Development Of Ternary Gold Labeled Immunochromatograph Kit For Colorectal Cancer Screening

Background and ObjectiveColorectal cancer(CRC,including rectal cancer and colon cancer)is one of the most common malignant tumors in the world,and severely influences the quality of life of patients.According to statistics,the incidence rate of colorectal cancer ranks second place of malignant tumors in Western countries.With the continuous improvement of people's living standards and dietary habits,the incidence of CRC in our country has obvious rising trend.Experts predict that the morbidity and mortality rates of colorectal cancer will steadily rise for a long period of time in the future and become one of the most common malignant tumors with the fastest rising incidence in China.However,those patients without nonspecific symptoms at early stage had mostly lost the best timing of treatment when colorectal cancer was detected at advanced stage due to lack technology with high sensitivity and specificity for early diagnosis for CRC.Thus,the development of non-invasive method for rapid diagnosis of colorectal cancer patients for diagnosis and treatment in early stage has important scientific significance,improving survival rates and cure rate of patients with malignant tumors.Tumor M2-pyruvate kinase,an isozyme belonging to pyruvate kinase family,is a newly marker for tumor,which has shown a good prospect in early diagnosis and prognosis of tumor in recent year.Current studies have shown that PKM2 can be used as a new biomarker for early diagnosis of colorectal cancer.Previous studies have shown that PKM2 was found in intestines,lung,embryos and proliferative or undifferentiated tissue.PKM2 can also be detected in blood,body fluids or feces of patients with colorectal cancer,the reason is that in the process of tumor tissue necrosis,transformation,proliferation,the cells secreted PKM2 and released to blood and other body fluids.In addition,due to decreased tumor cell adhesion and faster updates,shedding of tumor cells excreted with the feces,and then provides a source for the detection of PKM2 in colorectal cancer stool.Furthermore,some results from Elisa experiments showed that compared with normal subjects,PKM2 content in feces from patients with colon cancer was significantly higher,and providers a very promising new method of detecting PKM2 in feces from patients with CRC for colorectal cancer screening.Carbohydrate antigen 19-9(CA19-9),is 116NS199 monoclonal antibody obtained from Koprowski etal's experiments in 1979,hybridized with immunized mice with colorectal cancer cells and myeloma mice.CA19-9 is sialylated milk-N-fucose pentose ?,which is a kind of glycoprotein fraction of mucin.The molecular weight of CA199 is 1 million?5 million U,and its antigen molecular weight is 36kd,which remain unchanged under alkaline conditions 37 ?,sh antigenic and can be recognized by MeAb.CA199 is not only expressed in the pancreas,liver and gallbladder and intestine of the fetus,but also in the pancreas and gallbladder of the adult.However,some patients with rare blood(3?7%)could not express CA19-9.CA19-9 is a pancreatic cancer,gallbladder cancer,colon cancer and gastric cancer associated tumor markers,also known as gastrointestinal cancer-associated antigen,in a variety of carcinomas were increased.CA19-9 has been widely used in clinical practice,it has become the blood index of early diagnosis of colorectal cancer,but the effect of single detection in the blood of its sensitivity and specificity is not ideal and often combined with other markers to improve the diagnostic sensitivity.Fecal occult blood test(FOBT)is the most commonly method for clinical examination and screening for rectal cancer.FOBT is very sensitive to detect a small amount of bleeding from the digestive tract,except colorectal cancer,thus FOBT greatly reduces the specificity of screening for colorectal cancer.The principle of specific binding of antigen and antibody to FOBT method,the pearl protein components by using anti human hemoglobin antibody can be detected in feces unspoilt or other blood component and solves the problem that human hemoglobin by globin of upper digestive tract has been degraded,metabolism and lose the immunogenicity.Immunochemical detection of fecal occult blood of colorectal cancer hemorrhage is more specific,and immunochemical FOBT would not react with