Determination Of Chondroitin Sulfate By Chemiluminescence And The Establishment Of Double-antibody Sandwich ELISA

Lung cancer is one of the malignancies against human health, in the recent 10 years, the researches on lung cancer have made a lot of achievements. But so far lung cancer is still a big threat to human health. The incidence and mortality of lung cancer in China have significantly increased. According to statistics, about 70% of patients with lung cancer have been in the middle or advanced stage at diagnosis, and lost the chance of surgery, if they can be detected earlier, the 5-year survival rate of patients will be significantly improved. It has been known that some cancer cells can secrete specific substances, namely tumor markers, which could be detected by means of modern laboratory techniques. Tumor markers can be used for the early detection of cancer patients, determination of the incidence of tumor growth, invasion and metastasis, evaluation of the efficacy and. prognostic significance.Lung cancer markers currently used are neuron-specific enolase (NSE), carcinoembryonic antigen (CEA), Cytokeratin 19 fragments (CYFRA21-1), tissue polypeptide antigen(TPA), squamous cell carcinoma-associated(SCC-Ag), and so on. However, due to the low sensitivity and specificity, the clinical applications are restricted. So, investigators are striving to find tumor markers which have high sensitivity and specificity. Sulfate proteoglycan is a lung cancer marker studied more recently. Many studies have shown sulfate proteoglycan has a higher positive rate (86.20%), higher specificity and sensitivity in non-small cell lung cancer (specificity is 96.55%, sensitivity is 86.20%). Sulfate proteoglycan may be used in diagnosis of lung cancer, as a good lung cancer biomarker.The sulfate proteoglycan, also known as glycosaminoglycan (GAG), is uronic acid and acety1 amino sugars or sugar sulfate including two units of repetitive sequences. GAG in mammals, including chondroitin sulfate (CS), dermatan sulfate (DS), horny - sulfate (KS), heparin (HP), heparan sulfate or heparan sulfates (HS), and hyaluronic acid (HA). It was shown that sulfate proteoglycan was increased in the early stage of lung cancer, in primary or metastasis' tumor tissue GAG content was increased, mainly was the increase of hyaluronic acid (HA) and chondroitin sulfate (CS).Current assays for measurement of chondroitin sulfate have their own limitations. This study was designed to establish a better detection method of chondroitin sulfate and lay the foundation for the next study: establish the detection method of sulfate proteoglycan.Methods and results:1. Using Ce⁴⁺-Na₂SO₃ -H₂SO₄ redox system. When chondroitin sulfate is added to the system, its luminescence intensity markedly enhanced. Based on this phenomenon. The method was developed for determination of chondroitin sulfate. Results are as follows : regression equation y=0.4219x+3.3532 (y is the difference of sample' RLU and blank' RLU, x is the concentration of CS) , r = 0.9968. The linear range is 1.0 mg/L～8.0 mg/L, the detection limit is 0.5 mg/L. 2. CS acted as immunogen which immunized BALB/c mice and Japanese rabbits to produce antisera. The antiserum titer and specificity was obtained by indirect ELISA .The results showed: the four BALB/c mice antiserum end titer of CS were 1:6400,1:12800,1:6400,1:3200, respectively, and the specificity was good; Two Japanese rabbits end antiserum titer of CS were 1:64000,1:128000, respectively, and specificity was good.3. Optimizing conditions, initially established a chondroitin sulfate double-antibody sandwich ELISA. The optimum conditions for the detection is: antibody-coated is No. 2 mouse 1:200 antiserum, closed liquid is 1% casein, the second antibody is the No. 2 rabbit 1:20000 antiserum, goat anti-rabbit IgG horseradish peroxidase conjugates (HRP-IgG) concentration is 1 :4000. Results are as follows : regression equation is y=0.2945x+0.3968, the correlation coefficient is r =0.9819 (y is the difference of positive OD value and negative OD value, x is ln[CS]), the linear range is 2⁸00mg/L, the specificity of the method is good . Precision of the double-antibody sandwich ELISA indicated by coefficients of variation of intra-assay and inter-assay were 4.48% and 5.97%.4. The phloroglucinol spectrophotometry of chondroitin sulfate was studied. The phloroglucinol spectrophotometric method was used to detect chondroitin sulfate .The results are as follows: regression equation is y =0.0011 x-0.0106(y is absorbance, x is concentration of CS) .The correlation coefficient is r = 0.9918, the linear range is 200-800mg/L.Using phloroglucinol spectrophotometry as a standard reference method to test the reliability of double-antibody sandwich ELISA. The same solution was tested by the two methods, relevance and paired-t test were done to test the reliability of the double-antibody sandwich ELISA, the results are: t =0.247, P>0.05, r =0.999, P < 0.01, this showed a good correlation between the two methods, the double-antibody sandwich ELISA is reliable. Conclusion:1. The method was developed for determination of chondroitin sulfate by Chemiluminescence. The regression equation is y=0.4219x+3.3532 (y is the difference of sample' RLU and blank' RLU, x is the concentration of CS) , r = 0.9968. The linear range is 1.0 mg/L-8.0 mg/L, the detection limit is 0.5 mg/L.2. The method was developed for determination of chondroitin sulfate by double-antibody sandwich ELISA. The regression equation is y=0.2945x+0.3968, the correlation coefficient is r =0.9819 (y is the difference of positive OD value and negative OD value, x is ln[CS]), the linear range is 2 -800mg/L, the specificity and reliability of the method is good .