Abstract
MG7 is a gastric cancer–specific MAb with high specificity and sensitivity. By using MG7 MAb, we found that MG7Ag was increasingly detected in superficial gastritis, atrophic gastritis, intestinal metaplasia, atypical hyperplasia, and gastric cancer, indicating that MG7Ag could be considered an important early warning molecule of gastric cancer and MG7 MAb could be used as a tool for screening gastric cancer. We have developed a new and sensitive system, immuno-realtime PCR, for detection of MG7Ag in the serum. The use of qIPCR assays enabled the detection of MG7Ag in complex biological samples that were poorly accessible by conventional immunoassays.
Introduction
With the use of a homogenate preparation of the gastric cancer cell line MKN-45, Fan and colleagues have developed a series of gastric cancer–specific MAbs.(2) Among these antibodies, MG7 has the highest specificity and sensitivity, and it was ascertained that MG7Ag was a useful predictor of evolution of gastric dysplasia to carcinoma. By applying the immunohistochemical method to observe the expression of MGAgs in gastric mucosa tissue, we found that in those with positive MGAgs, the 5-year canceration rate was up to 30%, whereas those subjects without MGAgs rarely cancerated.(3) The results indicated that detection of MGAgs could forecast the canceration of gastric mucosa so that it would most likely become an important sign for precaution of gastric cancer. Whether or not MGAgs could be used as a gastric cancer marker of serum in the general investigation of high-risk groups was the significant criterion for evaluating its applicative value.
Although MGAgs were expressed in gastric cancer tissue, most of them were released into gastric lumen instead of blood. The small amount of MGAg that were released into blood were destroyed or metabolized by the immune or biochemical factors of the human body. In particular, in early tumors with fewer cancer cells, the few MGAgs that were secreted into the blood would be diluted several thousand times. Here, qIPCR assays were used to detect MG7Ag using MG7 antibody.
Materials and Methods
Cell line and antigens
The gastric cancer line MKN45 was routinely cultured in RPMI 1640 medium (Life Technologies, Inc., Gaithersburg, MD) supplemented with 10% fetal calf serum in a 37°C humidified incubator with a mixture of 95% air and 5% CO2. We used serial dilutions of supernatant from cultured MKN45 cells as the standard antigen. Serum samples were collected from gastric carcinoma patients admitted to our university hospital and physical examination serum sample were collected from our clinical laboratory.
Immunohistochemistry
Paraffin-embedded sections were cut from the representative formalin-fixed tissue blocks and then stained using the ABC technique; 3% hydrogen peroxidase in methanol was used to block the endogenous peroxidase activity in the tissue sections. The sections were then washed in PBS and incubated in a 1:10 dilution of normal horse's serum/PBS solution for 1 h to block non-specific binding. The primary monoclonal antibody MG7 was applied on the tissue samples at a dilution of 1:2000 and incubated at 4°C overnight. Peroxidase staining was done by using ABC kits (Vector Laboratories Inc. USA). The section of gastric tumor that stained positively for MG7 was used as a positive control and included in each staining batch. An irrelevant mouse monoclonal antibody was substituted for the primary antibody in test sections for negative control.
Preparation of biotinylated MAbs
A hybridoma cell line producing MAb MG7 against gastric carcinoma-associated antigen was developed in the study laboratory.(2) The antibody was purified from the induced ascites by DEAE-52 cellulose affinity chromatography. A sensitive method for in vitro labeling of MAbs using the photobiotin was introduced in our single determinant immuno-PCR method.(4–7) A solution of the appropriate MAb in PBS in a polypropylene tube was mixed with photobiotin in aqueous solution. The tube was placed in an ice water bath 10 cm below a sunlamp and illuminated for 15 min. The biotinylated MAb was purified on a sepharose G-50 minicolumn, (GZ Healthcare, USA), according to the manufacturer's instructions.
Preparation of biotinylated plasmid as specific marker
A full-length recombinant plasmid, pGAM-T, was used as the marker DNA that could be amplified by PCR. The labeling of pGAM-T (0.34 mg/mL) with photobiotin acetate (1 mg/mL) was completed while the same volume of each solution was pooled. The labeled pGAM-T was then extracted and concentrated with 2-butanol and precipitated by chilling and centrifugation, when a red pellet was obtained.(8,9)
ELISA
The plate was washed with PBS containing 0.05% Tween-20 (PBS-Tween) for 5 min twice. The plate was blocked with 3% BSA-PBS at 37°C for 1 h, washing three times for 5 min with PBS-T at room temperature. Then MG7 (MG7 5μg/mL in BSA-PBS) were added and incubated at 37°C for 1 h, washing three times for 5 min with PBS-T at room temperature. Next, biotinylated second antibody (2 μg/mL in 1% BSA-PBS) was added and incubated at 37°C for 1 h. Then 50 μL of the STV/alkaline phosphatase conjugate dilution was added to each well and incubated for 20 min at room temperature, stopped by adding 2 N HCL. Fluorescence was measured at 450 nm wavelength.
