PMID- 30445953
OWN - NLM
STAT- MEDLINE
DCOM- 20190808
LR  - 20191210
IS  - 1472-6750 (Electronic)
IS  - 1472-6750 (Linking)
VI  - 18
IP  - 1
DP  - 2018 Nov 16
TI  - Development of luciferase-linked antibody capture assay based on luciferase 
      immunoprecipitation systems for antibody detection of porcine reproductive and 
      respiratory syndrome virus.
PG  - 73
LID - 10.1186/s12896-018-0483-5 [doi]
LID - 73
AB  - BACKGROUND: Early detection of porcine reproductive and respiratory syndrome 
      virus (PRRSV) infection of swine is necessary to control this devastating 
      disease. By monitoring host serum antibodies to viral antigens, early virus 
      detection within herds is feasible. In this study, recombinant antigens were 
      generated using recombinant DNA techniques to fuse PRRSV structural protein (N) 
      or nonstructural protein 1alpha (nsp1alpha) with the Rellina luciferase gene. Next, fused 
      genes were cloned into plasmids and transfected into HEK-293 T cells for 
      transient expression. Upon co-incubation of lysates with pig sera, 
      antigen-antibody complexes formed that bound to Protein-G coated onto 
      microplates. By further measurement of luminance value, a modified form of 
      Luciferase Immunoprecipitation Systems, namely luciferase-linked antibody capture 
      assay (LACA) was developed for detection of PRRSV-specific antibodies. RESULTS: 
      Known anti-PRRSV antibody-positive or -negative serum samples (125 and 122 
      samples, respectively) were used to validate the LACA and compared it with IDEXX 
      PRRS x3 ELISA. Based on the result, N-Rluc and nsp1alpha-Rluc LACA results were 95.3 
      and 94.4% in agreement with IDEXX ELISA, suggested a similar specificity of LACA 
      to IDEXX ELISA. Moreover, when both LACA and IDEXX ELISA were used to evaluate 
      sequential serum samples obtained from PRRSV experimentally infected pigs, the 
      PRRSV-specific antibody response was detectable as early as 3 days 
      post-inoculation (dpi) using N-Rluc LACA, but undetectable until 7 dpi using 
      IDEXX ELISA, suggesting an improved sensitivity of LACA. Meanwhile, antibodies 
      specific for nsp1alpha were detected at higher levels overall, but were undetectable 
      until 10 dpi. Furthermore,. Notably, one IDEXX ELISA positive result was not 
      confirmed by LACA or IFA and was thus considered a false-positive result. 
      CONCLUSION: The LACA exhibited similar specificity but improved sensitivity to 
      that of the commercial IDEXX PRRS x3 ELISA kit for detection of PRRSV-specific 
      antibodies in pig serum. Importantly, LACA could be adapted for detecting 
      antibodies against other PRRSV targets, such as nsp1alpha, to achieve earlier 
      detection of PRRSV infection.
FAU - Li, Jie
AU  - Li J
AD  - Department of Preventive Veterinary Medicine, College of Veterinary Medicine, 
      Northwest A&F University, Yangling, 712100, Shaanxi, China.
AD  - Scientific Observing and Experimental Station of Veterinary Pharmacology and 
      Diagnostic Technology, Ministry of Agriculture, Yangling, 712100, Shaanxi, China.
FAU - Wang, Gang
AU  - Wang G
AD  - State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research 
      Institute of Chinese Academy of Agricultural Sciences, Harbin, 150001, China.
FAU - Yang, Di
AU  - Yang D
AD  - Department of Preventive Veterinary Medicine, College of Veterinary Medicine, 
      Northwest A&F University, Yangling, 712100, Shaanxi, China.
AD  - Scientific Observing and Experimental Station of Veterinary Pharmacology and 
      Diagnostic Technology, Ministry of Agriculture, Yangling, 712100, Shaanxi, China.
FAU - Zhao, Bao
AU  - Zhao B
AD  - Shaanxi Animal Disease Control Center, Xi'an, 710016, Shaanxi, China.
FAU - Zhao, Yongpan
AU  - Zhao Y
AD  - Shaanxi Domestic Animal Improving Station, Xianyang, 713702, Shaanxi, China.
