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(A) Schematic illustration of the workflow for cell-free production of nanobody-coiled coil conjugates for RAMONA. (B) Cell-free screen of nanobody-coiled coil conjugate pairs against various target antigens, prepared as 200nM pure proteins dissolved in PBS. Each antigen was co-incubated with crude cell-free lysates containing the functionalized nanobodies in equivolumetric parts for 15 minutes at room temperature before addition to lateral flow strips for readout. (C) Performance of purified and cell-free produced nanobody-coiled coil conjugates Nb11 and Nb19 against serially titrated concentrations of <t>ApoA1.</t> (D) Performance of purified and cell-free produced nanobody-coiled coil conjugates VF1 and VF2 against serially titrated concentrations of Fib. Error bars indicate standard deviation across n=3 independent replicate experiments, and statistical significance was determined by two-way ANOVA.
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(A) Schematic illustration of the workflow for cell-free production of nanobody-coiled coil conjugates for RAMONA. (B) Cell-free screen of nanobody-coiled coil conjugate pairs against various target antigens, prepared as 200nM pure proteins dissolved in PBS. Each antigen was co-incubated with crude cell-free lysates containing the functionalized nanobodies in equivolumetric parts for 15 minutes at room temperature before addition to lateral flow strips for readout. (C) Performance of purified and cell-free produced nanobody-coiled coil conjugates Nb11 and Nb19 against serially titrated concentrations of <t>ApoA1.</t> (D) Performance of purified and cell-free produced nanobody-coiled coil conjugates VF1 and VF2 against serially titrated concentrations of Fib. Error bars indicate standard deviation across n=3 independent replicate experiments, and statistical significance was determined by two-way ANOVA.
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(A) Schematic illustration of the workflow for cell-free production of nanobody-coiled coil conjugates for RAMONA. (B) Cell-free screen of nanobody-coiled coil conjugate pairs against various target antigens, prepared as 200nM pure proteins dissolved in PBS. Each antigen was co-incubated with crude cell-free lysates containing the functionalized nanobodies in equivolumetric parts for 15 minutes at room temperature before addition to lateral flow strips for readout. (C) Performance of purified and cell-free produced nanobody-coiled coil conjugates Nb11 and Nb19 against serially titrated concentrations of <t>ApoA1.</t> (D) Performance of purified and cell-free produced nanobody-coiled coil conjugates VF1 and VF2 against serially titrated concentrations of Fib. Error bars indicate standard deviation across n=3 independent replicate experiments, and statistical significance was determined by two-way ANOVA.
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(A) Schematic illustration of the workflow for cell-free production of nanobody-coiled coil conjugates for RAMONA. (B) Cell-free screen of nanobody-coiled coil conjugate pairs against various target antigens, prepared as 200nM pure proteins dissolved in PBS. Each antigen was co-incubated with crude cell-free lysates containing the functionalized nanobodies in equivolumetric parts for 15 minutes at room temperature before addition to lateral flow strips for readout. (C) Performance of purified and cell-free produced nanobody-coiled coil conjugates Nb11 and Nb19 against serially titrated concentrations of <t>ApoA1.</t> (D) Performance of purified and cell-free produced nanobody-coiled coil conjugates VF1 and VF2 against serially titrated concentrations of Fib. Error bars indicate standard deviation across n=3 independent replicate experiments, and statistical significance was determined by two-way ANOVA.
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(A) Schematic illustration of the workflow for cell-free production of nanobody-coiled coil conjugates for RAMONA. (B) Cell-free screen of nanobody-coiled coil conjugate pairs against various target antigens, prepared as 200nM pure proteins dissolved in PBS. Each antigen was co-incubated with crude cell-free lysates containing the functionalized nanobodies in equivolumetric parts for 15 minutes at room temperature before addition to lateral flow strips for readout. (C) Performance of purified and cell-free produced nanobody-coiled coil conjugates Nb11 and Nb19 against serially titrated concentrations of <t>ApoA1.</t> (D) Performance of purified and cell-free produced nanobody-coiled coil conjugates VF1 and VF2 against serially titrated concentrations of Fib. Error bars indicate standard deviation across n=3 independent replicate experiments, and statistical significance was determined by two-way ANOVA.
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(A) Schematic illustration of the workflow for cell-free production of nanobody-coiled coil conjugates for RAMONA. (B) Cell-free screen of nanobody-coiled coil conjugate pairs against various target antigens, prepared as 200nM pure proteins dissolved in PBS. Each antigen was co-incubated with crude cell-free lysates containing the functionalized nanobodies in equivolumetric parts for 15 minutes at room temperature before addition to lateral flow strips for readout. (C) Performance of purified and cell-free produced nanobody-coiled coil conjugates Nb11 and Nb19 against serially titrated concentrations of <t>ApoA1.</t> (D) Performance of purified and cell-free produced nanobody-coiled coil conjugates VF1 and VF2 against serially titrated concentrations of Fib. Error bars indicate standard deviation across n=3 independent replicate experiments, and statistical significance was determined by two-way ANOVA.
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(A) Schematic illustration of the workflow for cell-free production of nanobody-coiled coil conjugates for RAMONA. (B) Cell-free screen of nanobody-coiled coil conjugate pairs against various target antigens, prepared as 200nM pure proteins dissolved in PBS. Each antigen was co-incubated with crude cell-free lysates containing the functionalized nanobodies in equivolumetric parts for 15 minutes at room temperature before addition to lateral flow strips for readout. (C) Performance of purified and cell-free produced nanobody-coiled coil conjugates Nb11 and Nb19 against serially titrated concentrations of ApoA1. (D) Performance of purified and cell-free produced nanobody-coiled coil conjugates VF1 and VF2 against serially titrated concentrations of Fib. Error bars indicate standard deviation across n=3 independent replicate experiments, and statistical significance was determined by two-way ANOVA.

