Improvement And Application Of Glypican-3 Enzyme-linked Immunoassay

Proteoglycans(PGs)is a biomacromolecule composed of a core protein and one or more covalently linked glycosaminoglycan(GAG)chains.Glypicans(GPCs)is a important family of proteoglycans attached to the cell surface by a glycophosphatidylinositol anchor(GPI).There are six main members(GPC1-GPC6)in the family,in which GPC3 is a member of the family.GPC3 is a heparan sulfate proteoglycan(HSPGs),which is attached to the cell membrane by GPI anchor.GPC3 is composed of a core protein of 70 kDa containing 580 amino acids and two side chains attached around the C-terminal.GPC3 are specifically expressed in a variety of tumors,including hepatocellular carcinoma(HCC),lung squamous cell carcinoma(SqCC),gastric cancer,ovarian cancer,melanoma,and childhood embryonic tumors,among which the expression in HCC is particularly high.Thus,GPC3 has become a typical biomarker for HCC diagnosis from the point of view of molecular mechanism and clinical sample research results.At present,whether domestic or foreign,a large number of studies have been conducted on the clinical diagnosis of HCC using GPC3 as the target,and a series of important research results have been obtained.The traditional sandwich ELISA method is widely used in the detection of GPC3 in patient serum due to its simple operation,no need for expensive supporting equipment,and easy popularization.However,the method has the disadvantages of low sensitivity,poor reproducibility and time-consuming.Especially in the early stage of HCC,the GPC3 content in serum is very low.In addition,the serum contains a large number of biological macromolecules such as plasma proteins,peptides,GAGs and nucleic acids that are easily combined with GPC3,which seriously interferes with the detection of GPC3,making the traditional sandwich ELISA method encounter great difficulties in the early clinical diagnosis of HCC with GPC3 as a marker.Therefore,there is an urgent need to improve the GPC3 enzyme-linked immunosorbent assay method.Based on the structure of GPC3,the presence of two polyanionic HS side chains located at the carboxyl end of its core protein can not only bind to a variety of proteins and other biological macromolecules,but also interfere with the recognition of core protein by antibodies.We speculate that the existence of HS polysaccharide chains may be the main reason for the time-consuming,low sensitivity and poor reproducibility of the traditional ELISA method to detect GPC3.Therefore,in this study,we propose to use glycosaminoglycan-degrading enzymes to specifically degrade and remove the HS side chains at the carboxyl end of the GPC3 core protein,thereby exposing the antibody binding site on it in order to improve the sensitivity,stability and speed of GPC3 detection by traditional ELISA,and solve the difficult problems faced by this method in the clinical diagnosis of HCC.The main findings of this paper are as follows:.1.The expression and purification of secreted GPC3.The full-length gene of cell-secreted GPC3(GPC3 ΔGPI),which was obtained by deleting the GPI binding site in the previous research of our laboratory,was constructed into the eukaryotic expression vector pTracer and transfected into HEK293T cells.The cells were cultured at 37℃ and 5%CO2,and the culture supernatant was collected.GPC3 was purified from the cell culture supernatant by chromatography on DEAE Sepharose Fast Flow,and detected and quantified by Western-blot.The result Shows that the purified GPC3 is consistent with the reports in the literature.2.Preparation and titer determination of antibodies.(1)Preparation of monoclonal antibodies:two monoclonal cell lines that can specifically recognize GPC3 amino-terminal antibody(αGCN)and carboxy-terminal antibody(αGCC)were inoculated into the abdominal cavity of BALb/c mice to form ascites tumors and the ascites was collected to purify the corresponding monoclonal antibody by Protein G/A affinity chromatography.SDS-PAGE analysis showed that the purified monoclonal antibody was separated into a heavy chain of 55 kDa and a light chain of 25 kDa by electrophoresis under reducing conditions,indicating that the two monoclonal antibodies aGCN and αGCC have good purity.