the non-human hemoglobin.Patients do not have to limit the diet,and the impact of upper gastrointestinal bleeding is also minimal,so FOBT can significantly improve the specificity of colorectal cancer detection and patient compliance.Our group uses GICA technology to detect the markers of fecal shedding cells.GICA is a promising technique for rapid immunoassay technique which was recently developed.GICA technology(Gold immunochromatography assay,GICA)is a solid-phase immunoassay technology which organically combines colloidal gold labeling technique,immunoassay technology and a variety of chromatographic analysis.The gold labeled antibody has the characteristics of good stability,easy preservation,high sensitivity,and is not susceptible to interference in a sample of enzymes and other factors,the analysis technology of GICA can be used for detection of fecal samples.Moreover,the technique has the advantages of simple and rapid operation,no special instrument,etc.,making it suitable for medical units at all levels,and can also be used in the diagnosis,health care,physical examination and other aspects of family members.The purpose of our study is to further the technology innovation,from the original which can only detect a single marker to the development that simultaneous detection of multiple markers,which means using a strip to carry out a multiple simultaneous detection of multiple significant markers testing.The technology improved the detection of multiple indicators which has great value,can greatly improve the efficiency and reliability of detection.Methods1.The expression of PKM2 in CRC tissues and cellsProteins from 8 kinds of CRC cell lines are extracted for detecting the expression level of PKM2 in CRC cell lines using Western Blotting and exploring its localization by immunocytochemistry and immunofluorescence.Proteins from 10 pairs of CRC tissue and normal colorectal mucosa are determined to figure out the difference of protein expression level by Western Blotting.Imnunohistochemical method was used to detect the PKM2 expression in colorectal tissues and match normal tissues.In order to investigate the possible effect of PKM2 in colorectal cancer,we analyzed the relationship between PKM2 and clinical pathological parameters.2.The synthesis of colloidal gold antibody complexThe development of PKM2,CA19-9 and FOBT colorectal cancer rapid screening test strip was developed by using colloidal gold as tracer and immuno-chromatographic strip for lateral mobility respectively.First,we used 1%citric acid three sodium reduction method to prepare colloidal gold particles with five different diameters(10nm,15nm,20nm,30nm and 40nm).The colloidal gold was scanned at the wavelength of 400nm-700nm using UV-VIS spectrophotometer,and the maximum absorption wavelength,absorbance and median peak width were acquired.The particle size of colloidal gold was calculated based on the maximum absorption wavelength.The average diameter of the colloidal gold particles was measured by transmission electron microscopy,and the uniformity of particle size distribution was evaluated.Colloidal gold particles with appropriate particle size were chosen for subsequent experiment.Colloidal gold particle surface has a layer of biological macromolecules such as protein,and the surface of this layer of protein can protect these gold particles,so as to avoid the influence of external electrolyte and interaction together.And the adsorption of protein by colloidal gold particles depends on the pH value,which is due to the electrostatic charge of the protein depends on the solution of PH.In practice,the pH value of the colloidal gold is adjusted to PI+0.5 when preparing the colloidal gold probe,so that this protein is positively charged,and more conducive to binding protein.First the optimal volume of K2CO3 was determined and added to the colloidal gold solution to adjust pH value.Then different volumes of antibodies were added for conjugation,and OD520nm/OD580nm ratio was measured.Based on the results,the antibody volume-absorbance curve was plotted to determine the appropriate amount of antibodies.After the antibodies were added to the colloidal gold,halos around the colloidal gold particles were observed under the transmission electron microscope.The presence of halos indicated that the antibodies were absorbed onto the colloidal gold particles.Next the optimal