PMID- 29945600
OWN - NLM
STAT- MEDLINE
DCOM- 20190710
LR  - 20190710
IS  - 1472-6750 (Electronic)
IS  - 1472-6750 (Linking)
VI  - 18
IP  - 1
DP  - 2018 Jun 27
TI  - Membrane-bound and soluble forms of an NMDA receptor extracellular domain retain 
      epitopes targeted in auto-immune encephalitis.
PG  - 41
LID - 10.1186/s12896-018-0450-1 [doi]
LID - 41
AB  - BACKGROUND: Anti-NMDA receptor encephalitis (ANRE) is a potentially lethal 
      disease attributed to auto-antibodies against the N-methyl-D-aspartate receptor 
      (NMDAR). Full recovery is possible if therapy is initiated early in the disease 
      course. Detection of ANRE antibodies in the cerebrospinal fluid (CSF) is 
      essential for diagnosis. The assays for ANRE-associated IgGs often rely on cells 
      transiently transfected with NMDAR genes. A cell line that stably expresses 
      pathogenic NMDAR epitopes could improve standardization of the assays and provide 
      antigen that could be used in commercial solid state assay systems. RESULTS: We 
      expressed the amino terminal domain (ATD) of the GluN1 NMDAR subunit (NR1) as a 
      fusion protein on the outer plasma membrane of 293T cells, creating a stable cell 
      population (293T-ATD) that is recognized by ANRE patient monoclonal antibodies in 
      flow cytometry and immunofluorescence assays. The ATD fusion protein also 
      contains a Myc tag and a 6XHIS tag, which provide functionality for immunoassays 
      and antigen purification, and a TEV protease site, which allows the ATD domain to 
      be specifically released from the cells in essentially pure form. ATD mobilized 
      from the 293T ATD cell line maintained the pathogenic ANRE epitopes in ELISA 
      binding assays. CSF (3/4) and sera (4/4) from ANRE patients also bound the 
      293T-ATD cell line, whereas normal CSF and sera did not. CONCLUSIONS: The 
      293T-ATD cell line is potentially adaptable to a variety of formats to identify 
      antibodies associated with ANRE, including cell-based and soluble antigen 
      formats, and demonstrates a useful method to produce complex proteins for 
      research, drug discovery, and clinical diagnosis.
FAU - Sharma, Rashmi
AU  - Sharma R
AD  - Lankenau Institute for Medical Research, 100 E. Lancaster Ave, Wynnewood, PA, 
      19096, USA.
FAU - Al-Saleem, Fetweh H
AU  - Al-Saleem FH
AD  - Lankenau Institute for Medical Research, 100 E. Lancaster Ave, Wynnewood, PA, 
      19096, USA.
FAU - Puligedda, Rama Devudu
AU  - Puligedda RD
AD  - Lankenau Institute for Medical Research, 100 E. Lancaster Ave, Wynnewood, PA, 
      19096, USA.
FAU - Rattelle, Amy
AU  - Rattelle A
AD  - Division of Neurology, Children's Hospital of Pennsylvania, Philadelphia, PA, 
      19104, USA.
FAU - Lynch, David R
AU  - Lynch DR
AD  - Division of Neurology, Children's Hospital of Pennsylvania, Philadelphia, PA, 
      19104, USA.
FAU - Dessain, Scott K
AU  - Dessain SK
AUID- ORCID: 0000-0002-4331-6799
AD  - Lankenau Institute for Medical Research, 100 E. Lancaster Ave, Wynnewood, PA, 
      19096, USA. dessain@limr.org.
LA  - eng
GR  - R21 NS088148/NS/NINDS NIH HHS/United States
PT  - Journal Article
PT  - Research Support, N.I.H., Extramural
PT  - Research Support, Non-U.S. Gov't
DEP - 20180627
PL  - England
TA  - BMC Biotechnol
JT  - BMC biotechnology
JID - 101088663
RN  - 0 (Antibodies, Monoclonal)
RN  - 0 (Epitopes)
RN  - 0 (Membrane Proteins)
RN  - 0 (Receptors, N-Methyl-D-Aspartate)
RN  - 0 (Recombinant Proteins)
RN  - EC 3.4.- (Endopeptidases)
RN  - EC 3.4.- (TEV protease)
SB  - IM
MH  - Anti-N-Methyl-D-Aspartate Receptor Encephalitis/*diagnosis/immunology
MH  - Antibodies, Monoclonal/*immunology
MH  - Cell Line
MH  - Endopeptidases/genetics
MH  - Epitopes
MH  - HEK293 Cells
MH  - Humans
MH  - Membrane Proteins/immunology
MH  - Receptors, N-Methyl-D-Aspartate/*genetics/*immunology
MH  - Recombinant Proteins/immunology
PMC - PMC6020338
OTO - NOTNLM
OT  - ANRE
OT  - Anti-N-methyl-D-aspartate receptor encephalitis
OT  - Antigen
OT  - Autoimmune encephalitis
OT  - Autoimmunity
OT  - Conformational epitope
OT  - Monoclonal antibody
OT  - NMDA receptor
OT  - Recombinant protein expression
OT  - TEV protease
COIS- ETHICS APPROVAL AND CONSENT TO PARTICIPATE: CSF and patient sera were collected 
      at the Children's Hospital of Pennsylvania (CHOP), Philadelphia, PA, with full 
      informed consent and protocols approved by the CHOP Institutional Review Board. 
      No animal studies were performed. COMPETING INTERESTS: The authors declare that 
      they have no competing interests.
EDAT- 2018/06/28 06:00
MHDA- 2019/07/11 06:00
PMCR- 2018/06/27
CRDT- 2018/06/28 06:00
PHST- 2017/11/30 00:00 [received]
PHST- 2018/05/22 00:00 [accepted]
PHST- 2018/06/28 06:00 [entrez]
PHST- 2018/06/28 06:00 [pubmed]
PHST- 2019/07/11 06:00 [medline]
PHST- 2018/06/27 00:00 [pmc-release]
AID - 10.1186/s12896-018-0450-1 [pii]
AID - 450 [pii]
AID - 10.1186/s12896-018-0450-1 [doi]
PST - epublish
SO  - BMC Biotechnol. 2018 Jun 27;18(1):41. doi: 10.1186/s12896-018-0450-1.