| Background and objectives:Ovariancancerranked third in gynecological tumor incidence second only to the uterine cervix cancer and uterine body cancer,while the death rate hasoccupied the first place.This is mainly due to the ovaries deep in the pelvis with unobvious early symptoms.Its performance is similar to normal digestive symptoms,often overlooked and was found until the tumor grows to a certain size,outside beyond the pelvis.Most patients have been in advanced stage,and easy toperitoneal metastasis and recurrence.According to some data to incomplete statistics,theovarian cancer patientsbefore diagnosis about 77%had abdominal symptoms,70%had gastrointestinal symptoms,58%had pain,50%had systemic symptoms,34%had urinary symptoms and only 26%had pelvic symptoms.Ovarian cancer 5-year survival rate is only 20%to 30%,while the survival rate of patients with early ovarian cancer up to 90%,so early diagnosis and early treatment of ovarian cancer is the key to improving prognosis.So far,carcinoembryonic antigen 125(cancer antigen,CA125)is the most important tumor marker of ovarian cancer for diagnosis and monitoring.Which with human epididymal secretory protein 4(human epididymis secretory protein 4,HE4)are the only two approved as a marker for monitoring ovarian cancer by the U.S.Food and Drug Administration(FDA).CA125 is the major serum marker for screening early ovarian cancer,have been widely used in clinical diagnosis,efficacy and disease monitoring.However,ovarian cancer patients with CA125 elevated in stage I detection rate was 50%,and in some benign diseases occurred,this false positive diagnosis is not easy to discharge through the images to lead to unnecessary surgery.So,as an early diagnostic marker for ovarian cancer,CA125 has lack of specificity.It urgent need for a higher sensitive and specific marker to assist CA125 or to improve the diagnosis of ovarian cancer.The HE4 gene product,also known as whey acidic protein four-disulfide core domain protein 2(WFDC2),was a cysteine-rich secreted protein of approximately 25 kD and was demonstrated as a novel promising biomarker for OC diagnosis in recent years.The gene was discovered in distal epithelial cells of the human epididymis by Kirchoff in 1991.Hellstrom,who combined gene and protein technology to filter out HE4 protein,also proved HE4 was WFDC2(whey acidic protein four-disulfide core domain protein 2)gene expression products.He proved the whey acid protein(whey acidic protein,WAP)has a ring structure of the inhibitory domain,which may be inserted into the structure of the protease active region to inhibit the action of proteases.Then he proved HE4 and CA125 contrast detection in postmenopausal women serum levels and raised HE4 can be as a diagnostic tumor marker for ovarian cancer and more advantages than CA125 by ELISA method.Drapkin's studies have shown that the serums HE4 level elevated in 100%endometrioid carcinoma ovarian cancer,93%of serous ovarian cancer and 50%of ovarian clear cell carcinoma,but not expressed in ovarian mucinous carcinoma and normal ovarian tissue.Moore develop a risk model(Risk of Ovarian Malignancy Algorithm)Act by detected 531 clinical serum samples of the ovarian cancer.It could distinguish the level of risk of ovarian cancer.September 2011,FDA approved the sale and use of HE4 and CA125 protein combined ROMA in blood tests.In summary,HE4 is a suitable marker for ovarian cancer,combined detection of CA125 and HE4 contribute to the early detection and risk assessment of ovarian cancer.With the in-depth study of HE4 and widely used in the diagnosis and treatment of ovarian cancer rates will improve.Several methods such as ELISA,time-resolved fluoroimmunoassay(TrFIA),and electrochemiluminescence enzyme immunoassay(CLEIA)have been developed for serum HE4 detecting in OC patient,to develop a more sensitive and accurate detection kit still has a broad clinical utility.Therefore,the development of high-end HE4 immunological detection method,and it is very necessary and has broad application prospects.Chemiluminescence is an analysis which based on light-emitting substances by chemical reactions produce visible light excitation phenomena and measured luminous intensity.It is a highly sensitive trace analysis,it avoid the interference of background light and stray light by eliminating the need of exogenous excitation,reducing the acoustic noise,greatly improving the signal to noise ratio.The CLIA has the advantages of greater sensitivity,low noise signal,broader linearity,less assay time,radioactivity-free,and easy-to-use.This method has been widely used in mineral analysis in rocks,materials analysis,environmental