amount of antibodies was added into the colloidal gold solution to react for 1h.Then with the addition of 10%BSA100ul,the solution was mixed for 30min and centrifuged at 12000r/min for 40 min,and the antibody-colloidal gold conjugates were resuspended in colloidal gold stock solution to obtain the colloidal gold probes.Finally,the stability of gold standard solution and the condition of its activity can be effectively maintained by optimizing the stabilizer and buffer solution of gold labeled antibody.3.Development of ternary gold labeled immunochromatograph kit for colorectal cancer screeningBy analyzing the feces in patients with CRC exfoliated cells of the cytology smear,the cancer cells found in colorectal cancer specimens as positive samples,the normal human specimens without cancer cells were used as negative controls.Design and preparation of fecal shedding cell collection and detection reagent bottle:Reagent bottle has a wide mouth,convenient to add the feces samples;in the center of the inner core tube with plug seal,at the bottom of inner core pipe set a 100 mesh screen separated used to filter waste residue of food;inner core pipe export department set 300 mesh separation to further filter after filter for the first time by the rest of the stool residue,outside the inner core pipe mouth with glue sealing cover.To ensure the reagent bottle clean and easy to use,the reagent bottle mouth with rubber sealing cover;bottle preloaded sample treating liquid,when used within the core tube pulled out,adding samples,then put into the inner core tube,tighten the inner core tube,covering the reagent bottle,shake specimens,and then open the inner core tube cover,drop from the sample to the kit for detecting groove detection.Be sure Optimization of treatment solution for fecal samples.Goat anti-mouse IgG secondary antibody,goat anti-rabbit IgG secondary antibody,PKM2 mouse monoclonal antibody,CA19-9 mouse monoclonal antibody and rabbit polyclonal antibody against FOBT were coated onto the nitrocellulose membrane control line and test line,respectively.The membranes were sealed with sealing liquid.The gold standard pad is soaked in the colloidal gold antibody complex,and the conditions for drying are selected.Then nitrocellulose membrane,absorbent paper,gold-labeled pad and sample pad were pasted to the PVC substrate successively and cut into test strip measuring 6cm in length and 0.4cm in width.The parameters of the PKM2,CA199 and FOBT immunochromatographic strips were optimized according to the result of stool test and IPP image analysis.4.Application of ternary gold labeled immunochromatograph kit for colorectal cancer screening in clinical patientStool specimens from 32 cases of normal human feces and 170 cases of colorectal cancer in the south hospital were collected.After treatment of feces specimens with self-made pyrolysis liquid,the samples were filtered and the food residue was removed.The PKM2,CA19-9 or FOBT colloidal gold immune chromatography test strip was placed into the stool sample suspension respectively,and the color development of the strip was observed in 5min.If the test line and control line simultaneously at the display of red bands,said detection of antigen in the sample which are to be measured,then the sentence is positive;if only in the control line shows a red band,then the sentence is negative;if the control line does not display the red band,said test strip production failure.The three strips were all positive specimens were judged to be positive,otherwise,the judgment was negative.The test strip was evaluated by strip/kit's sensitivity,and false negative rate(rate of missed diagnosis),false positive rate(misdiagnosis rate)and other effect evaluation index.The results of 3 strips were evaluated and compared using r2 test,with P<0.05 indicating significant difference.Results1.The expression of PKM2 in colorectal cancer tissues and cells in colorectal cancerThe expression of PKM2 in 8 colorectal cancer cell lines was detected by Western Blotting and the results showed that PKM2 had different degrees of expression in 8 colorectal cancer cell lines.Immunocytochemistry and immunofluorescence analysis showed that PKM2 was mainly expressed in cytoplasm.We detected the protein expression of PKM2 in 10 human paired CRC tissues and adjacent normal intestinal mucosa tissue by western blotting,the results showed that