FAU - Liu, Yonggang
AU  - Liu Y
AD  - State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research 
      Institute of Chinese Academy of Agricultural Sciences, Harbin, 150001, China.
FAU - Cai, Xuehui
AU  - Cai X
AD  - State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research 
      Institute of Chinese Academy of Agricultural Sciences, Harbin, 150001, China.
FAU - Nan, Yuchen
AU  - Nan Y
AD  - Department of Preventive Veterinary Medicine, College of Veterinary Medicine, 
      Northwest A&F University, Yangling, 712100, Shaanxi, China.
AD  - Scientific Observing and Experimental Station of Veterinary Pharmacology and 
      Diagnostic Technology, Ministry of Agriculture, Yangling, 712100, Shaanxi, China.
FAU - Zhou, En-Min
AU  - Zhou EM
AD  - Department of Preventive Veterinary Medicine, College of Veterinary Medicine, 
      Northwest A&F University, Yangling, 712100, Shaanxi, China. zhouem@nwsuaf.edu.cn.
AD  - Scientific Observing and Experimental Station of Veterinary Pharmacology and 
      Diagnostic Technology, Ministry of Agriculture, Yangling, 712100, Shaanxi, China. 
      zhouem@nwsuaf.edu.cn.
FAU - Wu, Chunyan
AU  - Wu C
AD  - Department of Preventive Veterinary Medicine, College of Veterinary Medicine, 
      Northwest A&F University, Yangling, 712100, Shaanxi, China. 
      chunyanwu@nwafu.edu.cn.
AD  - Scientific Observing and Experimental Station of Veterinary Pharmacology and 
      Diagnostic Technology, Ministry of Agriculture, Yangling, 712100, Shaanxi, China. 
      chunyanwu@nwafu.edu.cn.
LA  - eng
PT  - Evaluation Study
PT  - Journal Article
PT  - Research Support, Non-U.S. Gov't
DEP - 20181116
PL  - England
TA  - BMC Biotechnol
JT  - BMC biotechnology
JID - 101088663
RN  - 0 (Antibodies, Viral)
RN  - 0 (Antigens, Viral)
RN  - EC 1.13.12.- (Luciferases)
MH  - Animals
MH  - Antibodies, Viral/*blood/immunology
MH  - Antigens, Viral/blood/immunology
MH  - Enzyme-Linked Immunosorbent Assay
MH  - HEK293 Cells
MH  - Humans
MH  - Immunologic Tests/*methods
MH  - Immunoprecipitation
MH  - Luciferases/genetics/metabolism
MH  - Porcine Reproductive and Respiratory 
      Syndrome/*blood/diagnosis/immunology/virology
MH  - Porcine respiratory and reproductive syndrome 
      virus/genetics/immunology/*isolation & purification
MH  - Sensitivity and Specificity
MH  - Swine
PMC - PMC6240198
OTO - NOTNLM
OT  - Antibody detection
OT  - ELISA
OT  - Luciferase immunoprecipitation systems, luciferase-linked antibody capture assay
OT  - PRRSV
COIS- ETHICS APPROVAL AND CONSENT TO PARTICIPATE: The animal protocol of this study was 
      approved by the animal welfare committee of Northwest A&F University. CONSENT FOR 
      PUBLICATION: Not applicable. COMPETING INTERESTS: The authors declare that they 
      have no competing interests. PUBLISHER'S NOTE: Springer Nature remains neutral 
      with regard to jurisdictional claims in published maps and institutional 
      affiliations.
EDAT- 2018/11/18 06:00
MHDA- 2019/08/09 06:00
PMCR- 2018/11/16
CRDT- 2018/11/18 06:00
PHST- 2018/08/02 00:00 [received]
PHST- 2018/10/29 00:00 [accepted]
PHST- 2018/11/18 06:00 [entrez]
PHST- 2018/11/18 06:00 [pubmed]
PHST- 2019/08/09 06:00 [medline]
PHST- 2018/11/16 00:00 [pmc-release]
AID - 10.1186/s12896-018-0483-5 [pii]
AID - 483 [pii]
AID - 10.1186/s12896-018-0483-5 [doi]
PST - epublish
SO  - BMC Biotechnol. 2018 Nov 16;18(1):73. doi: 10.1186/s12896-018-0483-5.