “capture particle” refers to a particle coated with a plurality of capture agents. In preferred embodiments, the capture particle is immobilized in a defined capture zone.

[0104] The term “capture zone” refers to a point on a membrane strip at which one or more capture agents are immobilized.

[0105] The term “sandwich assay” refers to a type of immunoassay in which the analyte is bound between a binding agent and a capture agent. The capture agent is generally bound to a solid surface (e.g., a membrane or particle), and the binding agent is generally labeled.

[0106] The term “antibody” refers to intact immunoglobulin molecules, fragments or polymers of immunoglobulin molecules, single chain immunoglobulin molecules, human or humanized versions of immunoglobulin molecules, and recombinant immunoglobulin molecules, as long as they are chosen for their ability to bind an analyte.

[0107] The term “ap tamer” refers to an oligonucleoside acid or peptide molecule that binds to a specific target molecule. Aptamers are generally selected from a random sequence pool. The selected aptamers are capable of adapting unique tertiary structures and recognizing target molecules with high affinity and specificity.

[0108] The term “specifically binds” or “selectively binds” refers to a binding reaction which is determinative of the presence of the analyte in a heterogeneous population. Generally, a first molecule that “specifically binds” a second molecule has an affinity constant (Ka) greater than about 105M-1(e.g., 106M"1, 107M"1, 108M"1, 109M"1, 1010M"1, 1011M"1, and 1012M-1or more) with that second molecule.

[0109] The term “detectable label” refers to any moiety that can be selectively detected in a screening assay. Examples include radiolabels, (e.g.,3H,14C,35S,125I,131I), affinity tags (e.g., biotin / avidin or streptavidin), binding sites for antibodies, metal binding domains, epitope tags, fluorescent or luminescent moieties (e.g., fluorescein and derivatives, green fluorescent protein (GFP), rhodamine and derivatives, lanthanides), colorimetric probe, and enzymatic moieties (e.g., horseradish peroxidase, β-galactosidase, β-lactamase, luciferase, alkaline phosphatase).

[0110] The term “biological sample” refers to a tissue (e.g., tissue biopsy), organ, cell, cell lysate, or body fluid from a subject. Non-limiting examples of body fluids include blood, urine, plasma, serum, tears, lymph, bile, cerebrospinal fluid, interstitial fluid, aqueous or vitreous humor, colostrum, sputum, amniotic fluid, saliva, anal and vaginal secretions, perspiration, semen, transudate, exudate, and synovial fluid.

[0111] 45808996.1 12 ATTORNEY DOCKET NO. KAUST 2025-014-02 PCT

[0112] A “sample collection apparatus,” as used herein, refers to an apparatus that can be used for collection of a biological sample or into which a collected biological sample can be deposited or stored.

[0113] “Not in fluid contact,” as used herein, indicates that fluid will not flow passively from the sample collection apparatus onto / into application point. For example, physical separation or separation by a physical component can be used.

[0114] B. Point-of-Care Assay

[0115] A rapid, reliable, sensitive, qualitative, and quantitative point-of-care assay is provided, developed to quantitatively measure analytes in a biological sample from a patient, including human and veterinary subjects. The point-of-care assay can be used in combination with nobodies binding agents and capture agents that specifically the analyte and binding agents that specifically bind the nanobodies, such as antibodies, nucleic acid aptamers, and peptide aptamers.

[0116] The point-of-care assay described herein is preferably a lateral flow immunoassay, particularly, a nanobody-based heterologous sandwich lateral flow assay.

[0117] 1. Analytes to be Detected

[0118] Analytes which can be detected using the point-of-care assay described herein include, but are not limited to viral antigens such as SARS-CoV-2 Spike (S) Protein, SARS-CoV-2 Receptor Binding Domain (RBD), MERS-CoV Spike (S) Protein, MERS-CoV Receptor Binding Domain (RBD), membrane (M), envelope (E), and nucleocapsid (N) protein of SARS-CoV-2 or MERS-CoV.

[0119] Non limiting examples of analytes that can be detected using the disclosed assays include antigens from Respiratory syncytial virus (RSV) (Fusion (F) Glycoprotein); Zika virus (Nonstructural protein 1 (NS1) protein); Dengue virus (Nonstructural protein 1 (NS1) protein); Chikungunya virus (envelope E2 (E2) glycoprotein); Influenza virus (hemagglutinin (HA) protein); Porcine reproductive and respiratory syndrome virus (PRRSV) (receptor binding domain (RBD)) and Human immunodeficiency viruses (HIV) (p24).

[0120] In some embodiments, other analytes can include drugs, or drug metabolites, hormones, and heavy metals.

[0121] Nanobodies® specific for respiratory syncytial virus fusion protein protect against infection by inhibition of fusion are known (Schepens, et al., J Infect Dis. 2011 204(11): 1692-701). ALX-0171 is a trivalent Nanobody derived from monovalent Nb017 that binds to antigenic site II of the human respiratory syncytial virus (hRSV) fusion (F) glycoprotein (Palolo, et al., Antimicrob Agents Chemother. 2016 Oct 21;60(l l):6498-6509). Other examples of nanobodies 45808996.1 13 ATTORNEY DOCKET NO. KAUST 2025-014-02 PCT

[0122] include known in the art, reviewed in Mei, et al., Sec. Respiratory Pharmacology Volume 13:963978 (2022), see esp. Table 1, reproduced in pertinent part, below).

[0123] Nanobody Disease Target Structural features and variations

[0124] MR3 SARS-CoV-2 RBD Engineered VHH constructs: tandem VHH dimer (bivalent) and bispecific tandem VHH; VHH-Fc fusion

[0125] D7, D3 Ehrlichia infection T4SS effector CPP-VHH conjugate (cell-penetrating peptide linked to VHH)

[0126] CeVICA SARS-CoV-2 RBD His-tagged VHH (Nb-His tag)

[0127] Fu2 SARS-CoV-2 RBD Engineered VHH constructs: bispecific and multivalent (bivalent / trivalent) linked-VHH formats; VHH-Fc fusion

[0128] 125s chronic hepatitis B hepatitis B surface VHH-Fc fusion (Fc-mediated dimerization — > infection antigen bivalent format)

[0129] VUN100 Latent human signaling of the viral Photosensitizer-VHH conjugate; tandem VHH cytomegalovirus receptor US28 dimer (linked bivalent); His-tagged VHH (HCMV) infection

[0130] Nb113 and IB10 Shiga toxinthe B subunit of Bispecific tandem VHH construct (targets producing Shiga toxin 2 Stx2B + Intimin C-terminus) Escherichia col (Stx2B) and the C

[0131] (STEC) terminus of Intimin

[0132] (lntC280)

[0133] Nb13 Mai de Rio Cuarto the major viral VHH fusions: VHH-alkaline phosphatase;

[0134] virus (MRCV) viroplasm VHH-eGFP

[0135] component, P9-1

[0136] NB7-14 Influenza H7N9 virus HA Tandem VHH dimer (linked bivalent format) C5, H3, C1, F2 SARS-CoV-2 RBD Multimeric VHH constructs (e.g., homotrimeric linked-VHH); VHH-Fc fusion

[0137] aEv6 Ebola Virus EBOV GP VHH-Fc fusion

[0138] aRBD-2, aRBD-3, SARS-CoV-2 RBD Bispecific VHH constructs; His-tagged VHH; aRBD-5, aRBD-7, VHH-Fc fusion

[0139] aRBD-41, aRBD- 42, and aRBD-54

[0140] Nb1 bovine viral diarrhea The nonstructural VHH-eGFP fusion

[0141] virus protein 5

[0142]

[0143] 45808996.1 14 ATTORNEY DOCKET NO. KAUST 2025-014-02 PCT

[0144] NbCXCR4 human CXCR4 VHH fused to anti-FITC scFv for delivery of immunodeficiency FITC-labeled siRNA (VHH-scFv carrier fusion) virus

[0145] NbMSIO Middle East RBD Multimeric VHH constructs (linked bivalent and respiratory syndrome trimeric formats); His-tagged VHH; VHH-Fc (MERS) coronavirus fusion

[0146] (MERS-CoV)

[0147] SNB02 Severe fever with The extracellular VHH-Fc fusion

[0148] thrombocytopenia domain of SFTSV Gn

[0149] syndrome virus* (sGn)

[0150] (SFTSV)

[0151]

[0152] 2. Lateral Flow Assay Device

[0153] A lateral flow assay (LFA) is an assay in which the test sample flows along a solid substrate via capillary action, on a lateral flow device. In some forms, the LFA is a lateral flow immunoassay, where antibodies are employed for analyte capture. The disclosed LFA device is robust and sensitive nanobody-based heterologous sandwich lateral flow assay which uses a capture nanobody and a detection nanobody.

[0154] A typical lateral flow test strip.

[0155] As illustrated in FIGS. 1 and 13A-I, the lateral flow device includes overlapping membranes (herein membrane strip) that are mounted on a backing card (solid substrate) for better stability and handling, having: (i) an application point (or sample pad), (ii) a conjugate zone (or conjugate pad) which contains one or more conjugate nanobodies, (iii) a capture zone which includes a test line on which is immobilized one or more capture nanobodies, and (iv) a control zone which includes a control line on which is immobilized one or more binding agents (such as protein, e.g. nanobody, molecular binder, antibody) specific for the conjugate nanobody.

[0156] A first set of nanobodies specific for an analyte are immobilized in the conjugate zone (herein, conjugate nanobodies). A second set of nanobodies specific for the analyte are immobilized in the capture zone ((herein, capture nanobodies), which contains a capture line for detecting captured analyte. The conjugate nanobodies and the capture nanobodies bind to different epitopes on the analyte (FIG. 2).

[0157] In some forms, the device includes one membrane strip each having (i) an application point (or sample pad), (ii) a conjugate zone (or conjugate pad) which contains a plurality of a first conjugate nanobody specific for a first analyte and a plurality of a second conjugate 45808996.1 J 5 ATTORNEY DOCKET NO. KAUST 2025-014-02 PCT

[0158] nanobody specific for a second analyte, (iii) a capture zone (i.e., test line) for capture nanobody immobilization, and (iv) a control zone. FIGS. 11B and 13A.

[0159] In some forms, the device includes two membrane strips each having (i) an application point (or sample pad), (ii) a conjugate zone (or conjugate pad) which contains one or more conjugate nanobodies, (iii) a capture zone (i.e., test line) for conjugate nanobody immobilization, one which is immobilized one or more capture nanobodies, and (iv) a control zone. FIGS. 1 and 11 A. The conjugate and capture nanobodies in the first membrane strip are specific for a first analyte and the conjugate and capture nanobodies in the second membrane strip are specific for a second and different analyte.

[0160] The solid substrate, such as a membrane strip, can be made of a substance of sufficient porosity to allow movement of antibodies and analyte by capillary action along its surface and through its interior. Examples of suitable membrane substances include: cellulose, cellulose nitrate, cellulose acetate, glass fiber, nylon, polyelectrolyte ion exchange membrane, acrylic copolymer / nylon, and polyethersulfone. In a one embodiment, the membrane strip is made of cellulose nitrate (e.g., a cellulose nitrate membrane with a Mylar backing) or of glass fiber.

[0161] In a preferred embodiment, the membrane strip is FUSION 5™ material (Whatman), which is a single layer matrix material that performs all of the functions of a lateral flow strip..

[0162] The solid substrate in some forms preferably contains a wicking pad. If a wicking pad is present, it can similarly be made from such absorbent substances as are described for an application pad. A wicking pad allows continuation of the flow of liquid by capillary action past the capture zone and facilitates the movement of non-bound agents away from the capture zone.

[0163] In some forms, the lateral flow assay device is made by 3D printing. The development of the housing for the Lateral Flow test followed a rigorous design and fabrication process to ensure precision, durability, and compatibility with testing protocols. The initial concept was designed using SolidWorks (Version 2022, from Dassault Systemes, France) software, allowing precise modeling based on predefined geometric and functional parameters.

[0164] For fabrication, the finalized design was exported in standard tessellation language (STL) format and processed using Bambu Studio (Version 2.0.3.54, from Bambu Lab All, China) slicing software. The housing was produced using Bambu Lab X1E (also from Bambu Lab All) Fused Filament Fabrication (FFF) 3D printer, utilizing Polylactic acid (PLA) material. Printing parameters, such as layer height (0,12 mm), infill density (15%), and print speed (45 mm / s), were carefully optimized to achieve high precision and consistency.

[0165] 45808996.1 16 ATTORNEY DOCKET NO. KAUST 2025-014-02 PCT

[0166] Due to the nature of the FFF technique, no post-processing was required, as the printed housing already exhibited the necessary dimensional stability and surface finish. The final housing met all technical requirements.

[0167] i. Application Point / Sample Pad

[0168] The solid substrate includes an application point, which can optionally include an application pad. For example, if the sample containing the analyte contains particles or components that should preferentially be excluded from the immunoassay, an application pad can be used. The application pad typically can filter out particles or components that are larger (e.g., greater than approximately 2 to 5 microns) than the particles used in the disclosed methods. The application pad may be used to modify the biological sample, e.g., adjust pH, filtering out solid components, separate whole blood constituents, and adsorb out unwanted antibodies. If an application pad is used, it rests on the membrane, immediately adjacent to or covering the application point. The application pad can be made of an absorbent substance which can deliver a fluid sample, when applied to the pad, to the application point on the membrane. Representative substances include cellulose, cellulose nitrate, cellulose acetate, nylon, polyelectrolyte ion exchange membrane, acrylic copolymer / nylon, polyethersulfone, or glass fibers. In one embodiment, the pad is a Hemasep™-V pad (Pall Corporation). In another embodiment, the pad is a Pall™ 133, Pall™ A / D, or glass fiber pad.

[0169] ii. Conjugate Zone / Pad

[0170] The solid substrate contains a conjugate zone which contains a nanobody specific for the analyte (herein, conjugate nanobody), as the binding agent, preferably attached to a colored particle (herein, tether particle), which can be a detectable label or include a detectable label. When the sample migrates through the conjugate zone containing conjugate nanobody, the analytes in the sample interacts with the conjugate nanobody to form capture complexes, which will be captured by the capture nanobody located at the test line in the test zone.

[0171] Detectable labels for lateral flow assays (LFAs) are in some forms, visual or instrument-readable particles conjugated to antibodies / antigens. Examples include gold nanoparticles (GNPs) for red lines, colored latex / polystyrene beads, cellulose nanobeads, carbon nanoparticles, magnetic nanoparticles, quantum dots, and fluorescent / luminescent labels (like Europium), used to create colored bands

[0172] Preferably, the conjugate nanobodies are conjugated to colored or fluorescent particles, for example, colloidal gold and latex microspheres.

[0173] Tether particles are physically trapped within the membrane. This allows for selection of optimal particle chemistry that is not influenced by the need for chemical immobilization.

[0174] 45808996.1 17 ATTORNEY DOCKET NO. KAUST 2025-014-02 PCT

[0175] Suitable tether particles include liposomes, colloidal gold, organic polymer latex particles, inorganic fluorescent particles, and phosphorescent particles. In some embodiments, the particles are polystyrene latex beads, and most particularly, polystyrene latex beads that have been prepared in the absence of surfactant, such as surfactant-free Superactive Uniform Aldehyde / Sulfate Latexes (Interfacial Dynamics Corp., Portland, Oreg.).

[0176] In some forms, the particles are monodispersed polymer microspheres based on melamine resin (MF) (e.g., available from Sigma-Aldrich). Melamine resin microspheres are manufactured by acid-catalyzed hydrothermal polycondensation of methylol melamine in the temperature range of 70-100 °C without any surfactants. Unmodified MF particles have a hydrophilic, charged surface due to the high density of polar triazine-amino and -imino groups. The surface functional groups (methylol groups, amino groups, etc.) allow covalent attachment of other ligands. For special applications, the MF particles can be modified by incorporation of other functionalities such as carboxyl groups. This increases possible surface derivatization such as chromophore or fluorophore labeling.

[0177] The particles can be labeled to facilitate detection by a means which does not significantly affect the physical properties of the particles. For example, the particles can be labeled internally (that is, the label is included within the particle, such as within the liposome or inside the polystyrene latex bead). Representative labels include luminescent labels; chemiluminescent labels; phosphorescent labels; fluorescent labels; phosphorescent labels; enzyme-linked labels; chemical labels, such as electroactive agents (e.g., ferrocyanide); and colorimetric labels, such as dyes. In one embodiment, a fluorescent label is used. In another embodiment, phosphorescent particles are used, particularly up-converting phosphorescent particles, such as those described in U. S. Patent No. 5,043,265.

[0178] iii. Test Zone / Test Line

[0179] The test zone / test line contains capture nanobody immobilized (e.g., coated on and / or permeated through the membrane) to the membrane strip. In preferred embodiments, the capture nanobody is immobilized in the test zone at the test line.

[0180] The test zone contains one or more test lines containing capture nanobody. Thus, the test zone can contain a plurality of test lines for multiplex analysis, i.e., detection of two or more analytes.

[0181] Because particle capture can be a rate limiting step in the assay, the distance between the application point and the capture zones (where particles are captured) must be sufficient to retard the speed of the liquid flow to a rate that is slow enough to allow capture of particles (i.e., capture complexes) when the liquid flow moves over the sample capture zone. The optimal 45808996.1 18 ATTORNEY DOCKET NO. KAUST 2025-014-02 PCT

[0182] distances between the components on the membrane strip can be determined and adjusted using routine experimentation

[0183] iv. Control Zone / Line

[0184] The control zone includes a control line, in which is immobilized a binding agent for the conjugate nanobody, such as anti-VHH (nanobody) antibody, or any other protein or molecular binder. Tether-particle-conjugate nanobodies not captured in the capture zone migrate to the control zone and are captured by an anti-VHH binding agent such as an antibody, indicating the proper function of the LFA strip.

[0185] v. Binding Agents and Capture Reagents

[0186] The disclosed assay preferably is a nanobody-based heterologous sandwich lateral flow assay in the sense that it uses a first nanobody (that specifically binds analyte) as the binding agent for the analyte to be detected and also uses a second nobody (that specifically binds analyte) at the capture agent to capture the first nanobody-analyte complex.

