| Objective:Alzheimer's disease(AD)is one of the major diseases that cause senile dementia.The etiology and pathological mechanism have not been fully elucidated so far.The difficulties in early diagnosis and early prevention are still hindering global sniper.The main reason for its rapid growth.Among them,exploring early diagnostic laboratory markers and constructing a diagnostic and differential diagnosis system with high sensitivity and high specificity may be the key to solving this dilemma.Considering that the A?cascade theory has been frustrated in the A?vaccine research,and another AD pathological marker,the neurofibrillary tangles,mainly appears in the cells in the early stage,while the other neurofilament protein light chain,which is also a neurofibrillary protein component,(NFL)is likely to be exposed to detectable extracellular fluid during the pathological process of AD.For this reason,we intend to detect AD,vascular dementia(VaD)patients and different enzymes by enzyme-linked immunosorbent assay(ELISA).Cerebrospinal fluid(CSF)and plasma NFL concentrations in normal age cognitive function(NC)were confirmed and analyzed by immunoblotting(WB)and immunohistochemical staining(ICH),respectively.method:(1)Grouping:Seventy-one subjects aged 1 to 86 years old were divided into three groups according to the disease diagnosis:19 in the AD group,9 in the VaD group,and 45 in the NC group;(2)Specimen collection:After routinely collecting relevant demographic data,clinical data,neuroimaging and cognitive neuropsychological examination data of all subjects,2 ml of whole blood was collected on an empty stomach in the morning to a test tube supplemented with EDTA anticoagulant,and centrifuged.After preparation into plasma samples.In some patients and NC group,2ml of cerebrospinal fluid was taken by routine disinfection and local anesthesia with lumbar puncture,and stored in a refrigerator at-80°C after being packed.(3)Experimental method:1.ELISA quantitative determination of NFL concentration:according to the SEEO38Hu 96T kit and IBL international kit instructions,respectively,the NFL concentration in all plasma and cerebrospinal fluid samples were tested;2.Immunoblotting(WB)qualitative analysis of autoanti-NFL antibodies in plasma and cerebrospinal fluid:First,the NFL concentrations detected by plasma and cerebrospinal fluid specimens ELISA were reclassified into four subgroups by quartile(NFL concentration<25%).Group,NFL concentration 25%to 50%group,NFL concentration 50%to 75%group,NFL concentration>75%group),followed by purified bovine spinal cord NFL protein(product code62008)as electrophoresis protein and target antigen,and Plasma(both 24 cases)and cerebrospinal fluid(16 cases)with NFL concentrations<25%and>75%were randomly selected as recognition antibodies,and polyclonal anti-NFL antibody(product code PAB12120)was set as positive control,phosphate buffer(PBS)was used as a negative control,and immunological characteristics of the anti-NFL antibody(nAb-NFL)were analyzed after immunochemical fluorescence imaging.3.Immunohistochemical staining(ICH):Randomly selected the above-mentioned ELISA test results in the quartile NFL concentration of<25%,>75%of plasma/cerebrospinal fluid specimens as primary antibody,goat anti-human IgG as secondary antibody The immunological recognition of NFL antigen in tissue-derived Tg2576 mouse sections was observed.result:1.ElISA test results1.1 Plasma NFL test results:plasma NFL concentration was ranked as VaD group,AD group and NC group(F=11.71,P=0.000).Subgroup analysis showed that VaD group and AD group were significantly higher than NC group,the difference was Statistical significance(t=4.132,P=0.000;t=2.079,P=0.081),but there was no significant difference between the VaD group and the AD group(t=0.878,P=0.410).1.2 CSF NFL test results:The concentration of CSF in the cerebrospinal fluid was ranked as VaD group,AD group and NC group(F=16.338,P=0.000).The subgroup analysis showed that the VaD group and the AD group were significantly higher than the NC group.Statistical significance,(t=32.116,P=0.000;t=4.130,P=0.009),but the difference between VaD group and AD group was not statistically significant(t=1.844,P=0.122).1.3 The age-related natural variation of plasma and cerebrospinal fluid NFL concentration:There was a statistically significant difference in plasma NFL concentration between the NC groups of different age groups(F=5.807,P=0.000),and showed a curve of overall inhibition.The upward trend,regression analysis showed that the age-determined coefficient of plasma NFL concentration in the NC group was 0.332;2 the NFL concentration of NC cerebrospinal fluid in different age groups showed an increasing trend of age-related curve similar to peripheral blood:the difference between different age groups Significant statistical significance was achieved(F=3.015;P=0.047),and the age determination coefficient was 0.297.The study also showed that the concentration of NFL in plasma and cerebrospinal fluid entered a steadily increasing phase from middle age in the age-related natural variation.1.4 Correlation analysis of NFL concentration in plasma/cerebrospinal fluid with population data and clinical variables:age,disease duration,education level,MMSE score,plasma S100B level and NFL concentration in plasma and cerebrospinal fluid were not found in the two groups.Statistically significant associations and differences(P>0.05).2.Results of immunoblotting(WB)study:2.1 24 samples of plasma were randomly selected according to the ratio of 1:2(11 cases with NFL concentration<25%,13 cases with NFL concentration>75%).Immunoblot analysis showed that 9 cases(9/24;37.5%)Plasma can recognize standard NFL protein(62008),including 1 case(1/11;9.09%)in the low concentration group and 8 cases(8/13;61.5%)in the high NFL concentration group.Fisher's exact probability analysis confirmed a statistically significant difference in the positive rate of nAb-NFL between the two groups(P=0.013).The results of immunoblotting were analyzed by disease group analysis.The positive rate of nAb-NFL in NC group(1/11)was significantly lower than that in AD group(7/8)and VaD group(1/1)(X2=11.67;P=0.003).).2.2 The same grouping and experimental methods were used to analyze 16 cases of cerebrospinal fluid samples(9 cases with NFL concentration<25%group;7 cases with NFL concentration>75%group);4 cases(4/9;44.44%)were detected in low concentration group.Seven patients(7/7;100%)were detected in the high-concentration group,and the positive rate was statistically significant(P=0.034).The results of immunoblotting were analyzed by disease group analysis.The positive rate of nAb-NFL in NC group(4/9)was significantly lower than that in AD group(5/5)and VaD group(2/2)(X~2=7.509;P=0.023)..Conclusion:1.The concentration of NFL in plasma and cerebrospinal fluid was significantly higher in the AD and VaD groups than in the NC group,which could be used as a biomarker for the diagnosis of dementia.However,this study does not support its use as a diagnostic indicator for the etiology of dementia.2.The level of NFL in plasma/cerebrospinal fluid showed a similar age-related natural variation in the NC group from 1 to 86 years old.The middle-aged stage was the key segment of change.3.nAb-NFL antibody is ubiquitous in peripheral blood and cerebrospinal fluid of all subjects,and is highly prevalent in dementia population,and also has certain biomarker potential for dementia diagnosis. |
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