Quantitative immuno-realtime PCR
The plate was washed to remove unbound biotinylated second antibody, then free streptavidin at 1 μg/mL in 1% BSA-PBS was added and incubated at room temperature for 30 min. The plate was washed with PBS-T for 5 min four times, and biotinylated DNA molecules were added at the concentration of 105 copy/mL in 1% BSA-PBS. The pins were washed with PBS-T for 5 min five times. Realtime-PCR was carried out under the following conditions. Nucleotide sequences of the primers: 5'-GCTCTAGAGCATCCCTGTCTTAACTG-3' for the sense direction and 5'-GCTCTAGAGCAAACGCTTCTCAACAT-3' for the anti-sense direction. An automatic baseline correction was applied by the software of the instrument.
Results
Immunohistochemical expression of MG7Ag
There was a total of 357 cases of gastric cancer tissues and 446 cases of precancerous lesion tissues (26 cases of superficial gastritis, 87 cases of atrophic gastritis, 145 cases of intestinal metaplasia, and 188 cases of atypical hyperplasia). As shown in Table 1, the expression of MG7Ag was significantly lower in precancerous lesion tissues (36.3%) than that in gastric cancer tissues (91%). The expression of MG7Ag in superficial gastritis, atrophic gastritis combined with intestinal metaplasia, and atypical hyperplasia was 3.8% (1/26), 31.9% (74/232), and 46.3% (87/188), respectively (Fig. 1).
Immunohistochemical expression of MG7-Ag in different gastric tissues. (
Comparison of sensitivity between ELISA and quantitative immuno-realtime PCR
To allow comparison of the data obtained from qIPCR and conventional ELISA, it was recommended to calculate delta Ct (DCt) values by subtracting the Ct values obtained for individual samples from the total number of cycles (e.g., for a total of 40 cycles). The LOD was typically defined as the value of NC to which three times the value of the average standard deviation (SD) was added. In the experimental protocol described here, the typical average SD for the sequential qIPCR (intra-assay double determination) of the NC was 1%. For example, with an NC signal of DCt10, the cutoff value for the LOD calculated to 10.30. The reliable cutoff value of ELISA was 4.011. The results showed that immuno-realtime PCR could detect MG7Ag on MKN45 cells at a level as low as 101 and was therefore approximately 103 times more sensitive than ELISA (Fig. 2).
Comparison of sensitivity between ELISA and immuno-realtime PCR.
Quantitative immuno-realtime PCR
We detected the expression of MG7Ag in 50 cases of serum of patients with gastric cancer, including preoperational (20 samples), postoperational (20 samples), and normal serum (10 samples). In the experimental protocol described here, the typical average SD for the sequential qIPCR (intra-assay double determination) of the NC was 1%. LOD was used as the cutoff value. The results showed that the expression of MG7Ag was higher than normal in 20 cases of preoperational patients, while it was higher than normal in four cases of postoperational patients, indicating that qIPCR could be used as a valuable method for screening the genesis and prognosis of gastric cancer.
Discussion
Gastric carcinoma is one of the most significant malignancies causing morbidity and mortality in China and other Asian countries. Therefore early diagnosis is of great importance in improving survival. At the current time, the diagnosis of gastric carcinoma principally is based on endoscopy and the x-ray-based barium meal examination. We have been aware that endoscopy with pathologic examination is the most reliable methodology for these patients. Unfortunately, the majority of patients with early gastric carcinoma are asymptomatic with no clinical manifestations. The detection of TAAs in the serum currently plays an important role in the detection of certain tumors and in monitoring for canceration or metastasis. For gastric carcinoma the sensitivity of such assays in advanced disease is only approximately 40–50% and most likely is significantly lower for early disease. Therefore, the development of a more sensitive technique is of great interest.
By applying the immunohistochemical method to observe the expression of MG7Ag in gastric mucosa tissue, we found that MG7Ag was increasingly detected in superficial gastritis, atrophic gastritis, intestinal metaplasia, atypical hyperplasia, and gastric cancer, indicating that MG7Ag could be considered an important early warning molecule of gastric cancer and MG7 could be used for screening gastric cancer.
No doubt even antigens with the highest sensitivity were hard to detect. This antibody had already been used with encouraging results to detect its corresponding antigen MG7Ag in the sera of patients with gastric carcinoma and other malignancies by immuno-PCR techniques.(10) The need for improving diagnostic methods for the early detection of such tumors has become critical. The quantitative immuno-PCR (qIPCR) technology combines the advantages of flexible and robust immunoassays with the exponential signal amplification power of PCR. The qIPCR allows one to detect antigens using specific antibodies labeled with double-stranded DNA. The label is used for signal generation by quantitative PCR. Because of the efficiency of nucleic acid amplification, qIPCR typically leads to a 10- to 1,000-fold increase in sensitivity compared to an analogous enzyme-amplified immunoassay.(11) The use of qIPCR assays enables the detection of rare biomarkers in complex biological samples that are poorly accessible by conventional immunoassays. Therefore, we have developed a new and sensitive system, the immuno-realtime PCR, for the detection of MG7Ag in the serum. In conclusion, MG7 MAb might be used as a screening tool for gastric cancer.
Footnotes
Acknowledgment
This study was supported in part by grants from the National Scientific Foundation of China (30600737, 30770958 and 30871141) and 863 programs (2006AA02A249, 2006AA02A402 and 2007AA02Z470) and National Key Basic Research (973) plan (2004CB518700).