[0187] One approach to enhancing the performance of LFAs involves the use of nanobodies (Nbs), also known as single variable domain on heavy chain antibodies (VHH)s. Nanobodies are the antigen-recognizing domains derived from a distinct class of single-chain antibodies found in camelids, with llamas and camels being the most common sources. They are small in size (~15 kDa, 4 nm long and 2.5 nm wide), highly soluble, stable under a wide range of conditions, and can bind their targets with high affinity and specificity.30Unlike regular antibodies, nanobodies can be produced more quickly, inexpensively, and in larger quantities from Escherichia coli (E. coli) bacterial cultures.

[0188] Single variable domain of heavy chain (VHH) antibodies, also referred to as NANOBODY® (a registered trademark of Ablynx N. V) molecules (Nbs), are antibody fragments derived from heavy-chain only IgG antibodies found in the Camelidae family.

[0189] Conventional antibodies consist of two heavy chains and two light chains adding up to a total molecular mass of 150 kDa in the case of IgGs. Each heavy chain consists of three constant domains (CHI, CH2, and CH3) and a variable domain (VH), while each light chain consists of a constant domain (CL) and a variable domain (VL). Heavy chain only antibodies (He Ab) are naturally produced by camelids and sharks. The antigen binding portion of the HcAb includes VHH fragment.. In 1989, Professor Raymond Hamers and his team at Vrije Universiteit Brussel found a unique type of antibody in camels infected with Trypanosoma evansi. Unlike conventional antibodies composed of heavy and light chains, these antibodies lacked light chains, consisting solely of heavy-chain fragments. These were later classified as Heavy-Chain Only Antibodies (HCAbs). In 1994, they pioneered the term VHH or VHH, which refers to the 45808996.1 19 ATTORNEY DOCKET NO. KAUST 2025-014-02 PCT

[0190] isolated variable domain of a heavy chain-only antibody. In VHH antibodies, the antigenbinding sites consist of only three CDRs (Hl, H2, and H3). CDR-H3 is formed around the junction of the VH, DH, and JH segments, and hence CDR-H3 is the most diverse and important for antigen recognition (Reviewed in Kuroda, et al. Methods Mol Biol. 2023:2552:61-79. doi: 10.1007 / 978-l-0716-2609-2_2).

[0191] Antigen-specific nanobodies can be selected from three different sources, namely immune, naive, and synthetic libraries. Immune libraries are typically obtained by immunizing a domesticated animal of the Camelidae family (such as alpacas or dromedaries) with a target antigen, a minimum of four times, generally over the course of two months. It is also possible to use transgenic mice that have been transformed to produce HCAbs in situations where the target antigen is limited. Lymphocytes are then purified from extracted blood for mRNA extraction, before cDNA conversion. A two-step polymerase chain reaction (PCR) approach is then applied by first amplifying from the leader sequence to a conserved region within the CH2 domain of all IgGs, followed by agarose gel electrophoresis to select for sequences from HCAbs (smaller amplicons compared to those of regular antibodies are obtained due to the absence of the CHI domain), and finally amplifying the VHH sequence by primers targeting viable restriction enzyme sites. Lastly, the obtained amplicons are ligated into a vector and transformed into an appropriate expression system, typically E. coli although it is possible to use other expression systems such as yeast or mammalian systems. Once the Nb library is obtained, target antigenspecific Nbs must be selected and retrieved. The most common method employed is through the use of phage display. Reviewed in Jin, et al., Int. J. Mol Science, 24(6):5994 (2023).

[0192] Despite their potential to enhance LFAs, nanobodies are still rarely used in these tests. The small size of nanobodies allows for a high density of receptor units, which can potentially increase sensitivity and reduce background noise, thereby significantly improving the performance of LFAs.

[0193] The successful use of nanobodies both as the binding and capture reagent in a LFA as disclosed herein requires careful selection of nanobodies whose binding sites have compatibility in their binding of the same analyte.

[0194] In some forms, nanobodies that can be used in the compositions and methods include nanobodies as disclosed herein such as NM1226, NM1230, C5, and F2 for SARS-CoV-2, and VHH55, VHH1, VHH83, and VHH84 for MERS-CoV. Exemplary nanobody containing sequences are SEQ ID NO:2 (NM1266); SEQ ID NO:6 (NM1230); SEQ ID NO:4 (C5) and SEQ ID NO:6 (F2)..

[0195] 45808996.1 20 ATTORNEY DOCKET NO. KAUST 2025-014-02 PCT

[0196] Thus, in some forms, for SARS-CoV-2 detection, the binding agent (conjugate nanobody) is NM1230, preferably conjugated with AuNP, and the capture nanobody is NM1226, used for immobilization on the test line on the strip. In some forms, the conjugate and capture nanobody is an antigen-binding variant of NM1230 and NM1226 having an amino acid sequences having about 70%, about 80%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99% sequence identity to NM1230 and NM1226, respectively.

[0197] For the anti-MERS-CoV nanobodies, data in the present application shows that VHH1 (SEQ ID NO:6) (capture nanobody) and VHH84 (SEQ ID NO:9 )(capture nanobody) formed effective pairs with AuNP-conjugated VHH83(SEQ ID NO:8) (conjugate nanobody). In some forms, the conjugate and capture nanobody is an antigen-binding variant of NM1230 or NM1226 having an amino acid sequences having about 70%, about 80%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99% sequence identity to NM1230 and NM1226, respectively.

[0198] Thus in some forms, for MERS-CoV detection, the binding agent (conjugate nanobody) is VHH83, preferably conjugated with AuNP, and the capture nanobody is VHH1 / VHH84, used for immobilization on the test line on the strip. In some forms, the conjugate and capture nanobody is an antigen-binding variant of VHH83, VHH1 or VHH84 having an amino acid sequences having about 70%, about 80%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99% sequence identity to VHH83, VHH1 and VHH84, respectively.

[0199] In some forms, for RSV detection, the binding agent (conjugate nanobody) is VHH4, preferably conjugated with AuNP, and the capture nanobody is ALX0171, used for immobilization on the test line on the strip. In some forms, the conjugate and capture nanobody is an antigen-binding variant of VHH4 (for example, SEQ ID NO: 17) or ALX0171 (for example, SEQ ID NO: 19) having an amino acid sequences having about 70%, about 80%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99% sequence identity to VHH4 and ALX0171, respectively.

[0200] In some forms, for Zika detection, the binding agent (conjugate nanobody) is ZIKV_NbD6, preferably conjugated with AuNP, and the capture nanobody is ZIKV_Nb32, used for immobilization on the test line on the strip. In some forms, the conjugate and capture nanobody is an antigen-binding variant of ZIKV_NbD6 (for example, SEQ ID NO:23), or ZIKV_Nb32 (for example, SEQ ID NO:20) or having an amino acid sequences having about 70%, about 80%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99% sequence identity to ZIKV_NbD6, and ZIKV_Nb32, respectively.

[0201] 45808996.1 21 ATTORNEY DOCKET NO. KAUST 2025-014-02 PCT

[0202] A typical variant of a polypeptide differs in amino acid sequence from another, reference polypeptide. Generally, differences are limited so that the sequences of the reference polypeptide and the variant are closely similar overall and, in many regions, identical. A variant and reference polypeptide may differ in amino acid sequence by one or more modifications (e.g., substitutions, additions, and / or deletions). A substituted or inserted amino acid residue may or may not be one encoded by the genetic code. A variant of a polypeptide may be naturally occurring such as an allelic variant, or it may be a variant that is not known to occur naturally.

[0203] Modifications and changes can be made in the structure of the polypeptides disclosed herein and still obtain a molecule having similar characteristics as the polypeptide (e.g., a conservative amino acid substitution) as well as nucleic acids encoding the same. For example, certain amino acids can be substituted for other amino acids in a sequence, without appreciable loss of activity. Since it is the interactive capacity and nature of a polypeptide that defines that polypeptide’s biological functional activity, certain amino acid sequence substitutions can be made in a polypeptide sequence and nevertheless obtain a polypeptide with like properties.

[0204] In making such changes, the hydropathic index of amino acids can be considered. The importance of the hydropathic amino acid index in conferring interactive biologic function on a polypeptide is generally understood in the art. It is known that certain amino acids can be substituted for other amino acids having a similar hydropathic index or score and still result in a polypeptide with similar biological activity. Each amino acid has been assigned a hydropathic index on the basis of its hydrophobicity and charge characteristics. Those indices are: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine / cysteine (+2.5); methionine (+1.9); alanine (+1.8); glycine (-0.4); threonine (-0.7); serine (-0.8); tryptophan (-0.9); tyrosine (-1.3); proline (-1.6); histidine (-3.2); glutamate (-3.5); glutamine (-3.5); aspartate (-3.5); asparagine (-3.5); lysine (-3.9); and arginine (-4.5).

[0205] It is believed that the relative hydropathic character of the amino acid determines the secondary structure of the resultant polypeptide, which in turn defines the interaction of the polypeptide with other molecules, such as enzymes, substrates, receptors, antibodies, and antigens. It is known in the art that an amino acid can be substituted by another amino acid having a similar hydropathic index and still obtain a functionally equivalent polypeptide. In such changes, the substitution of amino acids whose hydropathic indices are within + 2 is preferred, those within + 1 are particularly preferred, and those within + 0.5 are even more particularly preferred.

[0206] Amino acid substitutions are generally based on the relative similarity of the amino acid side-chain substituents, for example, their hydrophobicity, hydrophilicity, charge, and size. 45808996.1 22 ATTORNEY DOCKET NO. KAUST 2025-014-02 PCT

[0207] Exemplary substitutions that take various of the foregoing characteristics into consideration are well known to those of skill in the art and include (original residue: exemplary substitution): (Ala: Gly, Ser), (Arg: Lys), (Asn: Gin, His), (Asp: Glu, Cys, Ser), (Gin: Asn), (Glu: Asp), (Gly: Ala), (His: Asn, Gin), (He: Leu, Vai), (Leu: He, Vai), (Lys: Arg), (Met: Leu, Tyr), (Ser: Thr), (Thr: Ser), (Tip: Tyr), (Tyr: Trp, Phe), and (Vai: He, Leu). The polypeptides can include variants having about 50%, 60%, 70%, 80%, 90%, and 95% sequence identity to the polypeptide of interest.

[0208] “Identity” and “similarity” can be readily calculated by known methods, such as those described in (Computational Molecular Biology, Lesk, A. M., Ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D. W., Ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, Griffin, A. M., and Griffin, H. G., Eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; and Sequence Analysis Primer, Gribskov, M. and Devereux, J., Eds., M Stockton Press, New York, 1991; and Carillo and Lipman, SIAM J Applied Math, 48: 1073 (1988).

[0209] Preferred methods to determine identity are designed to give the largest match between the sequences tested. Methods to determine identity and similarity are codified in publicly available computer programs. The percent identity between two sequences can be determined by using analysis software (i.e., Sequence Analysis Software Package of the Genetics Computer Group, Madison Wis.) that incorporates the Needelman and Wunsch, (J. Mol. Biol., 48: 443-453, 1970) algorithm (e.g., NBLAST, and XBLAST). The default parameters are used to determine the identity for the polypeptides of the present disclosure.

[0210] SEQ ID Nos: 2-9 and 13-22 include nanobody sequence and additional tags / modulators / flexible linkers as described in details in the Examples.

[0211] Each nanobody is conjugated is a tether particle via modulator domain. Preferably, the nanobody is conjugated to the modulator domain via a flexible linker.

[0212] The term “linker” as used herein includes, without limitation, peptide linkers. Any linker suitable for conjugating peptides or proteins to a carrier can be used. In some forms, the linker includes one or more glycine and / or serine amino acid residues. In some forms, the linker includes a glycine-glutamic acid di-amino acid sequence. For example, a linker can include 4-8 amino acids. For example, a linker includes the amino acid sequence GQSSRSS (SEQ ID NO:26) or SGGGS (SEQ ID NO:25). In another embodiment, a linker includes 15-20 amino acids, for example 18 amino acids. Various linker sequences are known in the art, including, without limitation, glycine serine (GS) linkers such as (GS)n, (GSGGS)n (SEQ ID NO:27), 45808996.1 23 ATTORNEY DOCKET NO. KAUST 2025-014-02 PCT

[0213] (GGGS)n (SEQ ID NO:28), and (GGGGS)n (SEQ ID NO:29), where n represents an integer of at least 1. Other flexible linkers include, but are not limited to, the amino acid sequences Gly-Ser, Gly-Ser-Gly-Ser (SEQ ID NO:30), Ala-Ser, Gly-Gly-Gly-Ser (SEQ ID NO:31), (Gly4-Ser)2 (SEQ ID NO:32) and (Gly4-Ser)4 (SEQ ID NO:33), and (Gly-Gly-Gly-Gly-Ser)3 (SEQ ID NO:34), TSGGGGSGGGSGGGS (SEQ ID NO:35), TRGGGGSGGGSGGGS (SEQ ID NO:36), GGGGSGGGSGGGSTG (SEQ ID NO:37), DQSNSEEAKKEEAKKEEAKKSNS (SEQ ID NO:38), SGGGSGGGSGGGSGGSGGSGGGSGGSGGSGGGSGGGSGGG (SEQ ID NO:39), and ESKYGPPAPPAP (SEQ ID NO:40). Additional non-limiting examples of linkers are disclosed in Shen et al., Anal. Chem. 80(6): 1910-1917 (2008) and WO 2014 / 087010, the contents of which are hereby incorporated by reference in their entireties.

[0214] vi. Anti VHH Binding Agents

[0215] Binding agents that specifically bind the nanobodies, such as antibodies, nucleic acid aptamers, and peptide aptamers.

[0216] Fragments of antibodies which have bioactivity can also be used. The fragments, whether attached to other sequences or not, include insertions, deletions, substitutions, or other selected modifications of specific regions or amino acids residues, provided the activity of the fragment is not significantly altered or impaired compared to the non-modified antibody or antibody fragment. Methods for the production of single-chain antibodies are well known to those of skill in the art. A single chain antibody can be created by fusing together the variable domains of the heavy and light chains using a short peptide linker, thereby reconstituting an antigen binding site on a single molecule. Single-chain antibody variable fragments (scFvs) in which the C-terminus of one variable domain is tethered to the N-terminus of the other variable domain via a 15 to 25 amino acid peptide or linker have been developed without significantly disrupting antigen binding or specificity of the binding. The linker is chosen to permit the heavy chain and light chain to bind together in their proper conformational orientation.

[0217] A “nucleic acid aptamer” is an oligonucleic acid that binds to a target molecule via its conformation. A nucleic acid aptamer may be constituted by DNA, RNA, or a combination thereof. Nucleic acid aptamers are typically engineered using SELEX (systematic evolution of ligands by exponential enrichment). Nucleic acid aptamers are typically oligonucleotides ranging from 15-50 bases in length that fold into defined secondary and tertiary structures, such as stemloops or G-quartets. The oligonucleotide may be DNA or RNA and may be modified for stability. A nucleic acid aptamer generally has higher specificity and affinity to a target molecule than an antibody. Nucleic acid aptamers preferably bind the target molecule with a Kd less than 10-6, 10-8, 10-10, or 10-12. Nucleic acid aptamers can also bind the target molecule with a very 45808996.1 24 ATTORNEY DOCKET NO. KAUST 2025-014-02 PCT

[0218] high degree of specificity. It is preferred that the nucleic acid aptamers have a Kd with the target molecule at least 10, 100, 1000, 10,000, or 100,000-fold lower than the Kd with other molecules. In addition, the number of target amino acid residues necessary for aptamer binding may be smaller than that of an antibody.

[0219] Nucleic acid aptamers are typically isolated from complex libraries of synthetic oligonucleotides by an iterative process of adsorption, recovery and reamplification. For example, nucleic acid aptamers may be prepared using the SELEX (Systematic Evolution of Ligands by Exponential Enrichment) method. The SELEX method involves selecting an RNA molecule bound to a target molecule from an RNA pool composed of RNA molecules each having random sequence regions and primer-binding regions at both ends thereof, amplifying the recovered RNA molecule via RT-PCR, performing transcription using the obtained cDNA molecule as a template, and using the resultant as an RNA pool for the subsequent procedure. Such procedure is repeated several times to several tens of times to select RNA with a stronger ability to bind to a target molecule. The base sequence lengths of the random sequence region and the primer binding region are not particularly limited. In general, the random sequence region contains about 20 to 80 bases and the primer binding region contains about 15 to 40 bases. Specificity to a target molecule may be enhanced by prospectively mixing molecules similar to the target molecule with RNA pools and using a pool containing RNA molecules that did not bind to the molecule of interest. An RNA molecule that was obtained as a final product by such technique is used as an RNA aptamer. Representative examples of how to make and use aptamers to bind a variety of different target molecules can be found in U. S. Patent Nos.

[0220] 5,476,766, 5,503,978, 5,631,146, 5,731,424, 5,780,228, 5,792,613, 5,795,721, 5,846,713, 5,858,660, 5,861,254, 5,864,026, 5,869,641, 5,958,691, 6,001,988, 6,011,020, 6,013,443, 6,020,130, 6,028,186, 6,030,776, and 6,051,698. An aptamer database containing comprehensive sequence information on aptamers and unnatural ribozymes that have been generated by in vitro selection methods is available at aptamer.icmb.utexas.edu. A multi-stage SELEX process is used to select aptamers that bind with high specificity and efficiency to an immunocomplex between an antibody and its target analyte or derivatives thereof. The preferred multi-stage SELEX process is required to: (1) differentiate between two related antibodies that have the capacity to bind the analyte or its derivatives; (2) differentiate between an antibody that is bound to the analyte or its derivatives and an antibody that is unbound by the analyte or its derivatives; (3) differentiate between a single antibody that is bound to either the analyte or the analyte’s derivatives; and (4) alter of the aptamer’s structure upon binding the desired target immunocomplex. The preferred multi-stage SELEX process is conducted in two stages, wherein 45808996.1 25 ATTORNEY DOCKET NO. KAUST 2025-014-02 PCT

[0221] each stage utilizes a different modified SELEX method. Stage 1 involves enrichment and recombination of the aptamer library using CE-SELEX. Stage 2 involves completing aptamer selection using Structure-switching SELEX. The specific details of this preferred multi-stage SELEX process are demonstrated in Examples 2 and 3.

