Possibility of In-House Preparation of Liver Cancer Diagnostic Kits Based on AFP ELISA Test
Radnaa Enkhtuya1, Damdindorj Lkhagvasuren1, Dondog Purev2 and Tsendsuren Oyunsuren1
1Laboratory of Molecular Biology, Institute of Biology, Mongolian Academy of Sciences, Ulaanbaatar, 210351, Mongolia
2Department of Biochemistry and Bioorganic Chemistry, National University of Mongolia, Ulaanbaatar, 210646, Mongolia
Our research was focused on the study of possibility of preparation the AFP Enzyme-Linked Immunosorbent Assay (ELISA) diagnostic kits using tumor marker protein named alpha-fetoprotein (AFP) for hepatocellular carcinoma (HCC), the most frequent cancer in Mongolia. It is important to prepare the diagnostic kits for detection of liver cancer early, simply and inexpensively in this country. Detection of the marker in human sera would signifi cantly help successful therapy, since the tumor could be tackled at an early stage in its development, often before metastases and other consequences. Therefore, level of AFP in sera would give possibility to control the progression of liver cancer. The micro titer plates were prepared for AFP ELISA diagnostic kits, and their sensitivity and specifi city were 94.4% and 0.4, respectively. Statistically, there were no differences between our prepared micro titer plates for AFP ELISA diagnostic kits and commercially available ones (t = 0.071, P = 0.94, df = 24). It was concluded that there is a possibility to prepare AFP ELISA diagnostic kits using our prepared micro titer plates in house.
Keyword: AFP,hepatocellular carcinoma,ELISA,kit
Liver cancer is the sixth most commonly diagnosed cancer, and the third most common cause of death from cancer worldwide with incidence rate of 626,162 cases and mortality of 598,321 cases per year (Jeffrey et al., 2009 & http://www-dep.iarc.fr). The incidence of HCC in Asia is high, particularly in Eastern and South Eastern Asia (Vanessa & Chung, 2007; Teo & Fock, 2001; David & Dirk, 2008). As it was reported by the International Agency for Research on Cancer, the highest rate of HCC occurred in Mongolia (Offi ce for National Statistics, 2008; Welsh Cancer Intelligence and Surveillance Unit, 2008; ISD Cancer Information Programme, 2008; Northern Ireland Cancer Registry and Cancer Incidence and Mortality, 2008; http://www-dep.iarc.fr). For instance, Oyunsuren et al. (2006) noted that 35.3% of all newly registered cancer cases were liver cancers, with an incidence rate of 51.3 per 100,000 populations in the period of 2000- 2005, and the National Cancer Research Center of Mongolia reported that incidence rate of 54 per 100,000 populations in 2007 (National Cancer Research Center of Mongolia, 2007). The main factors associated HCC are hepatitis C viruses (HCV) and hepatitis B viruses (HBV) infections, dietary afl atoxin B1 exposure and intake of alcohol in worldwide (Navas, 2007; Hwang & Hassan, 2009; Oyunsuren et al., 2006). Oyunsuren et al. (2007) defi ned most commonly occurring genotypes relating to hepatitis viruses in Mongolia: 1b genotype among HCV, D genotype among HBV and 1 genotype among HDV. AFP is the most common used tumor marker for monitoring the response to therapy (usually chemotherapy) of HCC (Beastall et al., 1991; Aburano et al., 1980; Czauderna & Perilongo, 2004; Mashayekhi et al., 2005; Gerald, 2001). Prior to metastasis, most cancers can be cured by localized treatments, such as surgery or radiotherapy. Unfortunately, about 70-80% of cancers has already metastasized by the time of diagnosis, and therefore, cannot be cured by surgery alone (Cooper, 1992). The best approach to reduce the morbidity and mortality from cancer is its prevention and early detection. Therefore, it is important to prepare the diagnostic kits for detection of liver cancer early, simply and inexpensively in Mongolia, where HCC incidence is very high. Besides of the diagnosis, the detection of AFP level in sera would help to control the stages and progression of liver cancer.
Material and Methods
In total sera of 25 patients were used in this study, which diagnosed that 7 sera with liver cancer and 18 sera with chronic hepatitis or non liver cancer by the National Cancer Research Center of Mongolia. AFP ELISA diagnostic kits (Code: EMK 003), uncoated micro titer plates obtained from the Institute of Atomic Energy Beijing, China used for comparison. This kit consisted of a series of standard solutions of AFP at different concentrations from 10 ng/ml to 400 ng/ml, an enzyme conjugate, substrate solutions, reaction stopper, anti-AFP coated plate and buffered washing solution. Polyclonal antibody against AFP (82μg/ml) purifi ed in house were used for coating process to micro titer plates. Polyclonal antibodies were coated to micro titer plates by two methods (method I and method II) which differ from each other for dilution buffer, blocking solutions and experimental periods. The concentrations of antibody and blocking solution for coating procedures of ELISA were modifi ed and diagnostic procedures was done as described (Alphafetoprotein (AFP) ELISA kit, My BioSource (Instrument)). Briefl y, for preparation of micro titer plates of ELISA kit, each well of plates was coated with 4 μg/ml of antibody diluted in 0.1M NaHCO3, pH 8.6 at 40C overnight. After rinsing, 3% BSA in PBS blocking buffer was added into wells and it was incubated at 370C for 1 hour. For the diagnosis, 50μl of standards, controls and sera were added per wells and incubated at 370C for 1 hour. 100μl of Conjugates were added after washing, and incubated at 370C for 1 hour. After washing, 100μl substrate solutions were added to wells and incubated 15 minutes at room temperature. Upon dropping 50μl Stop solution to each well, optical densities of reactants were measured by micro titer plate reader at 450 nm. Positive results were estimated that the level of AFP equals to 20 ng/ml or more. The comparison of AFP binding capabilities of our prepared micro titer plates to commercially available kits was made by sandwich-ELISA and by constructing standard curve to determine AFP level in unknown sera. Our prepared micro titer plates were compared with imported commercially available ones by not only their AFP binding capabilities, but also their sensitivities and specifi cities. Statistical analysis of the data calculated with paired t test to compare AFP binding capabilities between imported commercially available micro titer plates and our prepared micro titer plates for AFP ELISA diagnostic kits.
