Cloning and plant-based production of antibody MC10E7 for a lateral flow immunoassay to detect [4-arginine]microcystin in freshwater

Abstract

Antibody MC10E7 is one of a small number of monoclonal antibodies that bind specifically to [Arg4]-microcystins, and it can be used to survey natural water sources and food samples for algal toxin contamination. However, the development of sensitive immunoassays in different test formats, particularly user-friendly tests for on-site analysis, requires a sensitive but also cost-effective antibody. The original version of MC10E7 was derived from a murine hybridoma, but we determined the sequence of the variable regions using the peptide mass-assisted cloning strategy and expressed a scFv (single-chain variable fragment) format of this antibody in yeast and a chimeric full-size version in leaves of Nicotiana tabacum and Nicotiana benthamiana to facilitate inexpensive and scalable production. The specific antigen-binding activity of the purified antibody was verified by surface plasmon resonance spectroscopy and ELISA, confirming the same binding specificity as its hybridoma-derived counterpart. The plant-derived antibody was used to design a lateral flow immunoassay (dipstick) for the sensitive detection of [Arg4]-microcystins at concentrations of 100-300 ng/L in freshwater samples collected at different sites. Plant-based production will likely reduce the cost of the antibody, currently the most expensive component of the dipstick immunoassay, and will allow the development of further antibody-based analytical devices and water purification adsorbents for the efficient removal of toxic contaminants.

Keywords: lateral flow immunoassay; microcystin; molecular farming; plant-made antibody; water contamination.

Figure 1

PMF sequence coverage of the hybridoma‐derived MC10E7 mAb (MALDI‐TOF‐MS).

Figure 2

(a) Vector cassettes used for transient overexpression of the chimeric MC10E7 in plant leaves. The VL and VH sequences of MC10E7 were used to reconstruct a full‐size antibody based on constant regions of human IgG1. To express full‐size antibody, genes for the heavy and light chains were codelivered to plant cells on separate binary plasmids. CaMV2x35SP, enhanced cauliflower mosaic virus (CaMV) 35S promoter; 35S TT, cauliflower mosaic virus (CaMV) 35S terminator; UTR, untranslated region of the tobacco etch virus; SP, murine IgG signal sequence for entry into the endoplasmic reticulum; Cγ1‐3, human IgG1 constant domain for the heavy chain; Cκ1 human constant domain for the kappa light chain; VH/L region, variable region of the MC10E7 antibody heavy or light chain. (b) Left panel: SDS‐PAGE of the purified recombinant plant‐produced antimicrocystin antibody MC10E7 (5 μg) under reducing conditions. Staining with Coomassie Brilliant Blue R‐250 showing the heavy and light chains; right panel: immunoblot of the same sample (≥100 ng) probed with anti‐human IgG (H + L)‐AP conjugate. (c) Comparison of the average yields of recombinant full‐size antimicrocystin mAb achieved in Nicotiana benthamiana (NB) and in Nicotiana tabacum SR1 (NT) leaves after Protein G purification. Data from five independent infiltration experiments on each plant species. Error bars represent SD. (d) Size‐exclusion chromatography of the Protein G purified plant‐produced MC10E7. Main peak (1) corresponds to MC10E7; smaller peaks – protein impurities.

Figure 3

ELISA results obtained with purified antibodies on the same microtitre plate (m = 12 for plant‐produced antibody, m = 11 for monoclonal mouse‐antibody, n = 3). Error bars represent ±1 standard deviation about the mean.

Figure 4

Single kinetic curves for hybridoma MC10E7 and plant‐derived MC10E7 using MC‐LR immobilized sensor chip CM5 and Biacore X100. Carrier solution: running buffer, flow speed 30 μL/min, five concentrations of individual mAbs between 0.13 nm and 2.03 nm; contact time: 2 min at 37 °C.

Figure 5

(a) Schematic illustration of the immunological dipstick. C line: control line with immobilized goat‐anti‐human IgG antibodies; T line: test line with immobilized MC‐LR‐BSA conjugate; (b) cyanobacterial bloom at lake Rudower See. (c) Typical photograph image of the detection of MC‐LR in standards and freshwater samples. Dipsticks no. 1–6: MC‐LR standards (blank, 0.1, 0.3, 0.5, 0.7, 0.9 μg/L); no. 7 and 8: Rudower See water, Lenzen 1 and 2; no. 9: river Isar.