The Preliminary Establishment Of ELISA Detection Method For The Subunit Of Medium Neurofilament Protein

OBJECTIVENeurofilaments(NFs)are intermediate filament in mature neurons,and are major components of cytoskeletal neurons.And they are closely related to maintain the form of axons,signal transduction,and axoplasmic transport.Neurofilaments are heteropolymers that composed of five subunits:neurofilament heavy,medium and light polypeptides,as well asα-internexin and peripherin.Among all of them,NFM plays a key role in maintaining the form of NFs,and the diameter of axons are determined by it.Changes in metabolism and synthesis of NFM are associated with many diseases.And it is expected to be a biomarker for them.Therefore,the detection of NFM in blood or cerebrospinal fluid can help to early diagnosis and the prognosis of the disease.At present,although NFM has many methods of detection,it lacks an ideal method for clinical detection.So it is necessary to study the sensitivity and specificity detection method of NFM.In this study,the NFM antibody and its standard product was prepared.Then establish the ELISA detection method.METHODS1.The preparation,purification and labeling of anti-NFM monoclonal antibodies(McAb)and polyclonal antibodies(PcAb).To analyse the NFM amino acid sequences of human,rat and mouse,and then to select three peptides P1,P2,P3 of human.They were synthesized and coupled with KLH.Immune mice and pick its serum.Western blot and IHC were used to detect the reactivity of NFM in human,rat and mouse brain tissues.Choose PI and P2 to immunize New Zealand white rabbits with a large amount of serum.Two hybridoma cell lines P2,P3 had been prepared by our lab.McAbs were prepared by inducing ascites in vivo.ELISA was used to detect antibody titer and affinity chromatography to purify serum and ascites.And the HRP was marked with Abs.2.Expression and purification of NFM protein.The mRNA was extracted from human glioma and turned into cDNA by RT-PCR.The RT-PCR product was cloned into pMD19-T Simple.After transformation and selection by ampicillin and blue/white spotting.The target gene and pET-30a were cut by enzyme and linked.Then expressed it in prokaryotic cell.SDS-PAGE and Western blot were used to identify the proteins.The bacteria were collected and then been broken by ultrasonic.At last use affinity chromatography to purify NFM.3.The preliminary establishment of NFM double antibody sandwich ELIS A detection method.Applicated the double antibody sandwich method to screen the best pair of Abs and determind their working concentration.The NFM protein was used as the standard and establish the ELISA detection method.4.Sensitivity and specificity of NFM double antibody sandwich ELISA.The detection limit of the method was detected by dilution and the specificity was detected by BSA.RESULTS1.The preparation,purification and labeling of anti-NFM McAb and PcAb.The anti-NFM Abs were successfully prepared.The IHC results showed that all of the three Abs were able to respond to NFM in human glioma,rat and mouse brain tissue.The results of Wesntem blot showed that only the anti-P1 PcAb can reacted with NFM in human glioma,rat and mouse brain tissue,while others do not.The Abs titer in rabbit serum and mouse ascites were greater than 128000.Abs were successfully purified and labeled.Successfully purified and labeled anti-P1 PcAb,anti-P2 PcAb,anti-P2 McAb and anti-P3 McAb.2.Expression and purification of human NFM protein.The clone vector and expression vector were successfully constructed,and the NFM protein standard was obtained after expressing and purifying.The western blot results showed that all of the anti-NFM antibodies and anti-His antibody were able to react with NFM protein.The concentration was 500 mg/ml and the purity was 90.9%.3.The preliminary establishment of NFM double antibody sandwich ELISA detection method.When capture Ab is anti-P3 McAb and the detect Ab is anti-P2-HRP PcAb,there is linear relationship.And the concentration was 20 μg/mL and 1 μg/mL respectively.When the detection range from 3.9 to 62.5 μg/ml,there is a good linear relationship and the linear equation was Y=116.27X+16.895,R2=0.9934.4.Sensitivity and specificity of NFM double antibody sandwich ELISA.The limit of detection was 3.9 μg/mL and this method has good specificity with no reaction abou BSA.CONCLUSIONIn this study,NFM protein was successfully expressed and purified.Anti-NFM McAb and PcAb were prepared.In addition,a double antibody sandwich method was established with the antigen that we have prepared,which has high specificity.However,some conditions of this detection method need to be further optimized.