[0222] A “peptide aptamer” is a combinatorial peptide molecule with a randomized amino acid sequence that is selected for its ability to bind a target molecule. Peptide aptamers are typically selected from combinatorial peptide libraries using yeast two-hybrid or phage display assays. Peptide aptamers are small peptides with a randomized amino acid sequence that are selected for their ability to bind a target molecule. Peptide aptamer selection can be made using different systems, but the most used is currently the yeast two-hybrid system. Peptide aptamer can also be selected from combinatorial peptide libraries constructed by phage display and other surface display technologies such as mRNA display, ribosome display, bacterial display and yeast display. These experimental procedures are also known as biopannings. Among peptides obtained from biopannings, mimotopes can be considered as a kind of peptide aptamers. All the peptides panned from combinatorial peptide libraries have been stored in a special database with the name MimoDB.

[0223] 3. Sample Collection Apparatus

[0224] The quantitative point-of-care assay may involve the use of a sample collection apparatus that is not in fluid contact with the solid phase apparatus. The sample collection apparatus can be any apparatus which can contain binding agents and to which a measured volume of fluid sample can be added. Representative sample collection apparatus includes a sample tube, a test tube, a vial, a pipette or pipette tip, or a syringe. In a preferred embodiment, the sample collection apparatus is a pipette or pipette tip.

[0225] In one embodiment, the sample collection apparatus contains a population of binding agents. The binding agents can be stored within the sample collection apparatus in a stable form, i.e., a form in which the agents do not significantly change in chemical makeup or physical state during storage. The stable form can be a liquid, gel, or solid form. In preferred embodiments, the agents are evaporatively dried; freeze-dried; and / or vacuum dried. In one preferred embodiment, the sample collection apparatus contains a pipette tip having vacuum-dried binding particles within its tip. In another preferred embodiment, the sample collection apparatus contains a pipette tip having vacuum-dried analyte binding particles and vacuum-dried calibration analyte binding particles within its tip.

[0226] In other embodiments, the sample collection apparatus contains a population of drug binding particles and a population of calibration analyte binding particles. The sample collection 45808996.1 26 ATTORNEY DOCKET NO. KAUST 2025-014-02 PCT

[0227] apparatus may also contain calibration analyte. If so, the population of particles is located at a different place in the sample collection apparatus from the calibration analyte. The calibration analyte can also be evaporatively dried, vacuum-dried or freeze-dried in the sample collection apparatus. If the calibration analyte is not stored within the sample collection apparatus, then it can be present in the assay fluid.

[0228] In either embodiment, the population of particles varies, depending on the size and composition of the particles, the composition of the membrane of the solid phase apparatus, and the level of sensitivity of the assay. The population typically ranges approximately between 1 x 103 and 1 x 109, although fewer or more can be used if desired. In certain embodiments, the amount of particles is determined as an amount of solids in the suspension used to apply the particles for storage within the sample collection apparatus. For example, when applying the particles in solution for freeze- or vacuum-drying in the sample collection apparatus, a suspension of approximately 0.05% to 0.228% solids (w / v) in 5 µL of suspension can be used. Alternatively, other amounts can be used, including, for example, from approximately 0.01% to 0.5% (w / v).

[0229] The binding particles (coated with both drug binding agent and calibration analyte binding agent), or the analyte binding particles and the calibration analyte binding particles, can be stored within the sample collection apparatus in a stable form, i.e., a form in which the particles do not significantly change in chemical makeup or physical state during storage. The analyte binding particles and the calibration analyte binding particles are stored at the same location within the sample collection apparatus (e.g., applied as a homogeneous mixture to the location).

[0230] C. ASSAY METHODS

[0231] The lateral flow assay described herein can be used to detect antigens in a sample, such as viral antigens, in a biological sample. The assay generally involves combining the biological sample with an assay fluid, and a conjugate nanobody that specifically binds the analyte by adding the assay sample onto the sample pad on the LFA device. (FIG. 2); binding of the analyte to the conjugate nobody forms a capture complex which migrates on the assay device by capillary action to the conjugation zone. Contacted conjugate nanobodies particles may or may not have analyte bound thereto, depending on whether analyte is present in the fluid sample. The concentration of analyte bound to the conjugate nanobody increases proportionally with the amount of analyte present in the fluid sample, and the probability of a capture complex being arrested in the test line similarly increases with increasing amount of analyte bound to the conjugate nanobody. The population of tether particle-conjugate nanobodies at the conjugate pad 45808996.1 27 ATTORNEY DOCKET NO. KAUST 2025-014-02 PCT

[0232] and migrating therefrom, may contain particles having various amount of analyte bound to the conjugate nanobody, as well as particles having no analyte bound to the conjugate nanobody.

[0233] 1. Sample Preparation

[0234] In one embodiment, the biological sample is first combined with a binding agent in an assay fluid to produce a mixed fluid sample. If analyte is present in the mixed fluid sample, binding occurs between the analyte and the binding agent to produce capture complex. The degree of binding increases as the time factor of the conditions increases. While the majority of binding occurs within one-minute, additional incubation for more than one minute, 2 minutes, 5 minutes, 10 minutes, or 15 minutes results in additional binding. In some embodiments, the binding agent is present in the sample collection apparatus. The biological sample is preferably mixed with calibration analyte and particles coated with a calibration binding agent. In preferred embodiments, the binding particles contain detectable labels.

[0235] If there is no calibration analyte in the sample collection apparatus, then the assay fluid can contain calibration analyte. Therefore, the mixed fluid sample contains drug binding particles, calibration binding particles, calibration analyte and sample analytes (if present).

[0236] In still other embodiments, the binding agent, conjugate to a detectable label are present in the conjugation zone of the lateral flow membrane strip. In these embodiments, the sample is collected into any sample collection container used in the art to collect such samples, for example, any common laboratory container for collecting random urine samples can be used to collect urine. Samples should be collected following recommended guideline known in the art to avoid false negative results as described with respect to urine samples for example in Moeller et al., Mayo Clin. Proc., 83(l):66-76 (2008).

[0237] 2. Application of Sample

[0238] The sample is applied to the application point of the membrane strip, or to the application pad, if present. After the membrane strip is contacted with the sample, the membrane strip is maintained under conditions (e.g., sufficient time and fluid volume) which allow the labeled conjugate nanobodies to move by capillary action along the membrane to and through the test zone and subsequently beyond the test zones (e.g., into a wicking pad), thereby removing any non-bound labeled binding agents from the capture zones. In some embodiments, the sample migrates through the conjugate zone containing labelled conjugate nanobodies. The analyte in the sample interacts with the conjugate nanobodies to form capture complexes.

[0239] As the applied sample passed through the membrane strip, analyte bound (sample) to conjugate nanobody (capture complex) are immobilized by capture antibodies in the capture zone, which are preferably conjugated to immobilized capture particles. The capture zone is 45808996.1 28 ATTORNEY DOCKET NO. KAUST 2025-014-02 PCT

[0240] preferably organized into one or more capture lines in specific areas of the capture zone where they serve to capture the capture complexes as they migrate by the capture lines. The capture zone preferably contains a plurality of capture lines for multiplex analysis and quantification.

[0241] Capillary action subsequently moves any capture nanobodies that have not been arrested onwards beyond the capture zone, for example, into a wicking pad which follows the capture zone. If desired, a secondary wash step can be used. Assay fluid can be applied at the application point after the mixed fluid sample has soaked into the membrane or into the application pad, if present. The secondary wash step can be used at any time thereafter, provided that it does not dilute the mixed fluid sample. A secondary wash step can contribute to reduction of background signal when the capture particles are detected.

[0242] 3. Detection

[0243] The amount of analyte bound by conjugate nanobody arrested in the capture zone (sandwich complex) may then be detected. The sample, together with the conjugate nanobody bound to the target analyte, migrates along the strip into the test zone. This is a porous membrane (usually composed of nitrocellulose) with capture nanobodies immobilized in lines. Their role is to react with the analyte bound to the conjugate nanobody. Recognition of the sample analyte results in an appropriate response on the test line, while a response on the control line indicates the proper liquid flow through the strip. The read-out, represented by the lines appearing with different intensities, can be assessed by eye or using a dedicated reader. The labeled binding or capture agents are detected using an appropriate means for the type of label used. In some forms, magnetic particle detection methods as well as colorimetric methods can be utilized. In some forms, the labeled binding or capture agents are detected by an optical method, such as by measuring absorbance or fluorescence. In some forms, the particles are detected using an ESEQuant™ Lateral Flow Immunoassay Reader (Qiagen). Alternatively, labeled binding or capture agents can be detected using electrical conductivity or dielectric (capacitance).

[0244] Alternatively, electrochemical detection of released electroactive agents, such as indium, bismuth, gallium, or tellurium ions, or ferrocyanide can be used. For example, if liposomes are used, ferrocyanide encapsulated within the liposome can be released by addition of a drop of detergent at the capture zone, and the released ferrocyanide detected electrochemically. If chelating agent-protein conjugates are used to chelate metal ions, addition of a drop of acid at the capture zone will release the ions and allow quantitation by anodic stripping voltammetry.

[0245] 4. Interpreting Results

[0246] For non-competitive assays, the amount of analyte in the sample is directly related to the level of detection agent detected in a capture line. This value is in some forms normalized by the 45808996.1 29 ATTORNEY DOCKET NO. KAUST 2025-014-02 PCT

[0247] amount of another detectable label immobilized within the membrane (e.g., test zone) to account for variations in detection device and parameters (e.g., light intensity). This normalized value may then be plotted against a standard curve or response surface that correlates these normalized values to analyte concentration. For example, a standard curve or response surface may be prepared in advance using analyte standards. In addition, three or more internal standard analytes may be detected in the assay and used to adjust or select the standard curve or surface from reference curves or surfaces.

[0248] D. Kits

[0249] Kits for use in the disclosed methods are also provided. In one embodiment, the kit includes the lateral flow device disclosed herein, which optionally includes a conjugate zone, which preferably includes a binding agent. The kit optionally contains a sample collection apparatus.

[0250] In some embodiments, the sample collection apparatus which is not in fluid contains the lateral flow device. In some embodiments, the sample collection apparatus contains a population of binding agents which are preferably, evaporatively, freeze- or vacuum-dried onto the sample collection apparatus. Kit components additionally can include analytes at known concentrations for generating a standard curve, capture particles, particles and conjugation buffer for coating particles with binding agents, disposal apparatus (e.g., biohazard waste bags), and / or other information or instructions regarding the sample collection apparatus (e.g., lot information, expiration date, etc.).

[0251] In some embodiments, kits for use in the disclosed methods include magnetic beads / particles. The magnetic beads / particles can be pre-conjugated with the binding agent or the capture agent. Alternatively, the magnetic beads / particles are not pre-conjugated with the binding agent or the capture agent, and will be conjugated with the binding agent or the capture agent by the user. The magnetic beads / particles in the kits may be in the form of a liquid suspension or dry powder.

[0252] The disclosed devices, compositions and methods can be further understood in view of the following non-limiting examples.

[0253] 45808996.1 30 ATTORNEY DOCKET NO. KAUST 2025-014-02 PCT

[0254] EXAMPLES

[0255] Example 1: Development and Validation of Nanobody-Based Lateral Flow Assays for Rapid and Sensitive Detection of SARS-CoV-2 and MERS-CoV Antigen A. Materials and Methods

[0256] Materials

[0257] The His-tagged coronavirus proteins were purchased from SinoBiological (Beijing, China): MERS-CoV Spike protein (cat: 40069-V08B), MERS-CoV RBD (cat: 40071-V08B1), SARS-CoV-2 S1 subunit (cat: 40591-V08B1) and SARS-CoV-2 RBD (cat: 40592-V08B). The Fc-tagged SARS-CoV-2 proteins were purchased from ACRObiosystems (Newark, DE, USA): SARS-CoV-2 S1 protein (cat: S1N-C5255), SARS-CoV-2 RBD (cat: SPD-C5255). Fc-tagged MERS-CoV RBD (cat: Z01LS-0722-LS3) was purchased from Creative Biolabs (Shirley, NY, USA). Anti-VHH antibody (cat: 128-005-232) was purchased from Jackson ImmunoResearch (West Grove, PA, USA). Anti-mNeonGreen VHH0025 (cat: NT-250) and VHH0054 were purchased from Chromotek (Martinsried, Germany). All the components used in LFA strips (Product code: 07.700.30) were purchased from ClaremontBio (Upland, CA, USA). Gold nanoparticles (cat: AUNR40, AUXR40) were purchased from nanoComposix (San Diego, CA, USA). Inactivated viruses (cat: PROSARS(COV2)-587, PROSARS(COV2)-624, NATSARS-ST, NATMERS-ST) were purchased from ZeptoMetrix (Buffalo, NY, USA). All other chemicals and solvents in case not specified were purchased from Sigma-Aldrich (St. Louis, MO, USA) and ThermoFisher Scientific (Waltham, MA, USA).

[0258] Cloning, Expression, and Purification of Nanobodies

[0259] The sequences of anti-GFP, anti-SARS-CoV-2 and anti-MERS-CoV nanobodies were obtained from previous research (All nanobody sequences are attached in Supplementary 5).33"37Sequences were codon optimized, synthesized (Twist Bioscience) and cloned into pET29 vector for expression. Plasmids were heat-shock transformed into SHuffle® T7 Express Competent E. coli (New England Biolabs). The transformed cells were grown in Luria-Bertani (LB) media containing 50 pg / ml Kanamycin at 30 °C, and induced with 0.5 mM Isopropyl - d-1-thiogalactopyranoside (IPTG) when OD600 reached 0.6-0.8. After induction, the temperature was lowered to 16 °C and expression was carried out for another 36-48 h. The cells were harvested by centrifugation at 6000 xg at 4 °C for 1 h. Afterwards, the cells were resuspended in lysis buffer (50 mM Tris / HCl, pH 8.0, 150 mM NaCl, 10 mM MgCl₂, 10 mM imidazole, 10 pg / ml DNase (SIGMA DN25), 1 tablet protease inhibitor (ThermoScientific Cat: A32963) per 50 ml lysis buffer) and lysed by Cell Disruptor (CONSTANT SYSTEMS CF1). The cell debris was removed by subsequent centrifugation at 24000 xg at 4 °C for 1 h. The cleared supernatant 45808996.1 31 ATTORNEY DOCKET NO. KAUST 2025-014-02 PCT

[0260] was loaded onto HisTrap column pre-equilibrated with wash buffer (50 mM Tris / HCl, pH 8.0, 150 mM NaCl, 30 mM Imidazole), washed with wash buffer and gradually eluted by elution buffer (50mM Tris / HCl, pH 8.0, 150 mM NaCl, 400 mM Imidazole). Next, proteins were loaded to size exclusion column (HiLoad Superdex 75pg) and eluted in dialysis buffer (50 mM Tris / HCl, pH 8.0, 150 mM NaCl, 5 mM DTT). The purified proteins were then stored with 20% glycerol at -20 °C.

[0261] Cloning, Expression, and Purification of mNeonGreen

[0262] The sequence of GFP (SEQ ID NO: 11) was cloned into pET303 vector for expression. Sequence of mNeonGreen (SEQ ID NO: 12) was cloned into pET29 for expression. Both plasmids were transformed to E. coli BL21(DE3) by electroporation. The transformed cells were grown in LB containing 100 pg / ml Ampicillin or 50 pg / ml Kanamycin at 37 °C, and induced by 1 mM IPTG when OD600 reached 0.6-0.8. After induction the temperature was lowered to 20 °C and expression was carried out for another 16 h. The cells were harvested by centrifugation at 6000 xg at 4 °C for 1 h. Afterwards, the cells were resuspended in lysis buffer (50 mM Tris / HCl, pH 8.0, 150 mM NaCl, 10 mM MgCl₂, 10 mM Imidazole, 10 pg / ml DNase, 1 tablet protease inhibitor per 50 ml lysis buffer) and lysed by Cell Disruptor (CONSTANT SYSTEMS CF1). The cell debris was removed by subsequent centrifugation at 24000 xg at 4 °C for 1 h. The cleared supernatant was loaded onto HisTrap column pre-equilibrated with wash buffer (50 mM Tris / HCl, pH 8.0, 150 mM NaCl, 30 mM Imidazole), washed with wash buffer and gradually eluted by elution buffer (50 mM Tris / HCl, pH 8.0, 150 mM NaCl, 400 mM Imidazole). The purified proteins were dialyzed in dialysis buffer (50 mM Tris / HCl, pH 8.0, 150 mM NaCl, 5 mM DTT) and stored with 20% glycerol at -20 °C.

[0263] Protein Characterization

[0264] The purified proteins were analyzed by SDS-PAGE followed by western blotting.

[0265] Briefly, the protein samples were prepared with 2x SDS sample loading buffer and heated at 95 °C for 10 min. SDS-PAGE analysis was performed using the precast Novex® Tris-glycine gels (4-20%, Invitrogen, Carlsbad, CA, USA).

[0266] Proteins were then transferred to pre-cut PVDF membranes (Invitrogen cat: PB9220).. Membranes were blocked for 30 mins in Pierce Fast Blocking Buffer (ThermoFisher Scientific Catalog number 37575), incubated in blocking buffer supplemented with 6x-His Tag Monoclonal Antibody Alexa Fluor 488 (ThermoFisher Scientific Catalog # MA1-21315-A488) followed by three washing steps using Tris-buffered saline with Tween-20 (TBST). All images were taken on an iBright Imager system (ThermoFisher, Waltham, MA, USA)

[0267] 45808996.1 32 ATTORNEY DOCKET NO. KAUST 2025-014-02 PCT

[0268] Tryptic Digest and LC-MS / MS Analysis

[0269] The identification of corresponding peptides was performed by liquid chromatography with tandem mass spectrometry (LC-MS / MS) analysis. The pure sample (80 pg) was prepared with 50 mM tetraethylammonium bromide (TEAB) to final volume 100 pl, and 1 pl of 1 M Tris(2- carboxyethyl)phosphine (TCEP) was added. The sample was incubated at 37 °C for 30 min and afterwards 4 pl of 1 M indole-3 -acetic acid (IAA) was added and incubated at room temperature in dark for 1 h. The sample was diluted with 500 pl 50 mM TEAB. Then 2 pg trypsin was added and sample was incubated at 37 °C for 16 h. Trifluoroacetic acid (TFA) was added to final concentration 1 % TFA. Then the sample was completely dried in centrifugal vacuum concentrator and resuspend in 20 pl 0.1 % TFA for sequencing. The prepared peptides were measured using an LTQ-Orbitrap mass spectrometer (Thermo Fisher Scientific, Waltham, MA, USA) and analyzed by MASCOT v2.3 (Matrix Sciences Ltd, UK).