The values of optical densities (ODs) of AFP ELISA using micro titer plates prepared two kinds of method and commercially available ones were demonstrated in Table 1. Standard curves were drawn by using the ODs of each AFP standard (vertical axis) against the respective concentrations (horizontal axis) to support the determination of AFP level in the unknown sera (Fig. 1). For computerized calculations and quality assessment normalized specifi c binding values, rather than OD values are used. This standard curves indicate that optical densities (ODs) of micro titer plates prepared by method I were more taken in rather than method II. Thus, micro titer plates prepared by method I were preferred to appropriate to make AFP ELISA diagnostic kits for detection of marker protein named AFP of liver cancer. Furthermore, we purposed to compare the sensitivities and specifi cities of micro titer plates prepared by method I and imported commercially available ones for AFP ELISA diagnostic kits. Consequently, 25 sera were examined by sandwich-ELISA using the micro titer plates prepared by method I and commercially available ones (Table 2). Table 2 demonstrates that 18 sera were normal or up to 20 ng/ml of AFP (negative), and 7 sera were abnormal or over 20 ng/ml of AFP (positive) of all 25 sera. Normal rate of AFP in adult human blood serum should be up to 20 ng/ml (Visual ELISA test for the qualitative determination of alphafetoprotein (AFP) in human serum (instrument); Enzyme immunoassay for the quantitative determination of AFP in human serum or plasma (instrument); Alpha-fetoprotein antibody, Rabbit IgG polyclonal antibody (description); Dominique et al., 1984; Kew, 1974; Johnson et al., 1978; Deyashiki et al., 1989). The values of B/Bmax,% = 20.14 (of micro titer plates prepared by method I) and B/Bmax,% = 21.40 (of micro titer plates of
imported commercially available ones) were calculated using optical densities of 20 ng/ml standard solution (B) as presented in Table 1.
As stated in Table 2, sera examined in micro titer plates prepared by method I and calculated more than B/Bmax,% = 20.14 values were supposed to be positive (abnormal), less than B/Bmax,% = 20.14 values were supposed to be negative (normal). Whereas, sera examined in micro titer plates using the commercially available ones and calculated more than B/Bmax,% = 21.40 values were supposed to be positive (abnormal), and less than B/Bmax,% = 21.40 values were supposed to be negative (normal) as well. For statistical assessment the results illustrated in Table 2 were calculated to expose the AFP binding capabilities of micro titer plates if there are some differences. As a result of paired t - test calculations two tailed p value equals 0.94, t = 0.071 and df = 24 (standard error of difference = 1.298).
By conventional criteria, this difference is considered to be not statistically signifi cant. Sensitivities, which were equal to 94.4% of micro titer plates prepared by method I, 97.6% of imported commercially available ones and specifi cities, which were equal to same 0.4 of micro titer plates prepared by method I and commercially available ones, for AFP ELISA diagnostic kits, were calculated.
The AFP ELISA system provides a direct quantitative determination of AFP level in human sera for diagnosis of HCC. AFP is a glycoprotein with a molecular mass of approximately 70 kDa. During fetal development, AFP maintains high level in sera and drops to very low level throughout the remainder of life. As a tumor marker, AFP has applications in primary liver carcinoma in adults and hepatoblastomas in children, in germ cell tumors and it is also occasionally elevated in cancer of the gastrointernal tract (Beastall et al., 1991; Alpha-fetoprotein (AFP) ELISA kit (instrument); AFP ELISA kit (instrument)). The method to prepare micro titer plates of AFP ELISA diagnostic kits was elaborated to detect the level of AFP, liver tumor marker in human sera. The sensitivity of micro titer plates prepared by elaborated method was 94.4%, and the specifi city was 0.4, which indicate possibility to apply our prepared micro titer plates for HCC diagnostic procedure. In addition, there were no signifi cant statistically difference between our prepared micro titer plates and imported commercially available ones to determine the AFP level in human sera (t = 0.071, p=0.94). Furthermore, it would be possible to prepare internal products using our prepared micro titer plates for AFP ELISA diagnostic kits.
The authors are grateful to all members of Laboratory of Molecular Biology for critical comments and to National Cancer Research Center of Mongolia for generous supply of human sera.
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