Production and Characterization of Novel Fabs Generated from Different Phage Display Libraries as Probes for Immunoassays for Gluten Detection in Food
"> Figure 1
<p>Schematic workflow of the production and characterization of novel Fab as probes in ELISA methods for gluten detection in foodstuff.</p> "> Figure 2
<p>Western blot analysis of transformed <span class="html-italic">E. coli</span> RV308 culture supernatants after induction for expression of recombinant Fabs. Lane 1: control Fab previously produced; Lanes 2 and 3: Fab-C; Lanes 4 and 5: Fab-E; Lanes 6 and 7: Fab-H; Lanes 8 and 9: Fab8E-4.</p> "> Figure 3
<p>Recombinant production, purification, and identification of Fabs. (<b>A</b>) Example of the immobilized metal affinity chromatography (IMAC) profile for the purification of the recombinant Fabs (Fab-C in this image). In green, monitorization of the protein quantity (absorbance 280 nm) going through the column. In red, conductivity measures. In black, percentage of elution buffer pumped to the system. In blue, measure of the system pressure. (<b>B</b>) Analysis of the purification steps by SDS-PAGE electrophoresis. Lane 1: control Fab previously produced. Lanes 2–4: samples from different fractions representing the first peak of the chromatogram (*). Lanes 5–9: samples from different fractions representing the second peak of the chromatogram (**) containing the desired Fab.</p> "> Figure 4
<p>Indirect ELISA results for characterization of the four recombinant Fabs produced. (<b>A</b>) Specificity results against ethanolic extracts of gluten-containing (wheat, barley, and rye) and gluten-free (oats, corn, and rye) flours (diluted 1:5 in PBS). (<b>B</b>) Sensitivity evaluation by comparative dose-response curves obtained against gliadin-PWG (0–20 µg/mL) diluted in PBS. (<b>C</b>) Comparison of dose–response curves for detection of gluten in an experimental mixture (rice flour spiked with growing concentrations of wheat, rye, and barley flours). (<b>D</b>) Linear regression results obtained from the analysis of the gliadin-PWG standard (0.025–0.625 µg/mL) by indirect ELISA using Fab-C as primary antibody.</p> "> Figure 4 Cont.
<p>Indirect ELISA results for characterization of the four recombinant Fabs produced. (<b>A</b>) Specificity results against ethanolic extracts of gluten-containing (wheat, barley, and rye) and gluten-free (oats, corn, and rye) flours (diluted 1:5 in PBS). (<b>B</b>) Sensitivity evaluation by comparative dose-response curves obtained against gliadin-PWG (0–20 µg/mL) diluted in PBS. (<b>C</b>) Comparison of dose–response curves for detection of gluten in an experimental mixture (rice flour spiked with growing concentrations of wheat, rye, and barley flours). (<b>D</b>) Linear regression results obtained from the analysis of the gliadin-PWG standard (0.025–0.625 µg/mL) by indirect ELISA using Fab-C as primary antibody.</p> ">
Abstract
:1. Introduction
2. Materials and Methods
2.1. Bacterial Strains and Growth Media
2.2. Recombinant Antibodies Selection and Bacterial Transformation
2.3. Production of Recombinant Antibodies
2.4. IMAC-Purification and Analysis
2.5. Reference Materials and Food Samples Used for Analysis
2.6. Extraction of Gluten from Samples
2.7. Indirect Enzyme-Linked Immunosorbent Assay (ELISA) Method Based on the Recombinant Fabs
2.8. Assay Validation
3. Results and Discussion
3.1. Expression and Purification of Recombinant Fabs
3.2. Indirect ELISA Development and Validation for Gluten Detection
3.3. Detection of Gluten in Commercial Food Products by Indirect ELISA Based on the Recombinant Fab-C
4. Conclusions
Author Contributions
Funding
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Okra (Abelmoschus esculentus) | Lentil (Lens culinaris) |
