Development Of Diagnostic Reagents For Carcino-embryonic Antigen And Neuron Specific Enolase Detection Using Amplified Luminescent Proximity Homogeneous Assay

Labeling immunoassay is a progressively comprehensive discipline and is the mainstream core technology of modern immunoassay. Established by principle of labeling immunology, it's characterized by high sensitivity and specificity, which has been widely used in basic medicine research and clinical examinations. In recent years, the development of new theories and new techniques has enormously improved the techniques in labeling immunoassay. The most widely used immunolabeling techniques are enzymatic immunoassay (EIA), chemiluminescence immunoassay (CLIA), electrochemiluminescent immunoassay (ECLIA) and time-resolved fluoroimmunoassay (TRFIA). Although sensitive and specific, all of these assays have their inherent limitations in the labeling agent used. In EIA, the enzymes used are often easily inactivated to lower the sensitivity, and enzyme labeling of large molecules often results in alterations in the spatial structure of the labeled molecules to affect the sensitivity. In CLIA, lots of factors affect its luminescence, especially the environment. Because of the extremely short life time of luminescence, the samples cannot be repetitively measured. In addition, due to the un-open reagent, it is expensive. In ECLIA, it is also expensive as an un-open reagent. In TRFIA, the procedures are complicated. The instrument and maintenance are costly. Moreover, isotopes in the environment and samples can generate the background fluorescence. Besides, all of the assays have a common drawback:the separation of the labeled and free label by washing. Therefore, it is necessary to develop a new labeling immunoassay to overcome the limitations. The amplified luminescent proximity homogenous assay (AlphaLISA) technology employs luminescent oxygen channeling immunoassay and relies on PerkinElmer's exclusive AlphaScreen(?). The new and quantitative immunolabeling technique which emerges in the recent year provides a solution to the obstacle of the traditional labeling immunoassay. This technique does not require of washing away of the free label. In addition, AlphaLISA could overcome other limitations in EIA, CLIA and TRFIA. As a homogenous assay, As a homogenous assay, AlphaLISA combined with the laser technology, nano-scale microspheres and the half lifetime of singlet oxygen is characterized by high throughput, high sensitivity, no radio contamination, good repetition, simple process, amenability to automation and so on. It has been widely used in biomedical studies and represents the development trend of the analytical techniques.Carcino-embryonic antigen (CEA) is a traditional and broad-spectrum tumor marker and is commonly studied. It belongs to the group of carcinofetal antigens that are produced during the embryonic and fetal period. CEA was firstly found in the fetus and colorectal carcinoma. CEA is a soluble glycoprotein of 200KDa and expressed highly by the gastrointestinal epithelium, pancreas and hepar of fetuses. The expression level of CEA is very low or nearly zero in the normal tissue. It significantly increased in serum in patients with gastrointestinal cancer. Moreover, it also increased in serum of the patients with gynecological cancer, lung cancer and so on. Only using CEA as the sole tumor marker to diagnose one cancer, the sensitivity and specificity are low. But simultaneously examining other tumor markers it is of great signification in early diagnosis, monitoring and curative effect of the malignancy tumors. The aim of this study was to establish and validate characteristics of a double-antibodies-sandwiched immunoassay (CEA-AlphaLISA) for the potential for clinical use.Neuron specific enolase (NSE) represents the gamma gamma- and alpha gamma- isoforms of the dimeric glycolytic enzyme enolase and is found predominantly in neurons and neuroendocrine cells. High NSE concentration in serum and brain and spinal cord fluid are frequently found in cases of the tumors which are located in neuroectoderm or neuroendocrine tissue, especially neuroblastoma and small cell lung cancer. As a biochemical marker of the brain damage and the small cell lung cancer, it is widely accepted that NSE is high specific and sensitive, and is also of value as an aid in diagnosis, monitoring and curative effect. In this study, the aim was to establish and validate characteristics of a double-antibodies-sandwiched immunoassay (NSE-AlphaLISA) for the potential for clinical use.Method:In this study, AlphaLISA kits for detecting CEA and NSE using antibody-sandwich method were developed. 