Establishment Of Detective Method By Enzyme Linked Immunosorbent Assay For Lectin Affinity Of Human Serum AFP-L3

ObjectiveHepatocellular carcinoma?HCC?is the sixth most common malignancy in the world,and it ranks third in lethality among cancers.Early diagnosis and early treatment is an effective means to improve five-year survival rate and quality of life.At present,there are two kinds of serum tumor markers:alpha-fetoprotein?AFP?and lens culinaris agglutinin-reactive alpha-fetoprotein?AFP-L3?,which are used to detect hepatocellular carcinoma.The sensitivity and specificity of AFP are insufficient.Clinically,about 40%of patients with HCC do not have elevated,while chronic hepatitis,cirrhosis and other chronic liver diseases increased in different degrees.AFP-L3 is a subtype of AFP,which is a glycosylated protein,mainly produced by hepatoma carcinoma cells.AFP-L3 can be used not only for early diagnosis,but also for efficacy evaluation and recurrence monitoring.In addition to AFP-L3,studies have found that other fucosylated proteins also play an important role in the occurrence and development of diseases.These glycoprotein molecules are also candidate markers for liver cancer and other diseases.The study intends to establish a lectin-based ELISA method for the direct detection of fucosylated proteins,which could lay a foundation for the detection of fucose glycosylated protein by using sugar affinity liquid chip in our later experiment.It can also provide a new method for detection of multiple fucosylation biomarkers for liver cancer and other diseases.We chose AFP-L3 as the target protein,because AFP-L3 is the only clinical tumor marker of fucose glycosylation,which is easy to compare with the clinical detection technology.Method1.Removing fucosyl group on the Fc segment of the coated AFP monoclonal antibody by using NaIO₄.2.Using AFP monoclonal antibody as coating antibody,biotin-labeled lectin recognizes fucosyl group on AFP-L3,using SA-HRP for detection.3.On the established lectin affinity of enzyme-linked immunosorbent assay each steps reaction condition was optimized,and the minimum detection limit,intra-assay and inter-assay precision,interference experiment,stability and other performance evaluation.Result1.The optimal treatment of aldehyde group formed by mild oxidation and blocking oxidation of antibody was as follows:25mmol/L NaIO4 dissolved in sodium acetate?PH4.5?,4?for 1.5h under darkness;1mmol/L MPBH,incubated at 18-22?for 2h without light;and 1mmol/L Cys-Gly solution,incubated at 4?for 12h under darkness.2.The optimal reaction conditions of ELISA for lectin affinity were as follows:1:800 diluted antibody coated,1:8000 diluted biotin-labeled lectin and SA-HRP;coating condition is 37?for 3h;blocking reagent and blocking condition is Carbo-free Blocking Solution,30min at room temperature;incubation condition for sample is 37?for 2h.The reaction conditions of biotin-labeled lectin and SA-HRP are 37?and 30min;the reaction conditions of TMB is at 37?for 20 min in dark.3.The evaluation results of the methodology are as follows:the absorbance at the lowest detection concentration is 0.1247,and the corresponding concentration is 18.23ng/ml;the intra-assay and inter-assay precision of high-value samples are 5.45%and6.47%,respectively.And those of low-value samples are 7.24%and 9.37%,respectively.The results are almost unaffected by bilirubin C,bilirubin F,hemoglobin,and chyme;the degree of instability increased with storage time,and the difference is statistically significant after 2 weeks?P<0.05?.The results obtained by this method are consistent with the clinical test result.Conclusion1.Established an effective method to remove fucosyl from the antibody.2.A method to detect AFP-L3 based on lectin affinity-based enzyme-linked immunosorbent assay was initially established.And the feasibility was confirmed by methodological evaluation.3.It is confirmed that detection results of the new method are in agreement with the clinical detection and it has the prospect of clinical application.