[0270] Binding Affinity Measurements using Microscale Thermophoresis (MST)

[0271] The dissociation constant (KD) was measured by microscale thermophoresis (MST) using a Monolith NT.115PICO instrument (Nanotemper Technologies). mNeonGreen measurement was under Nano-blue channel with 20% excitation power and medium MST power. mNeonGreen and nanobodies were dialyzed against MST optimized buffer (50 mM Tris / HCl, pH 7.4, 150 mM NaCl, 10 mM MgCl₂, 0.05% Tween-20). Then corresponding nanobodies were 16-point serial diluted from 100 pM to 3 nM and added to 200 nM mNeonGreen. Affinity measurements were conducted in Monolith Premium Capillaries and repeated three times.

[0272] The binding between coronavirus proteins and nanobodies were performed under Pico-RED channel with 5% excitation-power and medium MST power or Nano-RED channel with 40% excitation-power and medium MST power. All coronavirus proteins and the nanobodies were dialyzed against Phosphate-Buffered Saline (PBS, pH 7.4) with 0.05% Tween-20.

[0273] Nanobodies were labelled by His-tag Labeling Kit Red-tris-NTA 2nd Generation (Nanotemper SKU: MO-L018) following the provided protocol. Spike proteins or RBDs were 16-point serial diluted in different concentrations according to the binding affinity. After mixing with 50 nM labeled nanobodies, affinity measurements were conducted in Monolith Premium Capillaries and repeated three times. Each result was averaged and evaluated using the MO. Affinity Analysis 2.3.0.7385.

[0274] Binding Affinity Measurements Using Isothermal Titration Calorimetry (ITC)

[0275] The binding between GFP and its nanobody was defined by ITC. GFP and its nanobody were dialyzed in dialysis buffer (20 mM HEPES / NaOH, pH 7.4, 150 mM NaCl) at 4 °C 45808996.1 33 ATTORNEY DOCKET NO. KAUST 2025-014-02 PCT

[0276] overnight. Titrations consisted of 18 injections of 2 pl of 100 M nanobody into the cell containing 1 pM GFP with a 10-fold lower concentration. The concentrations were depending on the affinity. The titration was performed at 25 °C in a MicroCai PEAQ-ITC instrument (Malvern Panalytical).

[0277] Preparation of Protein- AuNP Biconjugate

[0278] The nanobodies were conjugated with either 40 nm colloidal gold (OD 1, ClaremontBio), Bioready 40 nm NHS Gold (OD 20, nanoComposix) or Bioready 40 nm Gold Nanospheres-Carboxyl (OD 20, nanoComposix). For all methods, proteins were previously dialyzed against 8 mM Potassium Phosphate buffer (pH 7.4).

[0279] Colloidal gold conjugation: 25 pl different pH buffers (100 mM Potassium Phosphate buffer, pH 7.4, pH 8.0, and 100 mM borate buffer pH 9.0) were added to the tubes, then 8 pg protein and 500 pl colloidal gold were added to each tube and mixed by vortex. The conjugation mixture was incubated with rotation for 10 min. 50 pl conjugation mixture was added to 50 pl 10% NaCl and incubated for 10 min. Color change after mixing with NaCl was observed. Only pH condition that did not cause color change was continued for another 20 min incubation. After the conjugation mixture was incubated for 30 min, 53 pl conjugate block buffer (10 mM potassium phosphate buffer, pH 7.5, 10% BSA) was added and incubated for another 30 min. Then the conjugation was centrifuged at 3800 xg for 10 min and carefully removed supernatant. The biconjugate was resuspended in 25 pl conjugate diluent buffer (10 mM potassium phosphate buffer, pH 7.5, 1% BSA) and stored at 4 °C.

[0280] NHS-gold conjugation: 1 ml reaction buffer (5 mM potassium phosphate, pH 7.4, 0.5% 20000 MW Polyethylene glycol (PEG)) was added to the small reaction aliquot, then 2.5 pg nanobody was added and mixed by vortex. After 1 h incubation, 5 pl quencher (50% (w / v) hydroxylamine) was added and incubated for another 10 min. The reaction mix was then centrifuged at 3800 xg for 10 min. The supernatant was removed, and pellet was resuspended in reaction buffer to wash the nanobody- AuNP biconjugate. The centrifuge and wash step were repeated twice. The pellet was at last resuspended in 50 pl conjugate diluent buffer (0.5x PBS, 0.5% BSA, 0.5% Casein, 1% Tween 20, 0.05% Sodium Azide pH 8) for use.

[0281] Carboxyl gold conjugation: sulfo- NHS and 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide-HCl (EDC) were made freshly at 10 mg / ml in water before use. Then required amount of sulfo-NHS and EDC were added to carboxyl AuNP solution. After 30 min incubation with rotation at room temperature, excess sulfo-NHS and EDC were washed by centrifugation at 3800 xg for 10 min. The supernatant was removed, and pellet was resuspended in reaction buffer (5 mM potassium phosphate, pH 7.4, 0.5% 12000 MW PEG). The wash step was repeated once.

[0282] 45808996.1 34 ATTORNEY DOCKET NO. KAUST 2025-014-02 PCT

[0283] Then 30 pg nanobody / ml AuNP was added for conjugation. After rotation at room temperature for 1 h, quencher (50% (w / v) hydroxylamine) was added. Then the reaction was incubated for another 10 min. The reaction mix was centrifuged at 3800 xg for 10 min. The supernatant was removed, and pellet was resuspended in reaction buffer to wash the nanobody- AuNP biconjugate. The centrifuge and wash step were repeated twice. Lastly, the pellet was resuspended in storage buffer (DDS Diagnostic Company). Minor modification of reaction buffer (5 mM potassium phosphate, pH 8.0, 0.5% 12000 MW PEG) was applied in VHH83 conjugation.

[0284] Preparation of LFA Strips

[0285] The conjugate pad (Glass fiber Ahlstrom 8964) was pretreated with conjugate blocking buffer (10 mM Phosphate buffer, pH 7.4, 2% (w / v) BSA, 2.5% (w / v) sucrose, 0.3% (w / v) PVP, l%(w / v) Triton X-100, and 0.02% (w / v) NaN3) and dried at 37 °C for 1 h. Then it was dispensed with nanobody-AuNP biconjugate using automated lateral flow reagent dispenser (ALFRD, ClaremontBio) at 10 pl / cm. Detection pad used in this assay was nitrocellulose membrane (MD100). Proteins were dispensed by ALFRD on the nitrocellulose membrane. The proteins for control line (0.6 mg / ml) and test lines (1.5 mg / ml) were dispensed at 0.83 pl / cm. The nitrocellulose membranes were dried at 37 °C for 30 min after printing. Then nitrocellulose membranes were blocked with blocking buffer (5 mM Potassium Phosphate, pH 8.0, different BSA%) for 30 min and dried again at 37 °C for another 30 min. For assembly, sample pad (Glass fiber Ahlstrom 8964), conjugate pad, nitrocellulose membrane and absorbent pad (AHL238 Cotton Fiber) were pasted on the backing plate (ClaremontBio Adhesive Backing Card) and cut into 3 mm using High Speed Test Strip guillotine Cutter (Werfen Equipment).

[0286] LFA Testing

[0287] Protein samples were 10x serial diluted with running buffer (20 mM Tris / HCl, pH 7.0 or pH 8.0, 0.5% BSA, 0.5% Triton X-100). Subsequent 50 pl of each sample was added to sample pad of LFA strips. For inactivated virus test, 50 pl of inactivated viruses were directly applied to LFA strips. The resulting readout is visible to the naked eye within 15 min.

[0288] B. Results and Discussion

[0289] Experimental Design and Working Principle

[0290] Complementary binding nanobodies were selected, and a robust and sensitive nanobodybased heterologous sandwich LFA (FIG. 1A-1C) was developed, where the capture nanobody and detecting nanobody are not interfering with each other's binding. The LFA strip includes of five distinct zones: (i) the sample pad, where the sample is added; (ii) the conjugate pad, which contains nanobody-gold nanoparticle (AuNP) conjugate and binds to the analyte; (iii) test line on 45808996.1 35 ATTORNEY DOCKET NO. KAUST 2025-014-02 PCT

[0291] detection pad, for which nitrocellulose membrane was used for nanobody immobilization, which then captures the antigen-nanobody-AuNP complex; (iv) control line, which the remaining nanobody- AuNP conjugate will be captured by an anti-VHH antibody, indicating the proper function of the LFA strip. In negative samples, the absence of nanobody-AuNP complexes results in a single band at the control line; and (iv) absorbent pad to which all the liquids are drawn, and drives the capillary action and accumulates excess samples or reagents.28

[0292] Proof of Concept

[0293] The design principles were tested with the green fluorescent protein mNeonGreen, as analyte, and commercially available anti-mNeonGreen nanobodies. All proteins used were characterized by SDS-PAGE (FIGS.11A-11E). Next, the binding affinities between mNeonGreen and anti-mNeonGreen nanobody were determined as follows, anti-mNeonGreen nanobody VHH0025 57.7±19.8 nM, and anti-mNeonGreen nanobody VHH0054 160.4+41.6 nM (Table 1). Moreover, the binding affinity between GFP and an anti-GFP nanobody was determined, which was used later as control, at 11.8+4.5 nM.

[0294] To create the lateral flow strips, anti-mNeonGreen nanobody VHH0025 was conjugated with carboxylated AuNP. The anti-VHH antibody was printed as the control line, and anti-mNeonGreen nanobody VHH0054 was printed as the test line. A serial dilution of mNeonGreen from 1.9 pM to 1.9 nM was successfully detected (FIG. 12A). Additionally, GFP and anti-GFP nanobodies were used as controls. GFP and anti-mNeonGreen nanobody VHH0025 were conjugated with colloidal gold. The anti-GFP nanobody was printed as the control line, and anti-mNeonGreen nanobody VHH0054 was printed as the test line. This setup also successfully detected a serial dilution of mNeonGreen from 1.9 pM to 1.9 nM (FIG. 12B). Thus, validating the working principle of nanobody-based LFA.

[0295] Nanobody Characterization

[0296] The spike protein was selected as the antigen, and suitable anti-SARS-CoV-2, and anti-MERS-CoV nanobodies were searched for in the Protein Data Bank (PDB), and in recent publications.33-37Nanobodies that had been characterized with respect to their structure and binding properties were chosen for this study. By focusing on well-characterized nanobodies, the accuracy of the detection method was ensured. The binding kinetics of SARS-CoV-2 nanobodies NM1226, NM1230, C5, and F2, as well as MERS-CoV nanobodies VHH55, VHH1, VHH83, and VHH84, have been investigated. Additionally, SARS-CoV-2 nanobodies NM1226, NM1230, C5, and F2, and MERS-CoV nanobody VHH55 have been crystallized. Epitope mapping has been conducted for all these nanobodies to further verify their specificity and binding efficacy.

[0297] 45808996.1 36 ATTORNEY DOCKET NO. KAUST 2025-014-02 PCT

[0298] Nanobody amino acid sequences include:

[0299] Anti-GFP nanobody:

[0300] MQVQLVESGGALVQPGGSLRLSCAASGFPVNRYSMRWYRQAPGKEREWVAGMSSAG DRSSYEDSVKGRFTISRDDARNTVYLQMNSLKPEDTAVYYCNVNVGFEYWGQGTQVT VGSSGSHHHHHH (SEQ ID NO:1);

[0301] NM1226:

[0302] MQVQLVESGGGSVQPGGSLRLSCLGSGSLDYYAIGWFRQAPGKEREGVSCIASSGDRTI YADSVKGRFTISRDYGKNTVYLQMNSLKPEDTAMYYCAALQGSYYYTGFVANEYDY WGQGAPVTVSSEQKLISEEDLKKKHHHHHH (SEQ ID NO:2);

[0303] NM1230:

[0304] MQVQLVESGGGLVRPGGSLRLSCVGSGFTFSGYAMNWYRQAPGKALELVAGISNAGD LTHYEEPMKGRVAISRANDKNTVYLQMDDLKPEDTAVYRCHAPGVRVGTGERKDVW GQGAQVTVSSEQKLISEEDLKKKHHHHHH (SEQ ID NO:3);

[0305] C5:

[0306] MQVQLVESGGGSVQAGGSLTLSCVASGVTLGRHAIGWFRQAPGKERERVSCIRTFDGIT SYVESTKGRFTISSNNAMNTVYLQMNSLKPEDTAVYFCALGVTAACSDNPYFWGQGTQ VTVSSKHHHHHH (SEQ ID NO:4);

[0307] F2:

[0308] MQVQLVESGGGLVQAGGSLRLACIASGRTFHSYVMAWFRQAPGKEREFVAAISWSSTP TYYGESVKGRFTISRDNAKNTVYLQMNRLKPEDTAVYFCAADRGESYYYTRPTEYEFW GQGTQVTVSSKHHHHHH (SEQ ID NO:5);

[0309] VHH1:

[0310] MQVQLQESGGGSVQTGGSLRLSCAASGSVYCMGWIRQAPGKEREGVARINSDGSVTY YARSAKGRFSISRDSAKNTVTLLMNSLKPEDTAIYTCTADTRKCMDLGSWIDSDYRGQG TQVTVSSKHHHHHH (SEQ ID NO:6);

[0311] VHH55:

[0312] MQVQLQESGGGSVQAGGSLRLSCVASGSIFSINAMDWYRQAPGKQRELVAGITSGGST NYGDFVKGRFTISRDNAKNTVYLQMDSLKPEDTAVYYCAAEVGGWGPPRPDYWGHG TQVTVSSGSLEVLFQSSKHHHHHH (SEQ ID NO:7);

[0313] VHH83:

[0314] MQVQLQESGGGSVQTGGSLRLSCAASGSVYCMGWIRQAPGKEREGVARINSDGSVTY YSRSAKGRFSISRDSAKNTVTLLMNSLKPEDTAIYTCTANLRECMDLGSWIDFDYRGQG TQVTVSSKHHHHHH (SEQ ID NO:8);

[0315] 45808996.1 37 ATTORNEY DOCKET NO. KAUST 2025-014-02 PCT

[0316] VHH84:

[0317] MQVQLQESGGGSVQAGGSLRLSCAASGSVYCMGWIRQAPGKEREGVARINSDGSVTY YARSAKGRFSISRDSAKNTVTLLMNSLKPEDTAIYTCTADTRKCIDLGSWIDSDYRGQG TQVTVSSKHHHHHH (SEQ ID NO:9).

[0318] Protein amino acid sequences used in this study include:

[0319] SUMO-GFP:

[0320] MGSSHHHHHHSSGLVPRGSHMSDSEVNQEAKPEVKPEVKPETHINLKVSDGSSEI FFKIKKTTPLRRLMEAFAKRQGKEMDSLRFLYDGIRIQADQTPEDLDMEDNDIIEAHREQ IGGSMSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVP WPTLVTTLSYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKF EGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKTRHNIED GSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITH GMDEEYN (SEQ ID NO: 10);

[0321] GFP:

[0322] MSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLP VPWPTEVTTESYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEV KFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKTRHNI EDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGIT HGMDEEYN (SEQ ID NO:11); and

[0323] mNeonGreen:

[0324] MVSKGEEDNMASLPATHELHIFGSINGVDFDMVGQGTGNPNDGYEELNLKSTK GDLQFSPWILVPHIGYGFHQYLPYPDGMSPFQAAMVDGSGYQVHRTMQFEDGASLTVN YRYTYEGSHIKGEAQVKGTGFPADGPVMTNSLTAADWCRSKKTYPNDKTIISTFKWSY TTGNGKRYRSTARTTYTFAKPMAANYLKNQPMYVFRKTELKHSKTELNFKEWQKAFT DVMGMDEEYKGSSGSHHHHHH (SEQ ID NO: 12).

[0325] Next, SHuffle® T7 Express Competent E. coli was used for the production of nanobodies with a specifically designed and improved protocol for the expression of high-yield of functional nanobodies for subsequent experiments. The yield of nanobodies ranged from 2.4 mg / L to 12.0 mg / L of cell culture. All purified proteins were characterized by SDS-PAGE (FIGS. 11A-11E).

[0326] The nanobodies were also confirmed by western blotting (FIGS. 11A-11E) and tryptic digestion followed by mass spectrometry (SEQ ID NO: 1-12). Next, the binding between Receptor Binding Domain (RBD) / spike protein and the corresponding nanobodies were confirmed using Microscale Thermophoresis (MST). Table 1 presents their KD values, and the binding curves. The binding between SARS-CoV-2 RBD / spike protein and their nanobodies is in the lower nM range, with KD values ranging from 10"8to IO"10. The strongest binding was observed between 45808996.1 38 ATTORNEY DOCKET NO. KAUST 2025-014-02 PCT

[0327] NM1230 and SARS-CoV-2 RBD, with a KD of approximately 0.7+0.3 nM. In contrast, the binding between MERS-CoV RBD / spike protein and their nanobodies is weaker ranging from 39.7+7.2 nM for the interaction between VHH55 and MERS-CoV RBD to 645.1+219.2 nM for the interaction between VHH1 and MERS-CoV RBD. Overall, these binding affinities fall within the nanomolar range and are sufficiently robust for application in LFAs.38

[0328] Table 1. KD values of coronavirus proteins and their nanobodies

[0329] i i 1 | j

[0330] i — Nanobody; i; i

[0331] 1 - 1 NM1228 1 NM123O I F2 | C6 i Ugand i i i

[0332] R3D! 3 1±1 1 nM | 0?±03 nM! <6±1 2 nM | 13.4±56 nM SARS-CoV-2 SI 1.3=0.8 nM 5.1±1 1 nM 3.4W.5 nM 4.2*2.8 nM! Nanobody: i: i:! • VHH1 i VHH55 • VHH33; VHH84: ugand i i

[0333] MERS-CoV RBD 645.1i21S.2 nM 53.5*5 ■ nM 144.3*334 nM 430*140 nM

[0334]

[0335] MERS-CoV S1 257.4*34.8 nM 39.7*7.2 nM 502.5*430 nM 449.1*66.4 nM Nanobody Pairing for LFA

[0336] The binding epitopes of the anti-SARS-CoV-2 nanobodies have been previously determined from initial studies.33,34The Protein Data Bank (PDB) structures were used to analyze the nanobody binding sites and identify potential compatibilities, data not shown.