(2)Preparation of camel polyclonal antibody:camel was immunized using the fully emulsified purified antigen GPC3N,and the titer of the camel’s antiserum was determined by using the indirect ELISA method.The results show that the titer can reach more than 10,000;(3)Preparation of rabbit polyclonal antibody:after fully emulsifying the purified antigen GPC3ΔGPI were used to immunizer abbits.By titer determination:the titers of the serum from New Zealand white rabbit No.1 and No.2 can reached 10,000 and 20,000,respectively.3.Improvement of glypican-3 enzyme-linked immunoassay.The purified GPC3 was treated with different types of glycosaminoglycan-degrading enzymes at 30℃for 30 min,and then detected by double-antibody sandwich ELISA method.The experimental results showed that the detection effect of GPC3 was the best after the combined treatment with Hepase and EnCHase.The conditions in this method are further optimized:(1)Selection of capture antibody and detection antibody:monoclonal antibodies aGCN and aGCC were used as capture antibodies for plating,and camel polyclonal antibody or rabbit polyclonal antibody was used for antigen detection,and finally the capture antibody was determined to be monoclonal antibody aGCN and detection antibody camel polyclonal antibody.(2)Determination of the coating amount of the capture antibody:different concentrations of aGCN were prepared,plated 50 μl/well overnight,and then detect according to the sandwich ELISA method.The coating amount of the antibody was selected to be 0.25 μg/well according to the difference in absorbance value and the background value.(3)Screening of blocking solution:buffers containing different concentrations of BSA or skimmed milk powder and buffers of different concentrations of BSA or skimmed milk powder containing 5%sucrose or lactose were used as blocking solutions,and GPC3 detection was performed under the optimized other conditions.The results show that PBS buffer containing 5%skimmed milk powder is the best blocking solution.(4)Screening of antigen dilution buffer:GPC3 was dissolved in 10 mM NaAc-HAc,NaH2PO4-Na2HPO4,or Tris-HCl buffers with different pH,and treated with glycosaminoglycan-degrading enzymes at 30℃for 30 min.GPC3 was tested under optimized other conditions,and the results showed that in the pH 5.0 NaAc-HAc was the best buffer for GPC3 detection;then pH 5.0 NaAc-HAc buffers containing different concentrations of NaCl were prepared and the GPC3 detection was performed according to the previous method.the results show that the optimal buffer is NaAc-HAc containing 100 mM NaCI pH 5.0.(5)Determination of antigen incubation time:the antigen treated with glycosaminoglycan-degrading enzymes was reacted with the capture antibody for different times(0.5,1.0,2.0,3.0,and 4.0 h),and other analysis steps were the same as above.The results showed that the best incubation time for antigen was 1 h at room temperature.(6)Determination of the dilution factor of the detection antibody:The camel polyclonal antibody was diluted in a concentration gradient with PBS buffer containing 1%skimmed milk powder,and GPC3 was detected under the optimized other conditions.The results showed that the optimal dilution factor for camel polyclonal antibody was 2,000 times in PBS buffer containing 1%skimmed milk powder.(7)Determination of the dilution factor of enzyme-labeled antibody:the enzyme-labeled antibody was diluted in a concentration gradient of PBS buffer containing 1%skimmed milk powder,and then GPC3 was detected under the optimized other conditions.The results showed that the best dilution condition of enzyme-labeled antibody was 50,000 times in PBS buffer containing 1%skimmed milk powder.(8)Pretreatment of antigen-containing serum:experiments have showed that the pretreatment of diluted serum samples containing GPC3 at 40℃ for 30 min could significantly improve the sensitivity of GPC3 detection.Through the above improvements and optimization of conditions of the sandwich ELISA method,the sensitivity of the detection of GPC3 in serum was increased from the original ng level to the pg level,and the time was shortened from the original 9～10 hours to 5 hours,and the stability of the test results was significantly improved.4.Application of the improved GPC3 enzyme-linked immunosorbent assay method:to verify the feasibility of the improved sandwich ELISA method in detecting GPC3 in the serum of HCC patients,we pretreated the serum samples at 40℃ for 30 minutes,and then added glycosaminoglycan-degrading enzymes to further react at 30℃ for 30 min,and then tested according to the improved sandwich ELISA detection method.The experimental results show that the positive detection rate in clinical samples can reach 61%.Compared with the traditional enzyme-linked immunoassay method,the improved GPC3 enzyme-linked immunoassay method has simple operation,significantly shortened detection time,high sensitivity,good specificity,and high positive detection rate in patients with hepatocellular carcinoma.It has important application value in the early diagnosis of HCC,laying the foundation for clinical application.