Immunological and Metabolomic Impacts of Administration of Cry1Ab Protein and MON 810 Maize in Mouse

We have investigated the immunological and metabolomic impacts of Cry1Ab administration to mice, either as a purified protein or as the Cry1Ab-expressing genetically modified (GM) MON810 maize. Humoral and cellular specific immune responses induced in BALB/cJ mice after intra-gastric (i.g.) or intra-peritoneal (i.p.) administration of purified Cry1Ab were analyzed and compared with those induced by proteins of various immunogenic and allergic potencies. Possible unintended effects of the genetic modification on the pattern of expression of maize natural allergens were studied using IgE-immunoblot and sera from maize-allergic patients. Mice were experimentally sensitized (i.g. or i.p. route) with protein extracts from GM or non-GM maize, and then anti-maize proteins and anti-Cry1Ab–induced immune responses were analyzed. In parallel, longitudinal metabolomic studies were performed on the urine of mice treated via the i.g. route. Weak immune responses were observed after i.g. administration of the different proteins. Using the i.p. route, a clear Th2 response was observed with the known allergenic proteins, whereas a mixed Th1/Th2 immune response was observed with immunogenic protein not known to be allergenic and with Cry1Ab. This then reflects protein immunogenicity in the BALB/c Th2-biased mouse strain rather than allergenicity. No difference in natural maize allergen profiles was evidenced between MON810 and its non-GM comparator. Immune responses against maize proteins were quantitatively equivalent in mice treated with MON810 vs the non-GM counterpart and no anti-Cry1Ab–specific immune response was detected in mice that received MON810. Metabolomic studies showed a slight “cultivar” effect, which represented less than 1% of the initial metabolic information. Our results confirm the immunogenicity of purified Cry1Ab without evidence of allergenic potential. Immunological and metabolomic studies revealed slight differences in mouse metabolic profiles after i.g. administration of MON810 vs its non-GM counterpart, but no significant unintended effect of the genetic modification on immune responses was seen

Immunochemical characterisation of structure and allergenicity of peanut 2S albumins using different formats of immunoassays

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Enzyme immunoassay for a urinary metabolite of 4-hydroxynonenal as a marker of lipid peroxidation

International audienceFree radical reactions are involved in the pathogenesis of numerous diseases, so there is a real need to develop biomarkers that reflect these reactions in vivo. 4-Hydroxy-2-nonenal (HNE) is a major product of the lipid peroxidation process that is a consequence of free radical reactions. We present here the development and validation of an enzyme immunoassay (EIA) of the major urinary metabolite of HNE, namely 1,4-dihydroxynonane-mercapturic acid (DHN-MA). EIA allowed direct measurement of DHN-MA in rat urine with good sensitivity (0.02 ng/ml) and precision (intraassay CV = 5.7%). Recovery was complete (99-102%). Cross-reactivity was very low with 1,4-dihydroxynonene and with different mercapturic acids except with one other HNE urinary metabolite. Good correlation (EIA = 0.79 x LC/MS + 14.03, r = 0.877, p < 10(-8)) was obtained between EIA and liquid chromatography/mass spectrometry (LC/MS) quantitation when analyzing urine samples of rats with different oxidative status, due to treatment with either BrCCl(3) or trinitrobenzene sulfonic acid, which are known to induce hepatic lipid peroxidation or colon inflammation, respectively

Sandwich immunoassay of Cry1Ab.

<p><b>A.</b> Standard curve obtained with purified Cry1Ab protoxin in sandwich immunoassay using mAb#120 passively adsorbed on microplates as capture antibody and AChE-labeled mAb#95 as tracer. Mean +/− SD is represented. Each point is the result of duplicate measurements, except NSB (EIA buffer alone, n = 8). The limit of detection, defined as the lowest concentration of standard Cry1Ab inducing a signal statistically significantly higher than NSB (i.e. mean+3σ<i>_n</i>₋₁) is shown in the insert as a black line. <b>B.</b> Extracts from reference maize powders containing MON810 maize mass fraction level of 0.5% (●), 1% (▼) or 2.5% (◆) (2- to 250-fold dilutions) or standard Cry1Ab (10 ng/mL, 2- to 128-fold dilutions, ○) were assayed using this sandwich immunoassay. All dilution points were assayed in duplicate. Results are expressed as mean of absorbance values (mAU at 414 nm) +/− SD. No signal was detected for non-GM reference maize (not shown).</p

Metabolic biomarkers identified from discriminant analysis.

<p>Assignment of significant variables explaining the 4 first components displayed by a linear discriminant analysis performed on OSC-PLS–corrected data corresponding to the late urine collections performed on days 27 and 28.</p

Specific IgG1, IgG2a and IgE antibodies induced after i.p. administration of purified KLH, BLG, Ara h1 or Cry1Ab.

<p>Specific IgG1 (•, left axis), IgG2a (o, left axis) and IgE (*, right axis) antibodies were assayed in sera of individual BALB/cJ mice after two i.p. administrations of 1 (<b>A</b>) or 100 µg (<b>B</b>) of each protein, without adjuvant. Bars represent the mean of 5 mice per group. Nonspecific binding (NSB) was determined using EIA buffer instead of serum. Limit of detection was determined as the mean of NSB +3σ<i>_n</i>₋₁. a p<0.05 when comparing mice receiving protein with mice receiving PBS (not shown), nonparametric test.</p

Linear discriminant analysis performed on OSC-PLS–corrected data corresponding to the late urine collections performed on days 27 and 28.

<p>The four LD are highly significant (p<0.0001) and the 3 respective factorial maps are displayed here: 1×2 (<b>A</b>), 2×3 (<b>B</b>) and 3×4 (<b>C</b>). The cultivar names are displayed on the factorial maps with the .2 and .3 suffixes designating the urine collection performed on days 27 and 28, respectively.</p

Cytokines secreted by splenocytes from mice given KLH, BLG, Ara h1 or Cry1Ab by the i.p. route.

<p>Splenocytes from BALB/cJ mice after i.p. administration of PBS (white bars), 1 µg (gray bars) or 100 µg (black bars) of KLH, BLG, Ara h1 or Cry1Ab were reactivated <i>ex vivo</i> with 50 µg of the corresponding antigen. Expected results were obtained from positive (ConA) and negative controls (medium alone) (data not shown).</p

A–C: Electrophoretic pattern and IgE-binding analysis of protein extracts from MON810 and non-GM maize:

<p>Protein extracts from MON 810 and its conventional counterpart were separated on 12% acrylamide gel and proteins were stained (<b>A</b>). After transfer to PVDF membrane, IgE binding proteins (i.e. allergens) were revealed by western blotting with the sera of 2 maize-allergic patients, i.e. #20770-MH (<b>B</b>) and #19392-CS (<b>C</b>). Specific IgE concentrations (<b>D</b>) in the same sera were determined on GM (open bar) and non-GM (black bar) protein extract coated plates. Bars represent mean+/−SEM obtained for 3 different dilutions of each serum, each assayed in duplicate.</p