[0337] Nanobodies that bind to proximal sites, such as NM1226 and F2 or NM1230 and C5, present challenges when pairing them in an LFA.

[0338] For the detection of the SARS-CoV-2 spike protein or its RBD, four different nanobody combinations were tested (FIG. 2A). Among these, the NM1226+NM1230 pair produced the most distinct positive test bands. Consequently, this pair was chosen for further experiments, with NM1230 used for AuNP conjugation and NM1226 for immobilization on the test line on the strip.

[0339] For the anti-MERS-CoV nanobodies, only the binding complex of VHH55 to the MERS-CoV RBD is known.35and for the MERS-CoV nanobodies VHH1, VHH83, and VHH84 epitope mapping has been conducted. Consequently, it was essential to test all possible combinations to identify potential compatibilities. Among the four anti-MERS-CoV nanobodies, all exhibited aggregation when conjugated with AuNP under standard conditions. However, VHH83 successfully conjugated with AuNP when the reaction buffer was adjusted to pH 8.0. Thus, three possible combinations were tested. The results indicated a false positive in the blank control for the VHH55 and VHH83 pair (FIG.2B), while VHH1 and VHH84 formed effective pairs with AuNP-conjugated VHH83. Based on these findings, the VHH83 and VHH84 pair was selected for further experiments.

[0340] 45808996.1 39 ATTORNEY DOCKET NO. KAUST 2025-014-02 PCT

[0341] Design parameters selected to improve individual LFA strips

[0342] The performance of sandwich LFAs is influenced by various parameters, including the pH for protein- AuNP conjugation, the selection of nanobody pairs, the concentration of bovine serum albumin (BSA) for nitrocellulose membrane blocking, and the components of the running buffer.40-42To determine the optimal conditions for LFAs, different concentrations of BSA (0%, 0.25%, 0.6%, 1.0%) were tested in 5 m potassium phosphate buffer (pH 8.0) for blocking the nitrocellulose membrane. Small-sized, non-protein blocking agents such as, Polyvinylpyrrolidone (PVP) and Polyvinyl alcohol (PVA), at various concentrations were also tested (data not shown).48However, since neither PVP or PVA exhibited increased blocking abilities compared to BSA, BSA was opted for as the blocking agent in subsequent experiments. Simultaneously, running buffers with different pH levels (20 mM Tris / HCl, pH 7.0 or 8.0, 0.5% BSA, 0.5% Triton X-100) were tested.

[0343] First, the nanobody pair NM1226+NM1230 for SARS-CoV-2 detection was evaluated. The running buffer at different pH levels showed significant differences, whereas varying BSA concentrations did not greatly affect the blank test / non-analyte test. The running buffer at pH 7.0 resulted in clear false positive bands, indicating non-specific binding caused by an improper buffer, while the pH 8.0 buffer did not result in non-specific binding (FIGS.3A-3B).

[0344] Next, the nanobody pair VHH83+VHH84 for MERS-CoV detection was tested under the same conditions. The running buffer at pH 7.0 resulted in non-specific binding, especially when blocked with 0% BSA, while the pH 8.0 running buffer did not produce clear non-specific bands (FIGS.3C-3D). Based on these results, the pH 8.0 running buffer was selected for subsequent experiments. It is important to note that at pH 8.0, although 0% BSA LFA strips did not show non-specific binding.

[0345] In the next set of experiments, a nitrocellulose membrane blocking buffer was used with BSA concentrations of 0.25%, 0.6%, and 1.0%. The running buffer employed will have a pH of 8.0. This setup aims to improve the conditions for the lateral flow assays, ensuring minimal nonspecific binding and accurate detection.

[0346] Validation of LFA strips

[0347] In the subsequent experiments, the specificity and sensitivity of the LFA strips was evaluated, which are important properties for their performance. To assess specificity, the SARS-CoV-2 LFA strips were tested using the MERS-CoV SI protein and RBD. Sensitivity was determined through a 10-fold serial dilution of the SARS-CoV-2 SI protein and RBD protein. The results indicated that the limit of detection (LOD) for the SARS-CoV-2 SI protein was 3.27 nM, while the LOD for the RBD was 0.94 nM (FIGS.4A-4C). Among the three 45808996.1 40 ATTORNEY DOCKET NO. KAUST 2025-014-02 PCT

[0348] different BSA blocked LFA strips tested, no significant differences in performance were observed. The LOD is consistent with the range reported in other published lateral LFAs.45It is noteworthy that even under the same preparation conditions, some test runs using 0.6% or 1% BSA blocked LFA strips revealed smearing or tailing of the AuNP over the nitrocellulose membrane (data not shown), hindering accurate readout. However, this issue was mitigated through fresh preparation of the reagents. Following the protein tests, inactivated SARS-CoV-2 viruses were used to simulate patient samples. Both the wildtype and Delta variant were tested using 0.6% blocked SARS-CoV-2 LFA strips. The results showed that the wildtype SARS-CoV-2 was effectively detected, whereas the Delta variant was not (FIG.5). This inability to detect the Delta variant indicates that the LFA exhibits low cross-reactivity with the variants.

[0349] Similar protein tests were conducted on MERS-CoV LFA strips. The LOD for the MERS-CoV spike (S) protein was determined to be 1.75 nM, while the LOD for the RBD was 9 nM (FIGS.6A-6C). Among the three BSA blocking conditions tested, the strips blocked with 1% BSA exhibited lower resolution compared to the others. Additionally, inactivated MERS-CoV virus was tested using 0.6% blocked SARS-CoV-2 LFA strips, and the virus was successfully detected (FIG.7).

[0350] Next, the single-analyte SARS-CoV-2 and MERS-CoV LFA was tested using selfcollected, untreated saliva mimicking patient sample conditions, considering the established LOD of the SARS-CoV-2 and MERS-CoV LFA, samples were spiked with SARS-CoV-2 RBD or MERS-CoV spike protein within the LOD range (data not shown), achieving reproducible positive test result. When self-collected, untreated saliva spiked with heat-inactivated virus samples were tested on the respective LFA setup, reproducible positive test results were observed as well. However, the bands were slightly fainter than previously noted. Encouraged by the superior results and performance of the individual SARS-CoV-2 and MERS-CoV tests, the combination of both assays were evaluated on a single test strip.

[0351] Combined LFA strip for SARS-CoV-2 and MERS-CoV distinction testing

[0352] The SARS-CoV-2 and MERS-CoV nanobody pairs were combined into a single strip and evaluated under previously confirmed conditions. In the combined LFA, the distance between the MERS-CoV test line and the conjugate pad is shorter compared to the individual LFA(data not shown). To assess the performance of the shorter distance MERS-CoV LFA, additional tests were conducted where the test line was maintained at the same distance as in the combined LFA, data not shown. The results indicated that the distance had no significant effect on the outcomes (data not shown). The combined LFA strips displayed clear test bands at the correct positions, 45808996.1 41 ATTORNEY DOCKET NO. KAUST 2025-014-02 PCT

[0353] except when detecting SARS-CoV-2 proteins (FIGS.8A-8D). When SARS-CoV-2 proteins were added, false positive bands appeared at the MERS-CoV line, under low BSA blocking conditions (FIG. 8A). Some non-specific binding was suspected. MST measurements confirmed that anti-MERS-CoV nanobodies bind to the SARS-CoV-2 RBD. All four anti-MERS-CoV nanobodies exhibited low binding to the SARS-CoV-2 RBD in the higher micromolar range (IO-6) (Table 1), which explained the non-specific binding observed in the combined LFA. However, this could be mitigated by increasing BSA conditions. Among the three blocking conditions tested, 0.6% BSA provided better resolution with less non-specific binding. Various BSA blocking conditions were further tested (data not shown). However, 0.6% BSA provided the best performance. At higher BSA concentrations, a negative effect on the capillary action was observed. Additionally, inactivated viruses were tested using the 0.6% BSA blocked LFA strips. The inactivated wild-type SARS-CoV-2 produced a test band at the correct position, but the inactivated MERS-CoV virus was barely detectable (FIG.8D).

[0354] In addition to detection by a single-analyte LFA, the distinction between SARS-CoV-2 and MERS-CoV can also be achieved by combining two individual LFAs within a single detection cassette, or in a combined LFA (FIGS. 9A-9B). The performance of the single-analyte LFA (FIG. 9A) and the combined LFA (FIG. 9B) is comparable and falls within the previously established LODs. While combining two single LFAs within one cassette may incur higher costs, it has been proven to clearly test both protein antigens and inactivated viruses without false results.

[0355] Due to the doubling of AuNPs in the multiplexed LFA, selecting the blocking conditions is important to avoid false positive bands. However, multiplexed detection of biomarkers in LFAs permits simultaneous detection of multiple targets within a single assay, providing several advantages over traditional single-analyte tests. This approach streamlines diagnostic processes, reduces the volume of samples needed, and cuts down on overall testing time and costs.

[0356] Specifically for detecting SARS-CoV-2 and MERS-CoV, multiplexed LFAs (FIG. 9B) offer an advantage. These viruses exhibit similar symptoms but require different treatments and containment measures. A multiplexed LFA can differentiate between them quickly and accurately, ensuring timely and appropriate clinical responses. The multiplexed single-analyte test (FIG. 9A) can be advantageous when different sample preparations are required.

[0357] Storage and Stability

[0358] Maintaining cold chain transportation and storage conditions is a major concern for point-of-care diagnostics. The stability of LFAs blocked with 0.6% BSA under two conditions were tested: storage at 4°C and room temperature (RT). After four weeks, all the strips remained 45808996.1 42 ATTORNEY DOCKET NO. KAUST 2025-014-02 PCT

[0359] functional. However, the LFAs stored at 4°C exhibited improved bands compared to those stored at RT (FIG. 10A-10B).

[0360] Conclusion and Outlook

[0361] Even now, COVID-19 continues to pose a global threat, especially to the elderly.

[0362] Similarly, MERS-CoV is transmitted between camels and humans, putting those in contact with camels at risk. The hallmark symptoms of both coronaviruses-fever, cough, and shortness of breath-are also observed in normal influenza. Highlighting the necessity for accurate diagnosis and effective treatments remains important. Proper identification of diseases and timely interventions significantly impact public health outcomes.

[0363] The developed low-cost, easily producible and nanobody-based LFAs for the rapid detection of SARS-CoV-2 and MERS-CoV adhere to the essential criteria of the World Health Organization’s recommended ASSURED guidelines. Moreover, the results are noticeable to the naked eye, which is important for point-of-care diagnostic. Additionally, this is the first reported nanobody-based MERS-CoV LFA and the first nanobody-based sandwich LFA that can be observed with the naked eye. A reliable protocol was established for conjugating gold nanoparticles with carboxyl groups to nanobodies produced in E. coli. It is believed that this advancement will enhance the use of nanobodies in diagnostics and accelerate the development of new LFAs.

[0364] Material availability

[0365] Plasmids have been deposited to the Addgene repository (http: / / www.addgene.org; catalog numbers #206890 (pET29_anti-MERS-Nanobody-VHH-l), #209417 (pET29_anti-MERS-Nanobody-VHH-55), #209418 (pET29_anti-MERS-Nanobody-VHH-83), #209419 (pET29_anti-MERS -Nanobody- VHH- 84), #209420 (pET29_anti-SARS-CoV-2-Nanobody-C5), #209421 (pET29_anti-SARS-CoV-2-Nanobody-F2), #209422 (pET29_anti-SARS-CoV-2-NM1226), #209423 (pET29_anti-SARS-CoV-2-NM1230), #209425 (pET29b_mNeonGreen), #210393(pET303_sumocodo_GFP_codoopti)).

[0366] References

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[0383] 15. Larners, et al. Nature Reviews Microbiology 2022, 20 (5), 270-284.

[0384] 16. Chen, et al. Frontiers in Public Health 2021, 9.

[0385] 17. Mercer, T. R.; Salit, M., Testing at scale during the COVID-19 pandemic. Nature Reviews Genetics 2021, 22 (7), 415-426.

[0386] 18. Urusov, A. E.; Zherdev, A. V.; Dzantiev, B. B., Towards Lateral Flow Quantitative Assays: Detection Approaches. Biosensors (Basel) 2019, 9 (3).

[0387] 19. Guglielmi, et al. Nature 2020, 583 (7817), 506-509.

[0388] 20. Drame, et al. J Med Virol 2020, 92 (11), 2312-2313.

[0389] 21. Sharma, et al.. Journal of Health Management 2020, 22 (2), 248-261.

[0390] 22. Maluleke, et al. Scoping Review of Supply Chain Management Systems for Point of Care Diagnostic Services: Optimising COVID-19 Testing Capacity in Resource-Limited Settings Diagnostics [Online], 2021.

[0391] 23. Grzelak, et al. A comparison of four serological assays for detecting anti-SARS-CoV-2 antibodies in human serum samples from different populations. Sci Transl Med 2020, 12 (559).

[0392] 24. Ang, et al. Lateral Flow Immunoassays for SARS-CoV-2. Diagnostics (Basel) 2022, 12 (11).

[0393] 25. Kelly-Cirino, et al. BMJ Glob Health 2019, 4 (Suppl 2), eOOl 105.

[0394] 26. Chen, et al. J Infect 2016, 73 (1), 82-4.

[0395] 27. Liu, et al. ACS Nano 2021, 15 (3), 3593-3611.

[0396] 28. Koczula, et al. Essays Biochem 2016, 60 (1), 111-20.

[0397] 45808996.1 44 ATTORNEY DOCKET NO. KAUST 2025-014-02 PCT

[0398] 29. Van Audenhove, et al.. EBioMedicine 2016, 8, 40-48.

[0399] 30. Liang, et al. J Nanosci Nanotechno 2016, 16 (12), 12099-12111.

[0400] 31. Arbabi-Ghahroudi, et al. Cancer Metastasis Rev 2005, 24 (4), 501-19.

[0401] 32. Maher, et al. Sci Rep 2023, 13 (1), 10643.

[0402] 33. Huo, et al. Nat Commun 2021, 12 (1), 5469.

[0403] 34. Wagner, et al. EMBO Rep 2021, 22 (5), e52325.

[0404] 35. Wrapp, et al. Cell 2020, 181 (6), 1436-1441.

[0405] 36. Stalin Raj, et al. Sci Adv 2018, 4 (8), eaas9667.

[0406] 37. Kubala, et al. Protein Sci 2010, 19 (12), 2389-401.

[0407] 38. Jun, et al. Strategies for developing sensitive and specific nanoparticle-based lateral flow assays as point-of-care diagnostic device. Nano Today 2020, 30.

[0408] 39. Pinto Torres, et al. Sci Rep 2018, 8 (1), 9019.

[0409] 40. Anfossi, et al. Anal Chim Acta 2010, 682 (1-2), 104-9.

[0410] 41. Liu, Y.; Wu, A.; Hu, J.; Lin, M.; Wen, M.; Zhang, X.; Xu, C.; Hu, X.; Zhong, J.; Jiao, L.; Xie, Y.; Zhang, C.; Yu, X.; Liang, Y.; Liu, X., Detection of 3-phenoxybenzoic acid in river water with a colloidal gold-based lateral flow immunoassay. Anal Biochem 2015, 483, 7-11.

[0411] 42. Molinelli, et al. J Agric Food Chem 2008, 56 (8), 2589-94.

[0412] 43. Zhan, et al. Development and optimization of thermal contrast amplification lateral flow immunoassays for ultrasensitive HIV p24 protein detection. Microsyst Nanoeng 2020, 6, 54. 44. Zeng, et al. Am. J. Biomed. Sci. 2009, 1(1), 70-79.

[0413] 45. Lee, et al. Sample-to-answer platform for the clinical evaluation of COVID-19 using a deep learning-assisted smartphone-based assay. Nature Communications 2023, 14 (1), 2361.

[0414] EXAMPLE 2: Nanobody-Based Lateral Flow Immunoassay for Rapid RSV Respiratory syncytial virus (RSV) is a leading cause of acute respiratory infections in infants, young children, older adults, and immunocompromised individuals, often resulting in bronchiolitis and pneumonia.1Despite global efforts to improve surveillance, RSV continues to impose a substantial disease burden, particularly in low- and middle-income countries where the majority of RSV-related deaths occur.2These challenges highlight the need for rapid, accurate, and accessible diagnostic tools.

[0415] RSV is an enveloped, negative-sense, single-stranded RNA virus of the Pneumoviridae family.3It spreads readily through respiratory droplets or direct contact and primarily infects the respiratory tract, and may lead to bronchiolitis, pneumonia, and other complications depending on host susceptibility.4, 5Two viral surface glycoproteins are critical for initiating infection: the 45808996.1 45 ATTORNEY DOCKET NO. KAUST 2025-014-02 PCT

[0416] attachment glycoprotein (G) and the fusion glycoprotein (F).6The F protein, which facilitates viral fusion with host cells, is highly conserved and immunogenic and is therefore the primary target of most commercial antigen-based diagnostic assays.7

[0417] A nanobody-based LFA targeting the RSV F protein was developed to overcome limitations associated with conventional antibody-based assays. The small size of nanobodies allows higher-density immobilization on the test strip, potentially enhancing sensitivity.

[0418] Moreover, it has been shown that nanobody expression in the T7 SHuffle E. coli strain supports high yields and stable nanobodies. Together, these advantages support the development of a cost-effective and durable diagnostic tool suitable for high-burden, resource-limited settings, that can contribute to improved RSV detection and disease management.

[0419] A. Materials and Methods

[0420] Materials

[0421] The F protein antigens were purchased from SinoBiological (Beijing, China): prefusion F protein (cat: 11049-VNAS), postfusion F protein (cat: 11049-108B, 40627-V08B, 40832-V08B). Anti-VHH antibody (cat: 128-005-232) was purchased from Jackson ImmunoResearch (West Grove, PA, USA). All the components used in LFA strips (Product code: 07.700.30) were purchased from ClaremontBio (Upland, CA, USA). Gold nanoparticles (cat: AUNR40, AUXR40) were purchased from nanoComposix (San Diego, CA, USA). All other chemicals and solvents in case not specified were purchased from Sigma-Aldrich (St. Louis, MO, USA) and ThermoFisher Scientific (Waltham, MA, USA).

