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A two-hybrid antibody micropattern assay reveals specific in cis interactions of MHC I heavy chains at the cell surface.

ABSTRACT: We demonstrate a two-hybrid assay based on antibody micropatterns to study protein-protein interactions at the cell surface of major histocompatibility complex class I (MHC I) proteins. Anti-tag and conformation-specific antibodies are used for individual capture of specific forms of MHC I proteins that allow for location- and conformation-specific analysis by fluorescence microscopy. The assay is used to study the in cis interactions of MHC I proteins at the cell surface under controlled conditions and to define the involved protein conformations. Our results show that homotypic in cis interactions occur exclusively between MHC I free heavy chains, and we identify the dissociation of the light chain from the MHC I protein complex as a condition for MHC I in cis interactions. The functional role of these MHC I protein-protein interactions at the cell surface needs further investigation. We propose future technical developments of our two-hybrid assay for further analysis of MHC I protein-protein interactions.

SUBMITTER: Dirscherl C

PROVIDER: S-EPMC6125123 | biostudies-literature | 2018 Sep

REPOSITORIES: biostudies-literature

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A two-hybrid antibody micropattern assay reveals specific <i>in cis</i> interactions of MHC I heavy chains at the cell surface.

Dirscherl Cindy C Hein Zeynep Z Ramnarayan Venkat Raman VR Jacob-Dolan Catherine C Springer Sebastian S

eLife 20180905

We demonstrate a two-hybrid assay based on antibody micropatterns to study protein-protein interactions at the cell surface of major histocompatibility complex class I (MHC I) proteins. Anti-tag and conformation-specific antibodies are used for individual capture of specific forms of MHC I proteins that allow for location- and conformation-specific analysis by fluorescence microscopy. The assay is used to study the <i>in cis</i> interactions of MHC I proteins at the cell surface under controlled ...[more]

PMID: 30180933

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Project description:BackgroundBacterial surface contamination contributes to transmission of nosocomial infections. Chemical cleansers used to control surface contamination are often toxic and incorrectly implemented. Additional non-toxic strategies should be combined with regular cleanings to mitigate risks of human error and further decrease rates of nosocomial infections. The Sharklet micropattern (MP), inspired by shark skin, is an effective tool for reducing bacterial load on surfaces without toxic additives. The studies presented here were carried out to investigate the MP surfaces capability to reduce colonization of methicillin-sensitive Staphylococcus aureus (MSSA) and methicillin-resistant S. aureus (MRSA) compared to smooth control surfaces.MethodsThe MP and smooth surfaces produced in acrylic film were compared for remaining bacterial contamination and colonization following inoculation. Direct sampling of surfaces was carried out after inoculation by immersion, spray, and/or touch methods. Ultimately, a combination assay was developed to assess bacterial contamination after touch transfer inoculation combined with drying (persistence) to mimic common environmental contamination scenarios in the clinic or hospital environment. The combination transfer and persistence assay was then used to test antimicrobial copper beside the MP for the ability to reduce MSSA and MRSA challenge.ResultsThe MP reduced bacterial contamination with log reductions ranging from 87-99% (LR = 0.90-2.18; p < 0.05) compared to smooth control surfaces. The MP was more effective than the 99.9% pure copper alloy C11000 at reducing surface contamination of S. aureus (MSSA and MRSA) through transfer and persistence of bacteria. The MP reduced MSSA by as much as 97% (LR = 1.54; p < 0.01) and MRSA by as much as 94% (LR = 1.26; p < 0.005) compared to smooth controls. Antimicrobial copper had no significant effect on MSSA contamination, but reduced MRSA contamination by 80% (LR = 0.70; p < 0.005).ConclusionThe assays developed in this study mimic hospital environmental contamination events to demonstrate the performance of a MP to limit contamination under multiple conditions. Antimicrobial copper has been implemented in hospital room studies to evaluate its impact on nosocomial infections and a decrease in HAI rate was shown. Similar implementation of the MP has potential to reduce the incidence of HAIs although future clinical studies will be necessary to validate the MP's true impact.

Dataset Information

A two-hybrid antibody micropattern assay reveals specific in cis interactions of MHC I heavy chains at the cell surface.

Publications

A two-hybrid antibody micropattern assay reveals specific <i>in cis</i> interactions of MHC I heavy chains at the cell surface.

Similar Datasets

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