Dataset Information

Development of ?-lactoglobulin-specific chimeric human IgE? monoclonal antibodies for in vitro safety assessment of whey hydrolysates.

ABSTRACT:

Background

Cow's milk-derived whey hydrolysates are nutritional substitutes for allergic infants. Safety or residual allergenicity assessment of these whey hydrolysates is crucial. Currently, rat basophilic leukemia RBL-2H3 cells expressing the human IgE receptor ?-chain (huFc?RI?-RBL-2H3), sensitized with serum IgE from cow's milk allergic children, are being employed to assess in vitro residual allergenicity of these whey hydrolysates. However, limited availability and inter-lot variation of these allergic sera impede standardization of whey hydrolysate safety testing in degranulation assays.

Objective

An oligoclonal pool of chimeric human (chu)IgE antibodies against bovine ?-lactoglobulin (a major allergen in whey) was generated to increase sensitivity, specificity, and reproducibility of existing degranulation assays.

Methods

Mice were immunized with bovine ?-lactoglobulin, and subsequently the variable domains of dissimilar anti-?-lactoglobulin mouse IgG antibodies were cloned and sequenced. Six chimeric antibodies were generated comprising mouse variable domains and human constant IgE/? domains.

Results

After sensitization with this pool of anti-?-lactoglobulin chuIgEs, huFc?RI?-expressing RBL-2H3 cells demonstrated degranulation upon cross-linking with whey, native 18 kDa ?-lactoglobulin, and 5-10 kDa whey hydrolysates, whereas a 3 kDa whey hydrolysate and cow's milk powder (mainly casein) showed no degranulation. In parallel, allergic serum IgEs were less sensitive. In addition, our pool anti-?-lactoglobulin chuIgEs recognized multiple allergenic immunodominant regions on ?-lactoglobulin, which were also recognized by serum IgEs from cow's milk allergic children.

Conclusion

Usage of our 'unlimited' source and well-defined pool of ?-lactoglobulin-specific recombinant chuIgEs to sensitize huFc?RI? on RBL-2H3 cells showed to be a relevant and sensitive alternative for serum IgEs from cow's milk allergic patients to assess safety of whey-based non-allergic hydrolyzed formula.

SUBMITTER: Knipping K

PROVIDER: S-EPMC4143325 | biostudies-literature | 2014

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Development of β-lactoglobulin-specific chimeric human IgEκ monoclonal antibodies for in vitro safety assessment of whey hydrolysates.

Knipping Karen K Simons Peter J PJ Buelens-Sleumer Laura S LS Cox Linda L den Hartog Marcel M de Jong Niels N Teshima Reiko R Garssen Johan J Boon Louis L Knippels Léon M J LM

PloS one 20140825 8

<h4>Background</h4>Cow's milk-derived whey hydrolysates are nutritional substitutes for allergic infants. Safety or residual allergenicity assessment of these whey hydrolysates is crucial. Currently, rat basophilic leukemia RBL-2H3 cells expressing the human IgE receptor α-chain (huFcεRIα-RBL-2H3), sensitized with serum IgE from cow's milk allergic children, are being employed to assess in vitro residual allergenicity of these whey hydrolysates. However, limited availability and inter-lot variat ...[more]

PMID: 25153680

Similar Datasets

Project description:UnlabelledRecent experiments suggest that some glycoprotein (GP)-specific monoclonal antibodies (MAbs) can protect experimental animals against the filovirus Ebola virus (EBOV). There is a need for isolation of MAbs capable of neutralizing multiple filoviruses. Antibody neutralization assays for filoviruses frequently use surrogate systems such as the rhabdovirus vesicular stomatitis Indiana virus (VSV), lentiviruses or gammaretroviruses with their envelope proteins replaced with EBOV GP or pseudotyped with EBOV GP. It is optimal for both screening and in-depth characterization of newly identified neutralizing MAbs to generate recombinant filoviruses that express a reporter fluorescent protein in order to more easily monitor and quantify the infection. Our study showed that unlike neutralization-sensitive chimeric VSV, authentic filoviruses are highly resistant to neutralization by MAbs. We used reverse genetics techniques to replace EBOV GP with its counterpart from the heterologous filoviruses Bundibugyo virus (BDBV), Sudan virus, and even Marburg virus and Lloviu virus, which belong to the heterologous genera in the filovirus family. This work resulted in generation of multiple chimeric filoviruses, demonstrating the ability of filoviruses to tolerate swapping of the envelope protein. The sensitivity of chimeric filoviruses to neutralizing MAbs was similar to that of authentic biologically derived filoviruses with the same GP. Moreover, disabling the expression of the secreted GP (sGP) resulted in an increased susceptibility of an engineered virus to the BDBV52 MAb isolated from a BDBV survivor, suggesting a role for sGP in evasion of antibody neutralization in the context of a human filovirus infection.ImportanceThe study demonstrated that chimeric rhabdoviruses in which G protein is replaced with filovirus GP, widely used as surrogate targets for characterization of filovirus neutralizing antibodies, do not accurately predict the ability of antibodies to neutralize authentic filoviruses, which appeared to be resistant to neutralization. However, a recombinant EBOV expressing a fluorescent protein tolerated swapping of GP with counterparts from heterologous filoviruses, allowing high-throughput screening of B cell lines to isolate MAbs of any filovirus specificity. Human MAb BDBV52, which was isolated from a survivor of BDBV infection, was capable of partially neutralizing a chimeric EBOV carrying BDBV GP in which expression of sGP was disabled. In contrast, the parental virus expressing sGP was resistant to the MAb. Thus, the ability of filoviruses to tolerate swapping of GP can be used for identification of neutralizing MAbs specific to any filovirus and for the characterization of MAb specificity and mechanism of action.