1. Coupling of antibody to beads:0.2 mg of antibody was added to microspincolumn, and was centrifuged for 8 min at 9000 rpm. Then, the antibody was washed six times using 0.13 mol/L sodium phosphate buffer pH 8.0 repeatedly. The solution was added to 1 mg acceptor beads with 10μL of 25 mg/mL NaBH3CN and 1.25μL of 10% Tween-20 and then incubated at 37℃for 48 hours. The total volume of the reaction solution was 200μL. To block nonconjugated sites, a fresh carboxy-methoxy lamine (CMO) solution (65 mg/mL) was prepared in a 0.8 M NaOH.10μL of CMO solution was added to the reaction, and incubated for an hour at 37℃. The reaction solution was centrifuged and the supernatant was removed. Then the bead pellet was re-suspended in 200μL of 0.1 M Tris-HCl, pH 8.0, centrifuged and washed. After the last centrifugation, the beads were re-suspended in storage buffer (200μL of PBS+ 0.05% Proclin-300 as a preservative) at the concentration of 5 mg/mL.2. Biotinylated antibody:1 mg of antibody was added to microspincolumn, and was centrifuged for 8 min at 9000 rpm. Then, the antibody was washed six times using 0.1 mol/L carbonate buffer (pH 9.5) containing 0.1% NaN3 repeatedly. NHS-D-Biotin was dissolved in DMSO immediately prior to use in dark place at a concentration of 22 mg/mL. Then a volume equal to 10% of the total volume of the antibody solution was added into the NHS-D-Biotin solution in portions to the antibody solution with gentle stirring, and incubated at room temperature for 4 hours. The unreacted NHS-D-Biotin was filtered with microspincolumn. The biotinylated antibody was stored (0.5 mg/mL) at -20℃until use.3. CEA and NSE calibrations:For calibration purposes, the calibrator series were carried out by diluting antigen in the buffer. The desired standard concentrations for CEA were 0,2,5,20,100 and 500 ng/mL. The desired standard concentrations for NSE were 0,2,10,40,200 and 600 ng/mL.The standards prepared above were lyophilized according to 0.5 mL aliquot and then stored at 4℃until used.4. Evaluation of assay performance 4.1 Accuracy:The ratio of real concentration of standards determined to concentration marked is between 0.90 and 1.10, which were regarded as a criterion of the accuracy.4.2 Calibration curve:A linearized standard curve was obtained on a log-log plot with six concentrations including zero calibrator.4.3 The measurement range and the hook effect:The different concentrations of the CEA antigen and the NSE antigen were determined by the proposed method. 4.4 Sensitivity:The zero calibration was detected for 20 times repeatedly and the mean signal value and the standard deviation were calculated. The signal value, which was obtain by the mean signal value of zero calibration plus double standard deviation and minus the background value, was substituted into the standard curve equation to acquire the corresponding concentration. This concentration was defined as assay sensitivity.4.5 Analytical recovery:Spiking recovery was assessed by adding one of the calibrations to the control serum. 10μl of the calibration was spiked into 200μl of the control serum for the ratio of 1:20. The evaluations were done by calculating the ratio between the calculated and the expected values.4.6 Precision:The control serum was measured 10 times repeatedly. Each mean concentration and standard deviation was achieved. Thus, the coefficients of variation were calculated.4.7 Specificity:A certain amount of disturbing substances were regarded as samples and were detected. The gained concentration was served as the analysis of specificity.4.8 Interference:The effect of hemolysis, lipemia, and bilirubinemia was assessed by adding hemolysate, bilirubin (unconjugated), triglyceride and sodium ascorbate to the serum samples of the patient volunteers.4.9 Reference value:The healthy serum samples were determined by the proposed method. The statistical software is used for the analysis of distribution of data. 4.10 Comparison:The 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.Result:The ratio of real concentration of standards determined to concentration marked is between 0.92 and 1.09. The analytical sensitivity of the AlphaLISA kit for CEA was 0.11 ng/mL. The measurement range was from 0 to 500 ng/mL. The intra-assay coefficients of variation were 3.30%-6.83% and the inter-assay coefficients of variation were 5.52%-8.09%. No cross reactivity was found with AFP, CA125, CA19-9 and human serum albumin. The testing results were not subjected to interference by hemoglobin, triglycerides, or bilirubin. The reference value of CEA-AlphaLISA reagent is 0 to 6.5 ng/mL. The result of the Wilcoxon signed-rank of 100 serum samples involved was Z=1.644(P=0.1>0.05) and indicated that there is no statistical significance between CEA-AlphaLISA assay and commercial ECLIA CEA kit (Roche). The correlation coefficient was 0.976.For NSE detection, the measurement range was from 0 to 600 ng/mL. The analytical sensitivity was 0.149 ng/mL. The intra-assay coefficients of variation were 3.70%-4.29% and the inter-assay coefficients of variation were 3.90%-5.19%. No cross-reactivity with CYFRA21-1 and NNE was found. The testing results were not subjected to interference by hemoglobin, triglycerides, or bilirubin. The reference value of NSE-AlphaLISA reagent is 0 to 16.5 ng/mL. The result of the Wilcoxon signed-rank of 154 serum samples involved was Z=-1.140 (P=0.254>0.05) and indicated that there is no statistical significance between NSE-AlphaLISA assay and commercial ECLIA NSE kit (Roche). The correlation coefficient was 0.979.Conclusion:The results demonstrate that the AlphaLISA reagent for CEA and NSE we developed meet the demand for clinical application and could be the substitute of the import products for basic research and clinical examinations.