[0422] Cloning, Expression, and Purification of His tagged anti-RSV Nanobodies

[0423] The sequences of nanobodies were obtained from previous literature.21-23All nanobody sequences are shown below.

[0424] His-VHH-4

[0425] QVQLQESGGGLVQPGGSLRLSCAASGFTLDYYYIGWFRQAPGKEREAVSCISGSSGS TYYPDSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCATIRSSSWGGCVHYGMDY WGKGTQVTVSSGGGSWWW (SEQ ID NO: 13).

[0426] Number of amino acids: 135; Molecular weight: 14,67 kDa; Theoretical pl: 8.39

[0427] Extinction coefficients:31650.

[0428] His-VHH-L66 QVQLQESGGGLVQPGGSLRLSCAASGFTLDYYYIGWFRQAPGKEREGVSCISSSHGSTY YADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCATVAVAHFRGCGVDGMDYW GKGTQVTVSSGGGS WWW (SEQ ID NO: 14).

[0429] Number of amino acids: 135; Molecular weight: 14,59 kDa; Theoretical pl: 7.89

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[0431] Extinction coefficients:24660.

[0432] His-VHH-C1184 QVQLQESGGGLVQAGGSLRLSCAASGQTFSGYVTGWFRQAPGKEREFVALIAWSGGRL YYADSVQGRFTISRDNAETTVYLQMNSLKPEDTAVYYCAAKRGGAVTAAEWYDYWG OGTONTVSSGGGSHHHHHH (SEQ ID NO: 15).

[0433] Number of amino acids: 133; Molecular weight: 14,39 kDa; Theoretical pl: 7.18 Extinction coefficients: 34045.

[0434] ALX-0171-His EVQLVESGGGLVQAGGSLSISCAASGGSLSNYVLGWFRQAPGKEREFVAAINWRGDITI GPPNVEGRFTISRDNAKNTGYLQMNSLAPDDTAVYYCGAGTPLNPGAYIYDWSYDYW GRGTQVTVSSGGGSKKKWWWASEQ ID NO: 16).

[0435] Number of amino acids: 139; Molecular weight: 14,90kDa; Theoretical pl: 7.25; Extinction coefficients: 34045.

[0436] Strep-VHH-4 QVQLQESGGGLVQPGGSLRLSCAASGFTLDYYYIGWFRQAPGKEREAVSCISGSSGSTY YPDSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCATIRSSSWGGCVHYGMDYWG KGTOVTVSSGGGSAWSHPQ EK*(SEQ ID NO: 17).

[0437] Number of amino acids: 138; Molecular weight: 14,96 kDa; Theoretical pl: 8.37; Extinction coefficients: 37150.

[0438] Strep-VHH-L66 QVQLQESGGGLVQPGGSLRLSCAASGFTLDYYYIGWFRQAPGKEREGVSCISSSHGSTY YADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCATVAVAHFRGCGVDGMDYW GKGTOVTVSSGGGSAWSHPQ EK*(SEQ ID NO: 18).

[0439] Number of amino acids: 138; Molecular weight: 14884.51; Theoretical pl: 7.84; Extinction coefficients: 30160.

[0440] Strep-VHH-C1184 QVQLQESGGGLVQAGGSLRLSCAASGQTFSGYVTGWFRQAPGKEREFVALIAWSGGRL YYADSVQGRFTISRDNAETTVYLQMNSLKPEDTAVYYCAAKRGGAVTAAEWYDYWG QGTQVTVSSGGGSAWSHPQFEK*(SEQ ID NO:24).

[0441] Number of amino acids: 136; Molecular weight: 14,67 kDa; Theoretical pl: 6.78; Extinction coefficients: 39545

[0442] 45808996.1 47 ATTORNEY DOCKET NO. KAUST 2025-014-02 PCT

[0443] Strep-ALX-0171 EVQLVESGGGLVQAGGSLSISCAASGGSLSNYVLGWFRQAPGKEREFVAAINWRGDITI GPPNVEGRFTISRDNAKNTGYLQMNSLAPDDTAVYYCGAGTPLNPGAYIYDWSYDYW GRGTQVTVSSGGGKKKSA WSHPQFEK* (SEQ ID NO: 19).

[0444] Number of amino acids: 142; Molecular weight: 15,19 kDa; Theoretical pl: 6.86 Extinction coefficients: 39545.

[0445] Sequences were codon optimized, synthesized (Twist Bioscience, South San Francisco, CA, USA) and cloned into pET29 vector for expression. The expression and purification steps are as described before.18-20Plasmids were heat-shock transformed into SHuffle® T7 Express Competent E. coli (New England Biolabs, Catalog #C3026J). The transformed cells were grown in Luria-Bertani (LB) media containing 50 pg / ml Kanamycin at 30 °C, and induced with 0.5 mM Isopropyl P- d-1 -thiogalactopyranoside (IPTG) when OD₆₀₀ reached 0.4-0.6. After induction, the temperature was lowered to 16 °C and expression was carried out for another 40 h. The cells were harvested by centrifugation at 6000 xg at 4 °C for 30 min. Afterwards, the cells were resuspended in lysis buffer (50 mM Tris / HCl, pH 8.0, 150 mM NaCl, 10 mM MgCl₂, 20 mM imidazole, 10 pg / ml DNase (SIGMA DN25), 1 tablet protease inhibitor (ThermoScientific Cat: A32963) per 50 ml lysis buffer) and lysed by Cell Disruptor (CONSTANT SYSTEMS CF1). The cell debris was removed by subsequent centrifugation at 20000 xg at 4 °C for 1 h. The cleared supernatant was loaded onto HisTrap column pre-equilibrated with wash buffer (50 mM Tris / HCl, pH 8.0, 150 mM NaCl, 20 mM Imidazole), washed with wash buffer and gradually eluted by elution buffer (50mM Tris / HCl, pH 8.0, 150 mM NaCl, 400 mM Imidazole). Next, proteins were loaded to size exclusion column (HiLoad Superdex 75pg) and eluted in dialysis buffer (Phosphate-buffered saline (PBS), pH 7.4). The purified proteins were then stored with 20% glycerol at -20 °C.

[0446] Cloning, Expression, and Purification of Strep tagged anti-RSV Nanobodies

[0447] The sequences of nanobodies were obtained from published study.21-23All nanobody sequences are shown in SEQ ID Nos:13-18. Sequences were added with StrepTagll, codon optimized, synthesized (Twist Bioscience, South San Francisco, CA, USA) and cloned into pET29 vector for expression. Plasmids were heat-shock transformed into SHuffle® T7 Express Competent E. coli (New England Biolabs, Catalog #C3026J). The transformed cells were grown in Luria-Bertani (LB) media containing 50 pg / ml Kanamycin at 30 °C, and induced with 0.5 mM Isopropyl P- d-1 -thiogalactopyranoside (IPTG) when OD₆₀₀ reached 0.4-0.6. After induction, the temperature was lowered to 16 °C and expression was carried out for another 40 h. The cells 45808996.1 48 ATTORNEY DOCKET NO. KAUST 2025-014-02 PCT

[0448] were harvested by centrifugation at 6000 xg at 4 °C for 30 min. Afterwards, the cells were resuspended in lysis buffer (lx PBS, pH 7.4, 10 mM MgCl₂, 10 pg / ml DNase (SIGMA DN25), 1 tablet protease inhibitor (ThermoScientific Cat: A32963) per 50 ml lysis buffer) and lysed by Cell Disruptor (CONSTANT SYSTEMS CF1). The cell debris was removed by subsequent centrifugation at 20000 xg at 4 °C for 1 h. The cleared supernatant was loaded onto StrepTrap HP column pre-equilibrated with wash buffer (PBS, pH 7.4), washed with wash buffer and gradually eluted by elution buffer (PBS, pH 7.4, 2.5 mM desthiobiotin)._Next, proteins were loaded to size exclusion column (HiLoad Superdex 75pg) and eluted in dialysis buffer (PBS, pH 7.4). The purified proteins were then stored with 20% glycerol at -20 °C.

[0449] Tryptic Digest and LC-MS / MS Analysis

[0450] The identification of corresponding peptides was performed by liquid chromatography with tandem mass spectrometry (LC-MS / MS) analysis. The pure sample (80 pg) was prepared with 50 mM tetraethylammonium bromide (TEAB) to final volume 100 pl, and 1 pl of 1 M Tris(2-carboxyethyl)phosphine (TCEP) was added. The sample was incubated at 37 °C for 30 min and afterwards 4 pl of 1 M indole-3-acetic acid (IAA) was added and incubated at room temperature in dark for 1 h. The sample was diluted with 500 pl 50 mM TEAB. Then 2 pg trypsin was added and sample was incubated at 37 °C for 16 h. Trifluoroacetic acid (TFA) was added to final concentration 1 % TFA. Then the sample was completely dried in centrifugal vacuum concentrator and resuspend in 20 pl 0.1 % TFA for sequencing. The prepared peptides were measured using an LTQ-Orbitrap mass spectrometer (ThermoFisher Scientific, Waltham, MA, USA) and analyzed by MASCOT v2.3 (Matrix Sciences Ltd, UK).

[0451] Preparation of nanobody-AuNP biconjugate

[0452] The nanobodies were conjugated with either Bioready 40 nm NHS Gold (OD 20, nanoComposix, SKU: AUNR40) or Bioready 40 nm Gold Nanospheres-Carboxyl (OD 20, nanoComposix, SKU: AUXR40). For both methods, proteins were previously dialyzed against 8 mM Potassium Phosphate buffer (pH 7.4).

[0453] NHS-gold conjugation: 1 ml reaction buffer (5 mM potassium phosphate, pH 7.4, 0.5% 20000 MW Polyethylene glycol (PEG)) was added to the small reaction aliquot, then 2.5 pg nanobody was added and mixed by vortex. After 1 h incubation, 5 pl quencher (50% (w / v) hydroxylamine) was added and incubated for another 10 min. The reaction mix was then centrifuged at 3800 xg for 10 min. The supernatant was removed, and pellet was resuspended in reaction buffer to wash the nanobody-AuNP biconjugate. The centrifuge and wash step were repeated twice. The pellet was at last resuspended in 50 pl conjugate diluent buffer (0.5x PBS, 0.5% BSA, 0.5% Casein, 1% Tween 20, 0.05% Sodium Azide pH 8) for use.

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[0455] Carboxyl gold conjugation: sulfo-NHS and l-Ethyl-3-(3-dimethylaminopropyl) carbodiimide-HC1 (EDC) were made freshly at 10 mg / ml in water before use. Then required amount of sulfo-NHS and EDC were added to carboxyl AuNP solution. After 30 min incubation with rotation at room temperature, excess sulfo-NHS and EDC were washed by centrifugation at 3800 xg for 10 min. The supernatant was removed, and pellet was resuspended in reaction buffer (5 mM potassium phosphate, pH 7.4, 0.5% 12000 MW PEG). The wash step was repeated once. Then 30 pg nanobody / ml AuNP was added for conjugation. After rotation at room temperature for 1 h, quencher (50% (w / v) hydroxylamine) was added. Then the reaction was incubated for another 10 min. The reaction mix was centrifuged at 3800 xg for 10 min. The supernatant was removed, and pellet was resuspended in reaction buffer to wash the nanobody- AuNP biconjugate. The centrifuge and wash step were repeated twice. Lastly, the pellet was resuspended in conjugate diluent buffer (0.5x PBS, 0.5% BSA, 0.5% Casein, 1% Tween 20, 0.05% Sodium Azide pH 8) and stored at 4 °C until use.

[0456] Fabrication of LFA strips

[0457] The conjugate pad (Glass fiber Ahlstrom 8964) was pretreated with conjugate blocking buffer (10 mM Phosphate buffer, pH 7.4, 2% (w / v) BSA, 2.5% (w / v) sucrose, 0.3% (w / v) Polyvinylpyrrolidone, l%(w / v) Triton X-100, and 0.02% (w / v) NaNa) and dried at 37 °C for 1 h. The nanobody- AuNP biconjugate was applied using the automated lateral flow reagent dispenser (ALFRD, ClaremontBio) at 20 pl / cm. The detection pad used in this assay was a nitrocellulose membrane (VIV120) and the proteins for control line (0.6 mg / ml) and test lines (1.5 mg / ml) were dispensed on it at 0.83 pl / cm. The nitrocellulose membranes were dried at 37 °C for 30 min after printing. If blocked, the dried nitrocellulose membrane was incubated in blocking buffer (5 mM potassium phosphate, pH 8.0, 0.5% Polyvinylpyrrolidone) for 15 min and dried at 37 °C for another 30 min. For assembly, sample pad (glass fiber Ahlstrom 8964), conjugate pad, nitrocellulose membrane and absorbent pad (AHL238 cotton fiber) were pasted on the backing plate (ClaremontBio Adhesive Backing Card) and cut into 3 mm strips using the High-Speed Test Strip guillotine Cutter (Werfen Equipment). Freshly made LFA strips were stored at room temperature with desiccant packs in sealed foil bag.

[0458] Detection of antigens in buffer or spiked body fluids

[0459] Standard samples were prepared by diluting antigen with running buffer (20 mM Tris / HCl, pH 8.0, 0.5% BSA, 0.5% Triton X-100). F proteins spiked into artificial nasopharyngeal fluid (Biochemazone, cat no. BZ252) were used to simulate actual biological samples. After spiking, the nasopharyngeal fluid spiked with F protein was diluted 20 times with running buffer for LFA test run. 60 pl of each sample was added to the sample pad of LFA strips.

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[0461] B. Results and Discussion

[0462] Nanobody expression and characterization

[0463] For detecting RSV F protein, four nanobodies were selected from previous literature.21-23All four nanobodies were engineered to include either a 6xHis tag or a StrepTagll, generating a total of eight constructs. All constructs were successfully expressed and purified from E. coli expression system. The yield ranged from 1.1 mg / L culture to 22 mg / L culture, indicating the potential of E. coli for high-yield nanobody production. Purified nanobodies were characterized with SDS-PAGE, western blot, and tryptic digestion followed by LC-MS / MS peptide identification (Figure 18A-18b, and data not shown).

[0464] Table 2. Nanobody expression yields.

[0465] Nanobody Yield (mg per L of culture)

[0466] His VHH4 2.76

[0467] His VHH L66 2.79

[0468] His VHH Cl 184 5.4

[0469] His ALX-0171 22

[0470] Strep VHH4 1.5

[0471] Strep VHH L66 1.35

[0472] Strep VHH Cl 184 3

[0473] Strep ALX-0171 1.1

[0474]

[0475] Nanobody pairing investigation

[0476] The binding epitopes of the anti-RSV nanobodies used in this study have been previously determined.

[0477] Although VHH-4 and VHH-L66 differ in sequence, they target overlapping epitopes on the F protein. Since the LFA relies on a heterologous sandwich immunoassay design which requires two anti-RSV nanobodies to bind the antigen sequentially, VHH-4 and VHH-L66 cannot be paired. Although the remaining nanobodies recognize distinct epitopes, sequential binding can still be challenging due to steric hinderance, or conformational change induced by the binding of first nanobody.24

[0478] To evaluate all potential nanobody combinations, all eight nanobodies were used for gold nanoparticle (AuNP) conjugation. His-tagged nanobodies consistently failed to conjugate, showing severe aggregation within five minutes of mixing with AuNPs. In contrast, all four Strep-tagged nanobodies were successfully conjugated. Using these conjugates, every 45808996.1 51 ATTORNEY DOCKET NO. KAUST 2025-014-02 PCT

[0479] combination of AuNP-labeled detection nanobodies and immobilized capture nanobodies was examined (data not shown). As shown in Figure 14, many combinations showed non-specific aggregation which produced false positive test lines, particularly when His -tagged nanobodies were used as capture nanobody. Other combinations failed to generate a test line even at 5 pg / mL prefusion F (preF) protein, which is a relatively high antigen concentration. Benchmark results using commercial RSV test kit are shown in Figure 15. Among all tested pairs, only the combination of Strep- VHH-4 as the detection nanobody and Strep-ALX0171 as the capture nanobody yielded a clear and reliable signal for preF protein (Figure 16).

[0480] Validation of the nanobody-based LFA

[0481] To benchmark the assay, the commercial RSV LFA using preF protein, postF protein, and commercial controls were tested. All were correctly detected. The nanobody pairing identified above was then evaluated. Detection of postF protein using the Strep- VHH-4 / Strep-ALX0171 pair produced a limit of detection (LOD) of approximately 500 ng / mL (Figure 16), indicating substantially lower sensitivity compared to the commercial RSV test.

[0482] Nasopharyngeal fluid testing

[0483] After characterizing detection in running buffer, assay performance in nasopharyngeal fluid was assessed to determine the most clinically relevant sample type for RSV testing. Both preF and postF proteins exhibited markedly improved sensitivity when spiked into nasopharyngeal fluid compared to buffer alone, achieving LODs of 50 ng / mL for both proteins (Figure 17). To exclude the possibility that components of nasopharyngeal fluid caused false-positive signals, Zika NS1 protein was tested under identical conditions. No signal was observed, demonstrating the high specificity of the nanobody-based LFA (Figure 17).

[0484] References

[0485] 1. Staadegaard, et al., Open Forum Infect Dis 2021, 8 (7), ofabl59.

[0486] 2. Cedrone, et al., Diseases 2024, 12 (1).

[0487] 3. Rima, et al., J Gen Virol 2017, 98 (12), 2912-2913.

[0488] 4. Colosia, et al., Influenza Other Respir Viruses 2023, 17 (2), el3100.

[0489] 5. Kaier, et al., Cureus 2023, 15 (3), e36342.

[0490] 6. McLellan, et al., Curr Top Microbiol Immunol 2013, 372, 83-104.

[0491] 7. Magro, et al., Proc Natl Acad Sci USA 2012, 109 (8), 3089-94.

[0492] 8. Chartrand, et al., J Clin Microbiol 2015, 53 (12), 3738-49.

[0493] 9. Onwuchekwa, et al., J Inf ect Dis 2023, 228 (2), 173-184.