PKM2 expression in colorectal cancer tissue was significantly higher than that in normal mucosa.Immunohistochemical results also showed that PKM2 was not expressed or low expressed in normal intestinal mucosa,but was highly expressed in colorectal cancer tissues.2.The synthesis of colloidal gold antibody complexThe color and transparency of the colloidal gold and the presence of precipitate and floating materials in the colloidal gold were observed by naked eyes.The best prepared colloidal gold sample was scanned at 400-700nm using UV-VIS spectrophotometer.Citric acid three sodium reduction method was applied to prepare colloidal gold particles with 5 different diameters(34.09nm,10.68nm,10.68nm,12.74nm and 35.15nm).Scanning and transmission electron microscopy results showed that colloidal gold particles with uniform size,good dispersion and no polygonal particles or agglomeration of particles was observed.The colloidal gold with a wavelength of 520nm and particle size of 12.74nm was selected as a marker for subsequent experiments.The pH value of the monoclonal antibodies was 8.2 and that of polyclonal antibodies was 7.5,which measured by pH test strip.We also used UV spectrophotometer to detect the absorbance of OD520nm/OD580nmD ratio and made a curve.However,the best system for the preparation of colloidal gold antibody complex was:3ul 0.1mol/L K2CO3,4ug PKM2,18ug CA19-9 and 5ug FOBT antibody protein.The 0.01mol/L pH8.2 acid(0.5%BSA,0.5%PEG20000,15%sucrose,0.05%Tween-20)was selected as the buffer solution of colloidal gold antibody complex by centrifugal purification of colloidal gold antibody complex.3.Development of ternary gold labeled immunochromatograph kit for colorectal cancer screeningBy optimizing the fabrication method and production process of test strip,nitrocellulose membrane was used as the solid phase earrier.For the control Iine,the optimal dilution concentration was 1:4 for goat anti-mouse IgG secondary antibody and 1:8 for goat anti-rabbit IgG secondary antibody.For the test line,the optimal dilution concentration was 1:4 for PKM2 and CA19-9 mouse monoclonal antibody,and 1:8 for FOBT antibody.The membrane were sealed with 1%bovine serum albumin(BSA)for lh and dried at room temperature.The colloidal gold-antibody conjugates were coated onto the gold-labeled pad and dried at room temperature.The pad was treated with the pH 8.2 0.01mol/L boracic acid buffer(0.5%BSA,0.5%PEG20000,15%sucrose,0.05%Tween-20).Then,the nitrocellulose membrane,gold-labeled pad,sample pad and absorbent paper were assembled into test strips,and PKM2,CA19-9 and FOBT test strips were prepared by the above optimal conditions respectively.Finally,the PKM2,CA19-9 and FOBT test strips were combined together and assembled into a combined colorectal cancer rapid screening kit which can detect these 3 kinds of markers at the same time.The stability test showed that the strips could be preserved at 4? in the dark for 4 months.4.Application of ternary gold labeled immunochromatograph kit for colorectal cancer screening in clinical colorectal cancer patientsPKM2,CA19-9 and FOBT detection test strips were combined together and assembled into a kit to detect fecal samples from 32 cases of normal persons and 170 cases of colorectal cancer patients.The results showed that:the sensitivity of PKM2,CA19-9 and FOBT detection test strips was 80%,82.35%and 75.29%respectively,and the missed diagnosis rate was 20%,17.65%,24.7%,respectively;the rate of misdiagnosis were:9.38%,12.5%,12.5%.But the sensitivity of the kit combination of three indexes was 62.35%,the rate of missed diagnosis was 37.64%and the misdiagnosis rate was 3.12%.These results displayed that the sensitivity of the kit combination of three indexes decreased,but it can significantly reduce the misdiagnosis rate,indicating that can be used in clinical application.Conclusion1.PKM2,CA19-9 and FOBT immunochromatographic kit for colorectal cancer screening were developed successfully,which could be used to detect fecal samples of patients and had the possibility of quick observation results.2.High sensitivity,specificity and lower misdiagnosis rate of the test strips made them highly applicable for colorectal cancer screening.It is convenient for people to check in early stage of colorectal cancer,so as to enable early detection,early diagnosis and treatment,reduce the mortality rate of colorectal cancer.