ad1 1 10  (Bio-Rad)

Journal: bioRxiv

Article Title: OmniDetect: a plant-produced universal IgG binder replacing secondary antibodies in immunoassays

doi: 10.64898/2026.01.14.699418

Figure Lengend Snippet: a. Schematic representation of the construct, pOmniDetect-G1 . FM’M-UD, an artificial promoter; Z, γ-zein signal peptide (1-19 aa, Q548E8); CTB, cholera toxin-B subunit (WP_338286491.1); LK2, linker GGGGSx2; GB3, three copies of B1 domain of Streptococcal protein G; HRP, horseradish peroxidase; 6xHis, His-tag; HDEL, ER-retention signal; 3PRt, artificial composite terminator. b. Conceptual design of OmniDetect-G1 generated in BioRender using structures imported from the PDB database. c-e. Expression of Omnidetect-G1. Leaf samples were collected at 3, 5, and 7 dpi; total soluble protein was extracted, separated on 10% SDS-PAGE, and analyzed by CBB-staining and western blot using Anti-His IgG. Relative expression levels were quantified as mean gray values of the target band and plotted. f-h. Purification, quantification, and multimer analysis of OmniDetetc-G1 protein. M, protein marker; TSP; total soluble protein; UB, unbound fraction; W washing fraction; E, elution fractions (E1, 50 mM imidazole; E2, 100 mM; E3, 250 mM). The arrow indicates the target protein band. i-j. Side-by-side comparison of commercial HRP-conjugated anti-mouse and anti-rabbit secondary antibodies with OmniDetect-G1 in a western blot. k. ELISA comparing commercial anti-rabbit secondary antibodies with OmniDetect-G1. Data are represented as mean ± SE (n = 3). Error bars indicate standard error. P-values were calculated by Student’s t-test. The OmniDetect-G1 conceptual protein structure was created using Biorender.com . PDB ID: CTB: 1PZJ, GB1: 1GB1, HRP: 1HCH.

Article Snippet: Membranes were then incubated overnight at 4 °C with one of the following primary antibodies diluted 1:1,000 in TBST containing 3% non-fat dry milk: mouse anti-His (Novus, AD1.1.10), rabbit anti-GFP (GenScript, Cat# A01388, USA), mouse anti-GFP (Living Colors JL-8, Cat# 632381, USA), or chicken anti-His (abcam, Cat# ab9107, United Kingdom).

Techniques: Construct, Generated, Expressing, SDS Page, Staining, Western Blot, Purification, Marker, Comparison, Enzyme-linked Immunosorbent Assay

Journal: bioRxiv

Article Title: OmniDetect: a plant-produced universal IgG binder replacing secondary antibodies in immunoassays

doi: 10.64898/2026.01.14.699418

Figure Lengend Snippet: a. Detection of IgY by OmniDetect. Fifty ng of highly purified His-GFP was coated on an ELISA plate and probed with primary chicken anti-His antibodies, followed by detection with serially diluted either HRP-conjugated anti-IgY antibody or OmniDetect-G3. Colorimetric signals were measured at 450nm and plotted. Data represent mean ± SE (n = 3). P-values were calculated using Student’s t-test. b-c. OmniDetect exhibits stable chemiluminescence over time. Fifty ng of total soluble protein from N. bentaminana leaves expressing His-GFP was analyzed by western blot. Band intensity was quantified using ImageJ software, and mean gray values were plotted. Data represent mean ± SE (n = 5). Error bars indicate standard error. d. OmniDetect shows no cross-reactivity with plant or animal cell proteins. Total soluble protein extracted from N. benthamiana , A549, HEK293, and HeLa cells was separated on SDS-PAGE, followed by western blotting and probed with mouse anti-actin antibody, and then incubated with either a commercial HRP-conjugated anti-mouse antibody or OmniDetect-G3. Chemiluminescence images were acquired simultaneously under a single auto-exposure setting.

Article Snippet: Membranes were then incubated overnight at 4 °C with one of the following primary antibodies diluted 1:1,000 in TBST containing 3% non-fat dry milk: mouse anti-His (Novus, AD1.1.10), rabbit anti-GFP (GenScript, Cat# A01388, USA), mouse anti-GFP (Living Colors JL-8, Cat# 632381, USA), or chicken anti-His (abcam, Cat# ab9107, United Kingdom).

Techniques: Purification, Enzyme-linked Immunosorbent Assay, Expressing, Western Blot, Software, SDS Page, Incubation