[0494] 10. Popow-Kraupp, et al., Open Microbiol J 2011, 5, 128-34.

[0495] 11. Wang, et al., Small Methods 2022, 6 (1), e2101143.

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[0497] 12. Kanwar, et al., Journal of Clinical Virology 2015, 65, 83-86.

[0498] 13. Ince, et al., Trends Analyt Chem 2022, 157, 116725.

[0499] 14. Pedreira-Rincon, et al., Lab Chip 2025, 25 (11), 2578-2608.

[0500] 15. Hamers-Casterman, et al., Nature 1993, 363 (6428), 446-8.

[0501] 16. Alexander, et al., J Nanobiotechnol 2024, 22 (1).

[0502] 17. Jin, et al., International Journal of Molecular Sciences 2023, 24 (6).

[0503] 18. Gutu, et al., Curr Protoc 2025, 5 (10), e70213.

[0504] 19. Peng, et al., ACS Synth Biol 2025.

[0505] 20. Peng, et al., Nanobody-Based Lateral Flow Immunoassay for Rapid Antigen Detection of SARS-CoV-2 and MERS-CoV Proteins. ACS Synth Biol 2025.

[0506] 21. Detalle, et al., Antimicrob Agents Chemother 2016, 60 (1), 6-13.

[0507] 22. Rossey, et al., Nature Communications 2017, 8 (1), 14158.

[0508] 23. Rossey, et al., J Virol 2021, 95 (11).

[0509] 24. Bai, et al., Analytical Chemistry 2022, 94 (51), 17843-17852.

[0510] EXAMPLE 3: Nanobody-Based Lateral Flow Assay for Rapid Zika Virus Detection The Zika virus (ZIKV) belongs to the Flaviviridae family, which also includes Dengue, West Nile virus, Japanese encephalitis, and yellow fever. These viruses are primarily transmitted by ticks and mosquitoes and can cause similar symptoms, but each requires distinct treatments.1,2ZIKV infections often proceed asymptomatically or with mild symptoms. However, the virus can be transmitted through blood transfusions and vertically during pregnancy.1Studies have linked in-utero ZIKV infections with microcephaly, a condition characterized by an abnormally small head in newborns, leading to developmental and neurological impairments. ZIKV infections can also affect the nervous system or cause Guillain-Barre Syndrome in adults.3, 4In 2015, Brazil and several South American countries experienced a ZIKV epidemic which was later declared a global public health emergency by the World Health Organization (WHO).5To date, around 39 regions in North and South America continue to report consistent ZIKV transmission.3Additionally, in 2018, India reported a resurgence of infections.1The often asymptomatic representation complicates clinical surveillance and Zika remains underdiagnosed.6, 7For example, the mosquito vector, Aedes aegypti, is endemic to Saudi Arabia but no cases have been reported to date. However, in a study on 410 pregnant local women, all were negative for acute virus but 6% were positive for ZIKV IgM, suggesting previous infection.7The substantial underdiagnosis of ZIKV infections may lead to undiagnosed health complications in newborns.6, 7

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[0512] ZIKV infection is characterized by two distinct stages: the acute phase (within the first five days of infection) and the convalescence phase (extending six days after the acute phase).8, 9During the acute phase, the virus genome and antigens can be detected in various body fluids, including blood, serum, urine, saliva, semen, and amniotic fluid.10, 11Among these, semen has the highest virus load and the longest virus persistence, but serum and urine are most commonly used for virus detection.12 14In the convalescence phase, antibodies produced by the immune response, immunoglobulin M (IgM) and IgG, can be detected.9

[0513] NS 1 is a critical viral protein that has emerged as an important diagnostic biomarker for multiple flavivirus infections.38This multifunctional protein exists in two distinct forms: a membrane-associated form that is essential for viral replication and a soluble form that is actively secreted into the bloodstream.38The secreted NS1 can be readily detected in various non-invasive biological specimens, including blood serum, urine, and saliva. The protein begins to appear in patient samples within hours of initial viral infection, significantly earlier than detectable antibody responses. This early secretion pattern, combined with its presence in easily accessible body fluids, makes NS1 an ideal target for rapid diagnostic testing.39We here further characterize anti-NSl nanobodies and integrate them into a nanobody-based lateral flow immunoassay for rapid and efficient POC diagnostics of ZIKV during the acute infection phase.

[0514] A. Material and Methods

[0515] Materials

[0516] All target proteins were produced by commercial providers in HEK293 cells with C-terminal His-tag. The ZIKV SPH2015 NS1 target protein was purchased from Sinobiological (cat: 40544-V07H, Supplementary 7). All four of the Dengue NS1 target proteins were purchased from the Native Antigen Company (cat: DENVX4-NS1-100). Anti-VHH antibody (cat: 128-005-232) was purchased from Jackson ImmunoResearch (West Grove, PA, USA). All the components used in LFA strips (Product code: 07.700.30) were purchased from ClaremontBio (Upland, CA, USA). Gold nanoparticles (cat: AUNR40, AUXR40) were purchased from nanoComposix (San Diego, CA, USA). All other chemicals and solvents in case not specified were purchased from Sigma-Aldrich (St. Louis, MO, USA) and ThermoFisher Scientific (Waltham, MA, USA).

[0517] Expression, and purification of strep-tagged nanobodies

[0518] The nanobody constructs were designed with a C-terminal StrepTag-II in vector pJE411c (in-house modified from DNA 2.0 pJExpress411, Supplementary 6), codon optimized for E. coli expression using an in-house Python script based on DNAChisel. The constructs were gene-synthesized by Twist Bioscience.40, 41

[0519] 45808996.1 54 ATTORNEY DOCKET NO. KAUST 2025-014-02 PCT

[0520] Plasmids were transformed by heat shock into chemically competent E. coli BL21(DE3) cells. Overnight starter cultures were inoculated from a single colony and used to inoculate IL 2xYT production cultures at 1:100 ratio, supplemented with 50 mg / L kanamycin and 0.5% w / v glucose. Cultures were incubated at 37°C and 220-250 rpm, induced with isopropyl beta-D-1- thiogalactopyranoside (IPTG, 0.25 mM final concentration) between 0.6 to 0.8 OD₆₀₀ and expressed for 4h at 37 °C and 220-250 rpm. Cells were harvested by centrifugation for 10 min at 6000 g, washed twice with 20-30 mL ice-cold PBS and stored at -80°C.

[0521] Proteins were extracted from the periplasm using osmotic shock.42The pellet was thawed and resuspended in extraction buffer I (30 mM Tris-HCl pH 8.0, 20% w / v sucrose, 1 mM EDTA). Resuspended cells were stirred slowly at room temperature for 10 minutes until the solution turned homogenous. Supernatant I was collected by centrifugation (10 min @ 10,000 g, 4°C). The pellet was resuspended in 30 ml ice-cold 5 mM Mg2SC>4 and then stirred for 15 min on ice and supernatant II was again collected by centrifugation. Both supernatants were combined, centrifuged at 30,000 g for 45 min at 4°C and filtered through MiraCloth filter (Merck Millipore, Cat: 475855-1R). The clarified sample was applied to a 5 mL StrepXT column (Cytivia) washed with binding buffer (100 mM Tris-HCl pH 8.0, 150 mM NaCl, 1 mM EDTA, 10 % glycerol) and eluted after 20 min incubation with 50 mM D-biotin in binding buffer. The column was regenerated between purifications with 50 mM NaOH. Fractions were verified by SDS-PAGE, pooled and concentrated with lOkDa centrifugation filters (Amicon Ultra, Merck Millipore, Cat: UFC901024) and subjected to gel filtration on a 16 / 600 Superdex 75 size exclusion column (Cytivia) in gel filtration buffer (20 mM HEPES pH 7.4, 300 mM NaCl, 1 mM EDTA, 10% glycerol). Fractions were analyzed, pooled, concentrated and final concentrations determined by 280 nm absorbance (NanoDropOne, ThermoFisher) using the sequence-specific extinction coefficient. Samples were aliquoted, flash frozen in liquid N2 and stored at -80°C in gel filtration buffer.

[0522] Expression, and purification of GST-tagged nanobodies

[0523] The nanobody constructs were designed with an N-terminal GST- in vector pET29b, codon optimized for E. coli expression and synthesized by Twist Bioscience. Nanobody sequences are shown below.

[0524] NbD6-Strep KYLLPTAAAGLLLLAAOPAMAEVOLVESGGGSVOPGGSLRLSCAASGFAFSHY (pelB signal AMRWVRQAPGKGLEWVSVINSDGEDTWYADSVKGRFTISRDNAKNTLYLQM peptide NSLKPEDTGVYYCAIGRTHDTKGQGTQVTVSSSGLEVLFQGPTGSAWSHPQFE underlined) KV (SEQ ID NO:23).

[0525] Nb32-Strep KYLLPTAAAGLLLLAAQPAMAEVOLVESGGGLVQAGGSLRLSCAVSGIDFSRY

[0526] AITWNRQSPGNQRREWVATLPPADTTVYADAVKGRFTISRDNTKNTVYLQMN

[0527]

[0528] 45808996.1 55 ATTORNEY DOCKET NO. KAUST 2025-014-02 PCT

[0529] (pelB signal SLKPEDTAVYYCATSPRIHNWGQGTQVTVSSSGLEVLFQGPTGSAWSHPQFEK peptide V (SEQ ID NO:20).

[0530] underlined)

[0531] GST-NbD6 MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKKFELGLEFP NLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRYGVS RIAYSKDFTiTLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALD VVLYMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGG DHPPKSDLVPRGSSSGSGSGSGSGSTGEVQLVESGGGSVQPGGSLRLSCAASGF AFSHYAMRWVRQAPGKGLEWVSVINSRKSEDT WYADSVKGRFTISRDNAKNTL YLQMNSLKPEDTGVYYCAIGRTHDTKGQGTQVTVS (SEQ ID NO:21).

[0532] GST-Nb32 MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKKFELGLEFP NLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRYGVS RIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDHVILYDALD VVLYMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATPGGG DHPPKSDLVPRGSSGSGSGSGSGSTGEVQLVESGGGLVQAGGSLRLSCAVSGID FSRYAItWNRQSPGNQRREWVATLPPADTTVYADAVKGRFTISRDNTKNTVYL QMNSLKPEDTAVYYCATSPRIHNWGQGTQVTVS(SEQ ID NO:22).

[0533]

[0534] Plasmids were transformed into E. coli SHuffle T7 competent cells (New England Biolabs, C3026J) using heat shock according to the manufacturer. Overnight starter cultures were inoculated from a single colony and used to inoculate IL LB (Lennox) cultures at 1:100 ratio, supplemented with 50 pg / L kanamycin. Cultures were incubated at 30°C and 200 rpm, induced with 0.5mM IPTG between 0.4 to 0.6 OD₆₀₀ and expressed for 36h at 16°C. Cells were harvested by centrifugation at 6000 g at 4 °C for 30 min, resuspended in lysis buffer [lx phosphate-buffered saline (PBS), 10 mM MgCl₂, 10 mM Imidazole, 10 pg / ml DNase (SIGMA DN25), 1 tablet protease inhibitor (ThermoScientific Cat: A32963) per 50 ml lysis buffer] and lysed on a Cell Disruptor (Constant Systems, CF1). Cell debris was removed by centrifugation at 24000 g at 4 °C for 1 h.

[0535] GST-tagged nanobodies were purified using Glutathione MagBeads (GenScript L00895) following the procedures provided in the product manual. After a 2 h incubation with MagBeads at 4°C, nanobodies were eluted with 10 mM reduced glutathione in 50 mM Tris-HCl, pH 8.0. Proteins were further purified by size exclusion chromatography as described above albeit using PBS buffer. Purified proteins were stored at -80°C in PBS buffer supplemented with 20% glycerol.

[0536] BLI binding kinetics and competitive assay

[0537] Biolayer interferometry (BLI) binding kinetics was measured on an Octet RH96 (Sartorius), following the measurement protocol shown in Table 3, and analyzed in the Octet 45808996.1 56 ATTORNEY DOCKET NO. KAUST 2025-014-02 PCT

[0538] Analysis software version 13.0.0.32. Sensors tips were hydrated for 10 min in binding buffer (PBS, with 0.02% Tween-20 and 0.1% bovine serum albumin). Experiments were performed at 25°C and 1000 rpm shaking in 96 and 384 well plates in 200 pL and 80 pL volumes, respectively. Optimized loading conditions for NS 1 glycoprotein onto Ni-NTA sensor tips were 600 seconds, using a protein concentration of 100 nM, in binding buffer. Optimized conditions for loading GST-tagged nanobodies onto anti-GST sensor tips were 400 seconds, with a protein concentration of 100 nM, in binding buffer. Sensor tips were regenerated (up to five times) with 10 mM Glycine pH 2.0, neutralized in binding buffer and activated with 10 mM NiSO4.

[0539] For the competitive assay, Ni-NTA tips loaded with NS1 were first saturated with ZIKV_Nb32 and a regular binding experiment was then performed with different concentrations of ZIKV_NbD6 as the competing nanobody.

[0540] Table 3. Summary of the steps used for the Octet experiments.

[0541] Step Step name Time [s] Solution

[0542] 1 Baseline 60 buffer

[0543] 2 Loading 400 - 600 100 nM ligand protein

[0544] 3 Wash 30 buffer

[0545] 4 Baseline 2 120 buffer

[0546] 5 Association 450 variable analyte cone.

[0547] 6 Dissociation 600 buffer

[0548] 7 Regeneration / Neutralization 60 10 mM Glycine pH 2.0

[0549] 8 Activation 30 10 mM NiSO4

[0550]

[0551] Characterization of Zika NS1 by mass photometry

[0552] The molecular mass distribution of the Zika NS1 glycoprotein was measured on the Refeyn TwoMP Mass Photometer using the AcquireMP software (v2024 R2.1) following the manufacturer’s instructions. Per measurement, 5 pL of PBS buffer was mixed with 5 pL of sample in the 10 pl sample well and collision events were recorded for 60s. Zika NS1 glycoprotein was tested at four different final concentrations: 25, 50, 100, and 250 nM.

[0553] Commercially purchased proteins — ovalbumin (Cytiva, Gel Filtration Calibration Kit), bovine serum albumin (BSA, Sigma-Aldrich, 9048-46-8), and catalase (Sigma-Aldrich, C100-50MG) — were used for mass calibration at concentrations of 50 nM, 50 nM, and 17 nM, respectively. Data were analyzed in the manufacturer’s DiscoverMP software (v2024 R2.1).

[0554] 45808996.1 57 ATTORNEY DOCKET NO. KAUST 2025-014-02 PCT

[0555] Preparation of nanobody-AuNP biconjugate

[0556] Nanobodies were dialyzed overnight against 8 mM potassium phosphate buffer (pH 7.4) and conjugated with Bioready 40 nm Gold Nanospheres-Carboxyl nanoparticles (OD 20, nanoComposix) according to the manufacturer’s protocol with the following adjustments: Sulfo-NHS and l-ethyl-3-(3-dimethylaminopropyl) carbodiimide-HCl (EDC) were made freshly at 10 mg / ml in water before addition to the carboxyl AuNP solution. After 30 min incubation with 20 rpm rotation on a Tube Revolver Rotator (Thermo Scientific™) at room temperature, excess sulfo-NHS and EDC were washed by centrifugation at 3800 g for 10 min. The supernatant was removed, and the pellet was resuspended in reaction buffer (5 mM potassium phosphate, pH 7.4, 0.5% PEG 12000). This wash step was repeated once. Then 30 pg nanobody per ml AuNP was added for conjugation. After rotation at room temperature for 1 h, the reaction was quenched with 50% (w / v) hydroxylamine and incubated for another 10 min. The reaction mix was centrifuged at 3800 g for 10 min, the supernatant was removed and the pellet was resuspended in reaction buffer (5 mM potassium phosphate, pH 7.4, 0.5% 12000 MW PEG). This wash step was repeated twice. The washed pellet was resuspended in storage buffer (DDS Diagnostic Company, Catalog 1933) and stored at 4 °C.

[0557] Preparation of LFA strips

[0558] The conjugate pad (Glass fiber Ahlstrom 8964) was pretreated with conjugate blocking buffer (10 mM Phosphate buffer, pH 7.4, 2% (w / v) BSA, 2.5% (w / v) sucrose, 0.3% (w / v) PVP, l%(w / v) Triton X-100, and 0.02% (w / v) NaNq and dried at 37 °C for 1 h. The nanobody-AuNP biconjugate was applied using the automated lateral flow reagent dispenser (ALFRD, ClaremontBio) at 20 pl / cm. The detection pad used in this assay was a nitrocellulose membrane (MD100) and the proteins for control line (0.6 mg / ml) and test lines (1.5 mg / ml) were dispensed on it at 0.83 pl / cm. The nitrocellulose membranes were dried at 37 °C for 30 min after printing and then blocked with blocking buffer (5 mM potassium phosphate, pH 8.0, 0.6% BSA) for 30 min and dried at 37 °C for another 30 min. For assembly, sample pad (glass fiber Ahlstrom 8964), conjugate pad, nitrocellulose membrane and absorbent pad (AHL238 cotton fiber) were pasted on the backing plate (ClaremontBio Adhesive Backing Card) and cut into 3 mm strips using the High-Speed Test Strip guillotine Cutter (Werfen Equipment). Freshly made LFA strips were stored at 4 °C with desiccant packs.

[0559] Detection of antigens in buffer or spiked body fluids

[0560] Standard samples were prepared by diluting antigen with running buffer (20 mM Tris / HCl, pH 8.0, 0.5% BSA, 0.5% Triton X-100). ZIKV NS1 protein (strain: SPH2015) spiked into bovine urine (ERM-BB386) or Normal Human Serum (Sigma-Aldrich) were used to 45808996.1 58 ATTORNEY DOCKET NO. KAUST 2025-014-02 PCT

[0561] simulate actual biological samples. 50 pl of each sample was added to the sample pad of LFA strips.

