Establishment Of A ELISA Method For Detection Of Specific Tau Protein In CSF Samples And Preliminary Application For Patients With Prion Diseases

Prion Disease(PrD),also known as Transmissible Spongiform Encephalopathy(TSE),is a contagious,neurological invasion of the Central Nervous System(CNS)in humans and other mammals.Degenerative diseases have the characteristics of long incubation period and 100%mortality.At present,human prion diseases are mainly divided into: Creutzefeldt-Jakob Disease(CJD),Kuru,and Gerstmann-Straüssler-Scheinker syndrome(GSS).And Fatal Familial Insomnia(FFI),the most common CJD.CJD can be further divided into sporadic(sCJD),iatrogenic(iCJD),familiar or genetic(fCJD or gCJD)and variant Creutzfeldt-Jakob disease(vCJD).At present,the clinical detection method has a biopsy of the brain tissue of the patient,and the diagnosis of the disease is assisted by detecting the mutation of the PRNP gene in the blood to determine the genetic type of prion disease,and detecting the increase of the 14-3-3protein content in the cerebrospinal fluid of the sCJD patient.Brain biopsy has great physical damage to the patient.According to whether the PRNP gene in the blood is mutated,only the genetic type of Creutzfeldt-Jakob disease can be judged,there is a great limitation,and the detection of 14-3-3 protein in cerebrospinal fluid is not specific.Therefore,there is an urgent need for an effective diagnostic method for prion diseases.Microtubules are composed of tubulin and microtubule-associated protein(tau protein),of which tau is the highest content of microtubule-associated protein,and microtubule system is a neurocytoskeleton component that can participate in a variety of cellular functions.Studies have shown that tau protein is closely associated with a variety of neurological diseases and is significantly elevated in prion patients and rodent model brain tissues,including exon 2(tauexon 2)and exon 10(tau-exon 10)accounts for the majority of the total tau protein content,so this study established a tau protein ELISA method and applied it to cerebrospinal fluid of different types of prion patients.In the present study,the target protein GST tau-exon 2/10 was expressed using prokaryotic cells containing the p GEX-2T-E2 and p GEX-2T-E10 plasmids,and the target protein concentration was determined.The GST tau-exon 2 protein concentration was 0.166 ?g/?L,GST tau-exon 10 protein concentration was 0.473 ?g/?L,and monoclonal antibodies were prepared.The prepared GST tau-exon 2 protein monoclonal antibody number is 1-4(1.8 mg/mL),4-1(1.7mg/mL),GST tau-exon 10 protein monoclonal antibody number 6-4(2.6 mg/mL),5-6(1.8mg/mL).The GST tau-exon 2/10 protein was detected by Western blotting using the prepared mouse monoclonal antibody,and the specificity of the prepared antibody was confirmed.The tau protein was detected in the neural cell model and mouse brain tissue by Western blotting.Proveantibody sensitivity.Using the prepared antibody to establish a more sensitive double-antibody sandwich ELISA method,the conditions of the final GST tau-exon 2/10 protein-specific monoclonal antibody double-antibody sandwich ELISA method were determined as follows:coating antibody H-150 concentration was 8 ?g/mL,detection antibody GST tau-exon 2(1-4)protein murine monoclonal antibody,GST tau-exon 2(4-1)protein murine monoclonal antibody,GST tau-exon 10(6-4)The working concentration of the mouse monoclonal antibody and the GST tau-exon 10(5-6)protein murine monoclonal antibody was selected to be 1 ?g/mL,1?g/mL,0.8 ?g/mL,and 1 ?g/mL,respectively.The horseradish peroxidase-labeled goat antimouse secondary antibody dilution ratio was 1:5,000.The established double-antibody sandwich ELISA method was used to detect the content of tau-exon 2/10 protein in cerebrospinal fluid of gCJD and non-CJD patients including sCJD,GSS,FFI and T188 K mutations.The results showed that the cerebrospinal fluid in the sCJD patients contained explicit The tau protein of sub-2 was significantly higher than that of non-CJD patients,with a statistically significant difference(p<0.001).The tau protein containing exon 10 in cerebrospinal fluid of sCJD patients was significantly higher than that of non-CJD patients.Significant statistical difference(p<0.01).The tau exon 2/10 protein content in the cerebrospinal fluid of patients with t188 K mutation in gCJD was significantly higher than that in non-CJD patients,with statistically significant difference(p<0.01).The content of tau exon 2/10 protein in cerebrospinal fluid of patients with GSS and FFI was not significantly different from that of nonCJD patients,and there was no statistical difference(NS).The specific tau double-antibody sandwich ELISA method established in this study can effectively detect the level of tau-exon 2/10 protein in cerebrospinal fluid of patients with different types of prions,and provide an effective test method for the detection of prion diseases.The mechanism of prion disease research provides a theoretical basis.