Journal: bioRxiv

Article Title: A Rapid and Modular Nanobody Assay for Plug-and-Play Antigen Detection

doi: 10.1101/2025.03.01.640988

Figure Lengend Snippet: (A) Schematic illustration of the workflow for cell-free production of nanobody-coiled coil conjugates for RAMONA. (B) Cell-free screen of nanobody-coiled coil conjugate pairs against various target antigens, prepared as 200nM pure proteins dissolved in PBS. Each antigen was co-incubated with crude cell-free lysates containing the functionalized nanobodies in equivolumetric parts for 15 minutes at room temperature before addition to lateral flow strips for readout. (C) Performance of purified and cell-free produced nanobody-coiled coil conjugates Nb11 and Nb19 against serially titrated concentrations of ApoA1. (D) Performance of purified and cell-free produced nanobody-coiled coil conjugates VF1 and VF2 against serially titrated concentrations of Fib. Error bars indicate standard deviation across n=3 independent replicate experiments, and statistical significance was determined by two-way ANOVA.

Article Snippet: Human Apolipoprotein A-I (ApoA1) AssayMax ELISA Kit (AssayPro) and Human Fibrinogen ELISA Kit - high sensitivity (AbCam) were purchased for ELISA tests.

Techniques: Incubation, Purification, Produced, Standard Deviation

(A) Schematic comparing the approximate time required for an ELISA versus a RAMONA. (B) Comparison of the concentration of ApoA1 calculated from ELISAs versus the band intensities analyzed from RAMONA LFAs for 10 fresh saliva samples. Untreated saliva was diluted for ELISAs, while 10x concentrated, protein precipitated saliva was used in RAMONAs. Results show n=3 independent replicate experiments. (C) Correlation between mean ELISA and RAMONA results for the same 10 fresh saliva samples in (B), with a Pearson correlation coefficient of 0.772 for ApoA1 ( p =0.0090). (D) Representative RAMONA LFA images for a subset of fresh saliva samples #7-10, which were chosen to demonstrate variation in band intensities depending on ApoA1 abundance in each sample. “C” indicates control line, and “T” indicates test line. (E) Comparison of the concentration of fibrinogen calculated from ELISAs versus the band intensities analyzed from RAMONA LFAs for 10 fresh saliva samples. Untreated saliva was diluted for ELISAs, while 10x concentrated, protein precipitated saliva was used in RAMONAs. (F) Correlation between mean ELISA and RAMONA results for the same 10 fresh saliva samples in (E), with a Pearson correlation coefficient of 0.961 for Fib ( p <0.001). (G) Representative RAMONA LFA images for a subset of fresh saliva samples #7-10, which were chosen to demonstrate variation in band intensities depending on Fib abundance in each sample. “C” indicates control line, and “T” indicates test line.

Journal: bioRxiv

Article Title: A Rapid and Modular Nanobody Assay for Plug-and-Play Antigen Detection

doi: 10.1101/2025.03.01.640988

Figure Lengend Snippet: (A) Schematic comparing the approximate time required for an ELISA versus a RAMONA. (B) Comparison of the concentration of ApoA1 calculated from ELISAs versus the band intensities analyzed from RAMONA LFAs for 10 fresh saliva samples. Untreated saliva was diluted for ELISAs, while 10x concentrated, protein precipitated saliva was used in RAMONAs. Results show n=3 independent replicate experiments. (C) Correlation between mean ELISA and RAMONA results for the same 10 fresh saliva samples in (B), with a Pearson correlation coefficient of 0.772 for ApoA1 ( p =0.0090). (D) Representative RAMONA LFA images for a subset of fresh saliva samples #7-10, which were chosen to demonstrate variation in band intensities depending on ApoA1 abundance in each sample. “C” indicates control line, and “T” indicates test line. (E) Comparison of the concentration of fibrinogen calculated from ELISAs versus the band intensities analyzed from RAMONA LFAs for 10 fresh saliva samples. Untreated saliva was diluted for ELISAs, while 10x concentrated, protein precipitated saliva was used in RAMONAs. (F) Correlation between mean ELISA and RAMONA results for the same 10 fresh saliva samples in (E), with a Pearson correlation coefficient of 0.961 for Fib ( p <0.001). (G) Representative RAMONA LFA images for a subset of fresh saliva samples #7-10, which were chosen to demonstrate variation in band intensities depending on Fib abundance in each sample. “C” indicates control line, and “T” indicates test line.

Article Snippet: Human Apolipoprotein A-I (ApoA1) AssayMax ELISA Kit (AssayPro) and Human Fibrinogen ELISA Kit - high sensitivity (AbCam) were purchased for ELISA tests.

Techniques: Enzyme-linked Immunosorbent Assay, Comparison, Concentration Assay, Control