[0562] B. Results and Discussion

[0563] Experimental design

[0564] The nanobody-based LFA employs a heterologous sandwich immunoassay involving two anti-ZIKV nanobodies, ZIKV_NbD6 and ZIKV_Nb32.4The detection nanobody ZIKV_NbD6 is conjugated with AuNP and applied to the conjugate pad. ZIKV_Nb32 serves as the capture nanobody and is immobilized on the nitrocellulose (NC) membrane. In the presence of the ZIKV NS1 protein antigen, the AuNP-NbD6: NSl complex is immobilized by ZIKV_Nb32 at the test line, resulting in a visible red band. Residual conjugated ZIKV_NbD6 is captured by an anti-VHH antibody at the control line, producing a second band. In the absence of the antigen, only the control line will display a band. The absence of a band at the control line indicates an invalid result (Figure 1C).

[0565] Nanobody expression and binding characterization

[0566] Two different expression and purification strategies for each of the two previously reported anti-ZIKV nanobodies were evaluated: (i) cytoplasmic expression in E. coli T7 SHuffle using a GST-tag for solubilization and affinity purification, and (ii) periplasmic expression from E. coli BL21(DE3) based on the pelB secretion signal and the much smaller StrepTag II affinity tag (Figure 2).

[0567] Both methods are assumed to foster the formation of the disulfide bridge that is part of the nanobody core structure.43However, yields per liter of bacterial culture were generally low and did not indicate a clear preference for one or the other strategy (Table 4).

[0568] Table 4. Nanobody expression yields.

[0569] Nanobody Yield

[0570] (mg per L of culture)

[0571]

[0572] GST-Nb32 0.60

[0573] GST-NbD6 0.70

[0574] Nb32-Strep 1.50

[0575] NbD6-Strep 0.07

[0576] Periplasmic nanobody production is typically in the low mg range and usually lower than cytoplasmic expression.44Nb32-Strep fell within this expected range (Table 2) while NbD6-Strep yield was very low. Yields were also relatively low for the expression of the GST-tagged nanobody versions from the E. coli SHuffle cytosol, even though the identical protocol for high 45808996.1 59 ATTORNEY DOCKET NO. KAUST 2025-014-02 PCT

[0577] yield expression (up to 12 mg / L) of His-tagged anti-SARS-CoV-2 and anti-MERS nanobodies was used.45This suggests that Nb32 and NbD6 may be more demanding expression targets and may require further optimizations for production scale up.

[0578] The original report of Nb32 and NbD6 only provided relative affinities estimated by ELISA.4The binding kinetics for both proteins was tested by biolayer interferometry (BLI). A setup comparable to the planned LFA setup was tested initially whereby the nanobodies immobilized were exposed on sensor tips to the target protein in solution. The immobilization of GST-tagged nanobodies led to a surprisingly tight NS1 binding with unmeasurably low off rate resulting in a low KD below 1 pM (Figure 3A).

[0579] Deviations from the 1:1 binding model (data not shown) were noted and NS1 oligomerization contribution to binding artifacts was pondered. Various oligomer states have been observed for flavi virus NS1 glycoproteins.4, 47, 48Dengue NS1 adopts a hexamer state in blood.49, and a mixture of oligomeric forms, including tetramers and hexamers, has been reported in vitro.50By contrast, a tetrameric structure was proposed for the Zika NS I50and a recent cryo-EM study described two different tetrameric (dimer of dimers) conformations.51The Zika NS1 target protein was examined by mass photometry which estimates molecular mass from light scattering signals of single particle collisions with a glass surface (Figure 21 A, data shown). A dynamic mixture of oligomeric states across the 25 to 100 nM concentration range that is accessible to the method was observed. The monomer was prevalent at or below 50 nM whereas, in line with the previous report, the tetramer state dominated at 100 nM and above. However, indications of dimers and dimers of tetramers were found (Figure 2 IB).

[0580] Further, the likelihood that NS1 avidity, i.e., the simultaneous binding of a single NS1 protein multimer to multiple immobilized nanobodies, could lead to an over-estimation of affinity was considered. In addition, individually captured NS1 monomers could also oligomerize on the surface preventing their unbinding. Indeed, the reverse orientation, with His-tagged NS1 immobilized and GST-tagged Nanobodies in solution, increased koff by at least two orders of magnitude (Figure 20B). The result was a still very tight but now measurable equilibrium dissociation constant of 35 pM for Nb32 and 1.1 nM for NbD6. The potential GST homodimerization among NS 1 -bound nanobodies creating avidity effects which would artificially reduce the off rate was noted. This experiment was repeated with StrepTag-II nanobodies lacking the GST domain (Figure 20C). This further multiplied the unbinding rate by a factor of 15-fold (Nb32) or 26-fold (Nb6) suggesting that GST dimerization had indeed reduced unbinding in the previous experiment. Interestingly, also the on-rate was (moderately) accelerated by 3.2-fold (Nb32) or 3.6-fold (Nb6), likely owing to the faster diffusion of the 2.6- 45808996.1 60 ATTORNEY DOCKET NO. KAUST 2025-014-02 PCT

[0581] fold lighter nanobody-only protein (15 kDa versus 40 kDa). The combined effects resulted in a KD of 0.16 nM for Nb32 and 8 nM for NbD6 which we consider the best estimate for the interaction of the single nanobody with a single NS1 binding site.

[0582] These results confirm the tight interaction between both nanobodies and Zika NS1 antigen and indicate that the oligomeric state of NS1 should, in fact, further improve its capture on nanobody-coated surfaces. However, the precise structural arrangement remains unclear. Therefore confirmation that both nanobodies can bind simultaneously to distinct NS1 epitopes was explored. Indeed, the initial binding of NbD32 did not significantly affect the subsequent binding kinetics of NbD6 (Figure 22A-22C). This supports the use of both nanobodies in a sandwich assay. Based on the stronger binding and slower dissociation, continuation with Nb32 as the capture nanobody and NbD6 as the detection nanobody was chosen.

[0583] Validation of the nanobody-LFA

[0584] Inspired by a previous report on the modular assembly of GST-tagged proteins onto gold nanoparticles,52an LFA with the two GST-tagged nanobody versions was built. NbD32-GST was printed on the LFA nitrocellulose membrane and conjugated NbD6-GST with gold nanoparticles which were subsequently soaked into the LFA conjugate pad. Different concentrations of purified Zika NS1 were then added to the LFA sample pad and allowed to run for 15 minutes at room temperature. The resulting bands were visible to the naked eye (Figure 23A-23C). It should be noted that, when observed directly, the lines appear much clearer than in the photographs presented here. However, a distinct band appearing at the test line was observed even without the presence of antigen in the running buffer, indicating a false positive result (Figure 22A). The LFA using only Strep-tagged nanobodies was assembled next. As shown in Figure 22B, the buffer negative control sample displayed a distinct band only at the control line (printed with anti-VHH antibody). The NS1 sample showed both test and control lines indicating the specific binding between NbD6-Strep: AuNP, NS1 and immobilized NbD32.

[0585] To further explore the effect of the GST-tag, a mixed setup using a GST-tagged capture nanobody (NbD32-GST) alongside a Strep-tagged detection nanobody (NbD6-Strep: AuNP) was examined. This layout likewise eliminated the false positive results (Figure 22C). The initial false positive effect therefore likely stems from the dimerization between GST tags on both the detection and capture nanobodies.53The sensitivities for both the StrepTag-only and the mixed StrepTag / GST-tag LFA architecture were assessed.

[0586] A 10-fold dilution series of ZIKV NS1 protein ranging from 2.5 pg / ml to 0.25 ng / ml to LFA test strips was applied in order to determine the limit of detection (LOD) of both versions. Clear bands at the test line were visible at 25 ng / ml antigen concentration for both LFA versions 45808996.1 61 ATTORNEY DOCKET NO. KAUST 2025-014-02 PCT

[0587] (Figure 24A-24B). The LOD of the ZIKV nanobody-LFA falls within the typical range for such assays, and is comparable to the 18 ng / ml LOD reported in a previous monoclonal antibodybased sandwich LFA against ZIKV NS1 protein.31The Strep-tagged Nanobody LFA version was used in further experiments as it consistently yielded better visible bands with higher contrast.

[0588] Serum and urine testing

[0589] The Nanobody-LFA with body fluid samples was tested. Serum and urine are two commonly used body fluids for early-stage ZIKV detection.9, 54Among these, urine is the more easily accessible, non-invasive specimen. Moreover, urine has been reported to be more sensitive for Zika diagnosis than serum, with a longer detection duration.54-57However, ZIKV NS1 levels in body fluids are still largely unknown.58In a previous study using ZIKV-infected Vero cells, the extracellular concentration of ZIKV NS1 was found to be around 800 ng / ml.31Using a human cell line, secreted ZIKV NS1 levels were observed to be within 400-500 ng / ml.20Overall, high sensitivity is essential for ZIKV detection, and a suitable POC diagnostic should be in the nanogram detection range. To verify that the LFA can detect antigens not only in a defined running buffer but also in more complex physiological environments, different concentrations of recombinant NS1 protein were spiked into human serum and bovine urine. Despite the higher complexity of serum compared to running buffer, the test line did not show any false positive bands in the absence of antigen (Figure 25A). When the antigen was diluted in 100% human serum, a similar sensitivity to that in running buffer, 20 ng / ml, was observed (Figure 25 A). This result is comparable to a previous aptamer-based ELISA analysis of ZIKV NS1 protein, which reached 10 ng / ml in 100% human serum.18The Nanobody-LFA performed better in bovine urine than both in buffer and human serum, achieving a 20-fold lower LOD of about 1 ng / ml NS1 protein (Figure 25B). The conclusion that the nanobody-LFA detects the ZIKV NS1 protein in various physiological fluids at high sensitivity was observed.

[0590] Specificity

[0591] One challenge of ZIKV diagnosis is the potential cross-reactivity with other flaviviruses, particularly with Dengue virus.59-61The concentration of Dengue NS1 protein in patient serum is typically around 15 pg / ml two days post-infection.58ZIKV NS1 concentrations are not yet well established but expected to be lower.58BLI binding assays were performed between the recombinant NS1 proteins from all four Dengue serotypes and the two Zika nanobodies (data not shown). In line with the ELISA results from the original nanobody study,4any cross-reactivity between Zika nanobodies and Dengue NS1 proteins was not observed. The Nanobody-LFA with the four serotypes of Dengue NS1 at concentrations ranging from 8.5 pg / ml to 26.6 pg / ml was

[0592] 45808996.1 62 ATTORNEY DOCKET NO. KAUST 2025-014-02 PCT

[0593] tested. No positive bands were observed (Figure 26), demonstrating the high specificity of our LFA for ZIKV detection.

[0594] Stability

[0595] Storage stability is another critical feature for guaranteeing LFA affordability and accessibility. The functionality of the LFA after one month of storage at both room temperature and at 4 °C was tested. The lateral flow strips stored under both room temperature and refrigerated conditions were subjected to weekly functionality tests (Figure 28). All strips consistently demonstrated full functionality after storage for 28 days (Figure 27). However, the bands were not as clearly visible when stored at room temperature. This indicates that, while the LFA retains functionality over time. Possible approaches include the freeze-drying of decorated AuNPs or test strips in combination with buffer optimization and cryoprotectants.62, 63Nanobodies can typically be lyophilized64with some studies reporting that cryoprotectants are unnecessary.65, 66

[0596] Conclusion and Outlook

[0597] ZIKV infections have been a concern for several decades, yet low-cost rapid detection methods remain elusive.67A fully nanobody-based LFA for the detection of ZIKV NS1 was developed. Two previously developed anti-NSl nanobodies recognize NS1 with very high affinity and high specificity. In particular, the lack of cross-reactivity with Dengue NS1 bodes well for real-world diagnostic applications. Interestingly, the oligomerization of the NS1 target protein appears to stabilize its capture on densely coated nanobody surfaces. Avidity-supported capture could also be utilized as a generally applicable design strategy for other multimeric targets. Among different purification strategies and purification tags, periplasmic Strep-tagged nanobodies achieved the highest detection sensitivity.

[0598] Compared to other established ZIKV detection methods, such as nucleic acid amplification, ELISA, and surface-enhanced Raman spectroscopy immunoassay,68-70the nanobody-LFA offers the advantage of supporting rapid point of care or self-testing. The test is not only cheaper to manufacture but also easier to use, while still achieving relatively high sensitivity. Notably, the highest sensitivity was observed when the NS1 protein was detected from urine. Urine is a well-established medium for nucleic acid and antibody tests, and there is growing evidence that viral antigens can be carried over or even enriched in this easily accessible fluid.71, 72In fact, LFA-based detection of Zika antigens in urine could be compatible with standard pregnancy tests, suggesting the value of a combined test that could flag pregnancies at risk of Zika-related microcephaly. The ZIKV nanobody-LFA meets the World

[0599] 45808996.1 63 ATTORNEY DOCKET NO. KAUST 2025-014-02 PCT

[0600] Health Organization’s ASSURED guidelines, which emphasize low cost, the ability to detect from unprocessed samples, and results that can be read with the naked eye.

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[0675] 45808996.1 66

Claims

ATTORNEY DOCKET NO. KAUST 2025-014-02 PCTCLAIMSWe claim:

1. A lateral flow assay device for detecting the presence of an analyte in a sample, comprising a solid substrate comprising:(i) a sample application point,(ii) a conjugate zone comprising a plurality of a first nanobody (“conjugate nanobody”) specific for the analyte, wherein the conjugate nanobody comprising a detectable label, (iii) a capture zone comprising a test line on which is immobilized a plurality of a second nanobody specific for the analyte (capture nanobody), and(iv) a control zone comprising a test line on which is immobilized a plurality of binding partners specific for the conjugate nanobody,wherein the analyte is a SARS-CoV-2 antigen, MERS-CoV antigen, RSV antigen and / or Zika virus antigen.

2. The device of claim 1, wherein the plurality first nanobodies specific for the analyte are immobilized in the conjugate zone.

3. The device of claims 1 or 2, wherein the plurality of second nanobodies specific for the analyte are immobilized on a capture line in the capture zone.

4. The device of any one of claims 1-3, wherein the conjugate nanobodies and the capture nanobodies bind to different epitopes on the analyte.

5. The device of any one of claims 1-4, wherein the detectable label is selected from the group consisting of gold nanoparticles, latex microparticles (LMPs), magnetic nanoparticles (MNPs), quantum dots (QDs), carbon nanoparticles (CNPs), silica nanoparticles and europium nanoparticles.

6. The device of any one of claims 1-5, wherein the conjugate nanobody is conjugated to the detectable label via a modulator domain selected from the group consisting of strep-tag (WSHPQFEK (SEQ ID N:23) or AWAHPQPGG ((SEQ ID N:23) or a lysine rich protein tag selected from the group consisting of strep a spycatcher having at least 7 lysine residues, optionally, via a flexible linker.

7. The device of any one of claims 1-6, comprising a first and a second solid substrate comprising, each comprising:(i) an application point,(ii) a conjugate zone comprising a plurality of a first nanobody (“conjugate nanobody”) specific for the analyte, wherein the conjugate nanobody comprising a detectable label,45808996.1 57ATTORNEY DOCKET NO. KAUST 2025-014-02 PCT(iii) a capture zone comprising a test line on which is immobilized a plurality of a second nanobody specific for the analyte (capture nanobody), and(iv) a control zone comprising a test line on which is immobilized a plurality of binding partners specific for the conjugate nanobody,wherein the analyte is a SARS-CoV-2 antigen, MERS-CoV antigen, RSV antigen and / or Zika virus antigen,wherein the conjugate and capture nanobodies in the first a solid substrate are specific for a first analyte and the conjugate and capture nanobodies in the second solid substrate are specific for a second and different analyte.

8. The device of any one of claims 1-6, wherein(a) the conjugate zone comprises a plurality of a first conjugate nanobody specific for a first analyte and a plurality of a second conjugate nanobody specific for a second and different analyte,(b) a first capture zone comprising a first test line on which is immobilized a plurality of a first capture nanobody specific for the first analyte and a second capture zone comprising a first test line on which is immobilized a plurality of a second capture nanobody specific for the second analyte, and(c) a control zone comprising a test line on which is immobilized a plurality of binding partners specific for: (a) the first conjugate nanobody specific for a first analyte or the second conjugate nanobody.

9. The device of any one of claims 1-8, for detecting a SARS-CoV-2 antigen wherein the conjugate zone or test line comprises a nanobody is selected from the group consisting of NM1226, NM1230, C5, F2, VHH55, VHH1, VHH83, and VHH84.

10. The device of claim 9, wherein the conjugate zone or test line comprises NM1226 and / or NM1230, respectively.

11. The device of any one of claims 1-8, for detecting a MERS-CoV antigen, wherein the conjugate zone or test line comprises a nanobody selected from the group consisting of VHH83, and VHH84, optionally, wherein VHH83 is the conjugate nanobody.

12. The device of any one of claims 1-8, for detecting a RSV antigen, wherein the conjugate zone or test line comprises a nanobody selected from the group consisting of VHH4 and ALX0171, optionally, wherein VHH4 is the conjugate nanobody.

13. The device of any one of claims 1-8, for detecting a zika virus antigen, wherein the conjugate zone or test line comprises a nanobody selected from the group consisting of ZIKV_NbD6 and ZIKV_Nb32, optionally, wherein ZIKV_NbD6 is the conjugate nanobody.45808996.1 68ATTORNEY DOCKET NO. KAUST 2025-014-02 PCT14. A method of detecting an analyte in a sample comprising contacting the sample with the device of any one of claims 1-13.

15. The method of claim 14, wherein the analyte is a SARS-CoV-2 and / or a MERS-CoV antigen.

16. The method of claim 15, wherein the analyte is a SARS-CoV-2 antigen, and wherein the conjugate or capture nanobody is NM1226 or NM1230.

17. The method of claim 15, wherein the analyte is a MERS-CoV antigen, and wherein the conjugate nanobody is VHH83 and the capture nanobody is VHH1 orVHH84.

18. The method of claim 14, wherein the analyte is a SARS-CoV-2 antigen and a MERS-CoV antigen.

19. The method of any one of claims 14-18, comprising mixing the sample with a running buffer, wherein the running buffer is at a pH of about 8, optionally, wherein the running buffer comprises Tris / HCl.

20. The device of any one of claims 1-13, comprising the nanobody sequence in any one of SEQ ID Nos: 2-9 and 13-22 or a combination thereof, or an antigen-binding fragment thereof comprising amino acid sequences having about 70%, about 80%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99% sequence identity to the nanobody sequence in SEQ ID Nos: 2-9 and 13-22.45808996.1 69

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