monitoringand in the detection of antibiotics and other contaminants in foods.The detection sensitivity of chemiluminescence immunoassay method is much higher than the ELISA,immunofluorescence,etc.,and the effect is quite with isotope detection,security pollution,and easy use as the conventional ELISA.Therefore,more and more clinical test items will be carried out by chemiluminescence immunoassay to replace the traditional ELISA and radioimmunoassay methods.CLIA has also been widely used in the clinical detection of tumor markers,such as alpha-fetoprotein(AFP),prostate specific antigen(PSA),CA125 and human chorionic gonadotrophin(hCG),as well as for detection of diabetes,thyroid hormones and reproductive hormones.In addition,chemiluminescence immunoassay method has been widely used for diabetes,thyroid hormones,reproductive hormone testing.In this method,AE was used as luminescent agent which can emit a visible light signal(470 nm)when mixed with H2O2 under alkaline conditions.Labeling with AE requires relatively mild conditions,which does not damage antibody activity.Coating magnetic beads with antibody expands the reaction surface area;as a result,bound antigens can easily be detected with very low signal background due to non-specific signals.Furthermore,the coating magnetic beads could accelerate antibody-antigen interactions and shorter the wash steps.Philosophy of transformation medical is the bridge between the basic medicine and clinical medicine effective fast bridge.How will the research of previous large-scale gene functional genomics,proteomics and functional proteomics applied to clinical research and diagnosis and treatment of disease as soon as possible,is the problem the biological medicine and clinical inspection and other fields think and attention and expectations implemented to solve.In this paper,we selected human epididymal secretory protein 4(human epididymis secretory protein 4,HE4),a great potential and novel tumor marker,used an advanced new immunological detection technology--chemiluminescence immune analysis method to carry out high-end immune quantitative assay development and do clinical diagnosis value of applied research,to solve the early diagnosis of ovarian cancer and prognosis monitoring urgent clinical need to tackle the practical problems.Methods:1.Screening materials:1.1 The selection of magnetic particlesSelect the magnetic microsphere surface is smooth,uniform particle size,good dispersion properties.Adsorption force and the measured RLU value CV(%)between the tube are the two important parameters of their choice.It requires strong adsorption of magnetic microspheres,measured RLU value CV(%)between the tubes should not be higher than 10%.The HE4 antibody labeled acridinium of coated magneticmicrospheres,spincoated 16-20 hour,fully washing after use.Each tube is add 5?L of the coated magnetic microspheres respectively,used full automatic chemiluminescence analyzer named Caris200 to measure the RLU value of each tube.Repeated 10 tubes and calculated mean RLU value and CV%.1.2 The selection of antigen and paired antibodyUse obtained recombinant HE4 protein as the detection of antigen,7 HE4 monoclonal,polyclonal antibodies as capture antibodies and detection antibodiesto established detection mode.Luminescence values of different combinations two antibodies with high concentrations of antigen reactions detected by chemiluminescence immunoassay.Calculate the signal to noise(the highest concentration of standards and values than standard luminescence value of 0,ie,F/A),indicating that the higher signal to noise ratio the greater the specificity of antigen-antibody.Select the maximum signal to noise ratio as the capture antibody and detection antibody for the CLIA kit.1.3 The selection of AEIt requires high purity,high labeling efficiency,light intensity;good stabilityand stable supply.We bought two kinds of acridine salt Suzhou sinoera Chem Co.Ltd.By querying the literature and consulting provider.We taken 10?g acridinium salt on 0.5mg H5 labeled antibody labeled to a concentration of 100pmol/L of HE4 standard as a detection target,react with the package is the magnetic microspheres 6F8 antibody,compared with the same two manufacturers acridine performance piperidine salts.Repeat 10 to calculate the mean RLU and CV%.2 Established response modeThe HE4 recombinant protein was diluted to different concentrations as standards.Then a magnetic bead-based antibody and AE-conjugated antibody was prepared.Two antibodies and antigen formed antigen-antibody sandwich complexes,added pre-excitation and excitation liquid solution to make the complexes lighting,with Caris200 