Development of a Dot Blot Assay with Antibodies to Recombinant "core" 14-3-3 Protein: Evaluation of Its Usefulness in Diagnosis of Creutzfeldt-Jakob Disease

Overview

Journal Ann Indian Acad Neurol

Specialty Neurology

Date 2016 Jun 14

PMID 27293331

Citations 3

Authors

Sarada Subramanian

Anita Mahadevan

Parthasarathy Satishchandra

Susarla Krishna Shankar

Affiliations

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Abstract

Background And Purpose: Definitive diagnosis of Creutzfeldt-Jakob disease (CJD) requires demonstration of infective prion protein (PrP(Sc)) in brain tissues by immunohistochemistry or immunoblot, making antemortem diagnosis of CJD difficult. The World Health Organization (WHO) recommends detection of 14-3-3 protein in cerebrospinal fluid (CSF) in cases of dementia, with clinical correlation, as a useful diagnostic marker for CJD, obviating the need for brain biopsy. This facility is currently available in only a few specialized centers in the West and no commercial kit is available for clinical diagnostic use in India. Hence the objective of this study was to develop an in-house sensitive assay for quantitation of 14-3-3 protein and to evaluate its diagnostic potential to detect 14-3-3 proteins in CSF as a biomarker in suspected cases of CJD.

Materials And Methods: A minigene expressing the "core" 14-3-3 protein was synthesized by overlapping polymerase chain reaction (PCR) and the recombinant protein was produced by employing a bacterial expression system. Polyclonal antibodies raised in rabbit against the purified recombinant protein were used for developing a dot blot assay with avidin-biotin technology for signal amplification and quantitation of 14-3-3 protein in CSF.

Results: The results in the present study suggest the diagnostic potential of the dot blot method with about 10-fold difference (P< 0.001) in the CSF levels of 14-3-3 protein between the CJD cases (N= 50) and disease controls (N= 70). The receiver operating characteristic (ROC) analysis of the results suggested an optimal cutoff value of 2 ng/mL.

Conclusions: We have developed an indigenous, economical, and sensitive dot blot method for the quantitation of 14-3-3 protein in CSF.

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References

Parchi P, Giese A, Capellari S, Brown P, Schulz-Schaeffer W, Windl O . Classification of sporadic Creutzfeldt-Jakob disease based on molecular and phenotypic analysis of 300 subjects. Ann Neurol. 1999; 46(2):224-33. View

Fu H, Subramanian R, Masters S . 14-3-3 proteins: structure, function, and regulation. Annu Rev Pharmacol Toxicol. 2000; 40:617-47. DOI: 10.1146/annurev.pharmtox.40.1.617. View

van Hemert M, Steensma H, van Heusden G . 14-3-3 proteins: key regulators of cell division, signalling and apoptosis. Bioessays. 2001; 23(10):936-46. DOI: 10.1002/bies.1134. View

Yaffe M . How do 14-3-3 proteins work?-- Gatekeeper phosphorylation and the molecular anvil hypothesis. FEBS Lett. 2002; 513(1):53-7. DOI: 10.1016/s0014-5793(01)03288-4. View

Mahadevan A, Shankar S, Yasha T, Santosh V, Sarkar C, Desai A . Brain biopsy in Creutzfeldt-Jakob disease: evolution of pathological changes by prion protein immunohistochemistry. Neuropathol Appl Neurobiol. 2002; 28(4):314-24. DOI: 10.1046/j.1365-2990.2002.00399.x. View

Berg D, Holzmann C, Riess O . 14-3-3 proteins in the nervous system. Nat Rev Neurosci. 2003; 4(9):752-62. DOI: 10.1038/nrn1197. View

Van Everbroeck B, Boons J, Cras P . 14-3-3 {gamma}-isoform detection distinguishes sporadic Creutzfeldt-Jakob disease from other dementias. J Neurol Neurosurg Psychiatry. 2004; 76(1):100-2. PMC: 1739297. DOI: 10.1136/jnnp.2003.032037. View

Shiga Y, Wakabayashi H, Miyazawa K, Kido H, Itoyama Y . 14-3-3 protein levels and isoform patterns in the cerebrospinal fluid of Creutzfeldt-Jakob disease patients in the progressive and terminal stages. J Clin Neurosci. 2006; 13(6):661-5. DOI: 10.1016/j.jocn.2005.09.004. View

Cao L, Cao W, Zhang W, Lin H, Yang X, Zhen H . Identification of 14-3-3 protein isoforms in human astrocytoma by immunohistochemistry. Neurosci Lett. 2008; 432(2):94-9. DOI: 10.1016/j.neulet.2007.11.071. View

10.

Subramanian S, Divya Shree A . Enhanced Th2 immunity after DNA prime-protein boost immunization with amyloid beta (1-42) plus CpG oligodeoxynucleotides in aged rats. Neurosci Lett. 2008; 436(2):219-22. DOI: 10.1016/j.neulet.2008.03.024. View

11.

Subramanian S, Madhavadas S, Balasubramanian P . Influence of conformational antibodies on dissociation of fibrillar amyloid beta (A beta 1-42) in vitro. Indian J Exp Biol. 2009; 47(5):309-13. View

12.

Subramanian S, Bandopadhyay D, Mishra P, Mathew M, John M . Design and development of non-fibrillar amyloid beta as a potential Alzheimer vaccine. Biochem Biophys Res Commun. 2010; 394(2):393-7. DOI: 10.1016/j.bbrc.2010.03.031. View

13.

Satishchandra P, Shankar S . Creutzfeldt-Jakob disease in India (1971-1990). Neuroepidemiology. 1991; 10(1):27-32. DOI: 10.1159/000110244. View

14.

Satoh K, Tobiume M, Matsui Y, Mutsukura K, Nishida N, Shiga Y . Establishment of a standard 14-3-3 protein assay of cerebrospinal fluid as a diagnostic tool for Creutzfeldt-Jakob disease. Lab Invest. 2010; 90(11):1637-44. DOI: 10.1038/labinvest.2009.68. View

15.

Matsui Y, Satoh K, Miyazaki T, Shirabe S, Atarashi R, Mutsukura K . High sensitivity of an ELISA kit for detection of the gamma-isoform of 14-3-3 proteins: usefulness in laboratory diagnosis of human prion disease. BMC Neurol. 2011; 11:120. PMC: 3204235. DOI: 10.1186/1471-2377-11-120. View

16.

Foote M, Zhou Y . 14-3-3 proteins in neurological disorders. Int J Biochem Mol Biol. 2012; 3(2):152-64. PMC: 3388734. View

17.

Muayqil T, Gronseth G, Camicioli R . Evidence-based guideline: diagnostic accuracy of CSF 14-3-3 protein in sporadic Creutzfeldt-Jakob disease: report of the guideline development subcommittee of the American Academy of Neurology. Neurology. 2012; 79(14):1499-506. PMC: 3525296. DOI: 10.1212/WNL.0b013e31826d5fc3. View

18.

Paterson R, Torres-Chae C, Kuo A, Ando T, Nguyen E, Wong K . Differential diagnosis of Jakob-Creutzfeldt disease. Arch Neurol. 2012; 69(12):1578-82. PMC: 4401069. DOI: 10.1001/2013.jamaneurol.79. View

19.

Margalit H, Spouge J, Cornette J, Cease K, Delisi C, Berzofsky J . Prediction of immunodominant helper T cell antigenic sites from the primary sequence. J Immunol. 1987; 138(7):2213-29. View

20.

Schagger H, von Jagow G . Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa. Anal Biochem. 1987; 166(2):368-79. DOI: 10.1016/0003-2697(87)90587-2. View