Button mushroom (Agaricus bisporus) | Cassava (Manihot esculenta) |
Onion (Allium cepa) | Mango (Mangifera indica) |
Leek (Allium ampeloprasum) | Red banana (Musa acuminata) |
Cashew (Anacardium occidentale) | Myrtle (Myrtus communis) |
Peanut (Arachis hypogaea) | Olive (Olea europaea) |
Strawberry tree (Arbutus unedo) | Rice (Oryza sativa) |
Beetroot (Beta vulgaris) | Passion fruit (Passiflora edulis) |
Cabbage (Brassica oleracea) | Pepperomia (Peperomia pellucida) |
Chinese cabbage (Brassica rapa subsp. pekinensis) | Avocado (Persea americana) |
Pigeon pea (Cajanus cajan) | Common bean (Phaseolus vulgaris) |
Chili pepper (Capsicum annuum) | Runner bean (Phaseolus coccineus) |
Tabasco pepper (Capsicum frutescens) | Cape gooseberry (Physalis peruviana) |
Scotch bonnet pepper (Capsicum chinense) | Pea (Pisum sativum) |
Papaya (Carica papaya) | Almond (Prunus dulcis) |
Chickpea (Cicer arietinum) | Sweet almond (Prunus dulcis var. dulcis) |
Lemon (Citrus limon) | Plum (Prunus domestica) |
Mandarin orange (Citrus reticulata) | Raspberry (Rubus idaeus) |
Watermelon (Citrullus lanatus) | Atlantic salmon (Salmo salar) |
Cantaloupe (Cucumis melo var. cantalupensis) | Castor bean (Ricinus communis) |
Cucumber (Cucumis sativus) | Blackberry (Rubus fruticosus) |
Quince (Cydonia oblonga) | Sesame (Sesamum indicum) |
Persimmon (Diospyros kaki) | Tomato (Solanum lycopersicum) |
Teff (Eragrostis tef) | Cherry tomato (S. lycopersicum cerasiforme) |
Arugula (Eruca vesicaria subsp. sativa) | Spinach (Spinacia oleracea) |
Strawberry (Fragaria x ananassa) | Vanilla (Vanilla planifolia) |
European strawberry (Fragaria vesca) | Adzuki bean (Vigna angularis) |
Soybean (Glycine max) | Mung bean (Vigna radiata) |
Sunflower (Helianthus annuus) | Grape (Vitis vinifera) |
Walnut (Juglans regia) | Ginger (Zingiber officinale) |
Products | No. of Products | Indirect Fab-C | Sandwich R5 |
---|---|---|---|
(A) Products declared to contain gluten in the labeling (9) | |||
Pasta | 2 | +(2) | +(2) |
Soup or rice plates | 3 | +(3) | +(3) |
Oats flakes | 1 | +(1) | +(1) |
Cereal bars | 2 | +(2) | +(2) |
Dairy products | 1 | +(1) | +(1) |
(B) Products with “may contain” precautionary labeling (6) | |||
Cereal | 4 | +(3)/−(1) | +(3)/−(1) |
Flakes | 2 | −(2) | −(2) |
(C) Products that did not declare gluten or that did not specifically warn of the presence of gluten (21) | |||
Cereal products | 9 | +(7)/−(2) | +(7)/−(2) |
Oat drinks | 3 | +(1)/−(2) | +(1)/−(2) |
Oat flakes | 8 | +(2)/−(6) | +(5)/−(3) |
Meat | 1 | −(1) | −(1) |
(D) Products with “gluten free” labeling and/or certification (14) | |||
Cookies/cakes | 6 | −(6) | −(6) |
Pasta | 1 | −(1) | −(1) |
Baby food | 2 | −(2) | −(2) |
Corn flakes | 1 | −(1) | −(1) |
Oat flakes | 1 | −(1) | −(1) |
Meat products | 3 | −(3) | −(3) |
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Garcia-Calvo, E.; García-García, A.; Rodríguez, S.; Takkinen, K.; Martín, R.; García, T. Production and Characterization of Novel Fabs Generated from Different Phage Display Libraries as Probes for Immunoassays for Gluten Detection in Food. Foods 2023, 12, 3274. https://doi.org/10.3390/foods12173274
Garcia-Calvo E, García-García A, Rodríguez S, Takkinen K, Martín R, García T. Production and Characterization of Novel Fabs Generated from Different Phage Display Libraries as Probes for Immunoassays for Gluten Detection in Food. Foods. 2023; 12(17):3274. https://doi.org/10.3390/foods12173274
Chicago/Turabian StyleGarcia-Calvo, Eduardo, Aina García-García, Santiago Rodríguez, Kristiina Takkinen, Rosario Martín, and Teresa García. 2023. "Production and Characterization of Novel Fabs Generated from Different Phage Display Libraries as Probes for Immunoassays for Gluten Detection in Food" Foods 12, no. 17: 3274. https://doi.org/10.3390/foods12173274