automated chemiluminescence immunoassay system to detect luminous intensity.One-step and two-step methods were used for the optimization of reaction patterns,and ultimately determine the best detection system,complete the preparation of kit.3.HE4 chemiluminescence immunoassay kit prepared for laboratory analysis performance evaluation.3.1 Sensitivity,linearity range and effect HOOKThe sensitivity of the assay was defined as 3*SD/K,where SD is the standard deviation of chemiluminescence intensities obtained with 20 replicate blanks(zero-concentration),and K was the slope of the calibration curve obtained using average of the various concentrations of antigen(standards)and blanks.HE4 antigen was diluted into a series of concentrations were measured to the antigen concentration as the abscissa,the number of signal values 1 × 103 counts for the vertical axis,the double logarithmic mathematical model Log-Log function processing.3.2 AccuracyAccuracy used recoveries and dilution recoveries to determine.HE4 antigen was added("spiked in")to normal human serum at theoretical concentrations of 72.00 pmol/L,144.00 pmol/L,287.50 pmol/L,575.00 pmol/L,1150.00 pmol/Land 1725.00 pmol/L,and the increase in measured concentration of HE4 for each spiked serum was determined by comparison to normal serum.Recovery rates are calculated as the increase in the measured concentration divided by the theoretical increase in concentration x 100.In addition,each concentration of antigen was diluted 2-,4-and 8-fold in normal human serum to assess linearity of the assay.Precision refers reproducibility of measurement results,the evaluation coefficient of variation(coefficient of variation,CV).3.3 PrecisionThe HE4 antigen was diluted to three concentrations in normal human serum(700 pmol/L,300 pmol/L,and 100 pmol/L).These samples were measured 20 times to determine the intra-assay coefficient of variation(CV)and three times independently to determine the inter-assay CV,CV%= standard deviation(SD)/Mean.3.4 SpecificityThe cross-reactivity of this method was determined with several tumor markers associated with OC,including AFP,CA125,carcino-embryonic antigen(CEA),?-hCG.3.5 InterferenceTo determine factors of serum interference of the assay,antigen concentrations of 40 pmol/L,400 pmol/L,1500 pmol/L were prepared in normal serum with the addition of common serum constituents known to cause interference in immunoassays:hemolysate(500 ng/mL,1000 ng/mL),triglycerides(500 ng/mL,1000 ng/mL)and bilirubin(25 ng/mL,50 ng/mL),respectively.3.6StabilityDetection reagent placing the components 37 ? 7 days was measured and the correlation set 4? control curve for comparison.4 Preliminary evaluation of clinical performance4.1 Determine the normal reference rangeThe healthy serum samples were determined by the proposed method.The statistical software is used for the analysis of distribution of data.4.2The correlation with similar foreign agentsThe samples were determined simultaneously by the proposed method and control reagents.The data was analysed,including the Wilcoxon signed-rank test and the linear correlation.4.3An analysis of the clinical effects of reagents to detect HE4We use the HE4-CLIA detection reagent detected 40 cases of ovarian cancer,11 cases of ovarian cysts,9 cases of uterine endometriosis,7 cases of uterine fibroids,5 cases of uterine fibroids with ovarian cysts and cases of pelvic inflammatory disease and 49 cases of control serum.Then draw ROC curves and analysis.Result:Select 6F8 as capture antibody H5 as the detector antibody in the assay establish sandwich mode.The reaction use a two-step method,the optimal reaction temperature is 37 ?.The assay demonstrated a linear range from 2.5 to 2000 pmol/L,with an analytical sensitivity at a 2.5 pmol/L.The reproducibility,recovery,and specificity of the immunoassay were demonstrated to be acceptable.The normal reference range is 0-90pmol/L.Compared with the ECLIA kit in 124 serum samples(40 patients with OC,35 patients with benign gynecological diseases and 49 controls),there is a satisfied correlation coefficient of 0.875.And the area under the receiver-operating curve(ROC-AUC)was 0.903(95%CI was 0.839-0.966 P<0.001)for HE4,0.787(95%CI was 0.694-0.879 P<0.001)for CA125,and 0.914(95%CI was 0.866-0.962 P<0.001)for combined analysis of HE4 and CA 125.Conclusions:A quantitative method(HE4-CLIA)for detecting HE4 in serum was successfully established.The HE4-CLIA kit showed great promise with high clinical value in the screening and early diagnosis of OC.Furthermore,HE4 and CA125 could be a valuable combination for the diagnosis of OC. |
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