• Journal of Periodontal and Implant Science
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• v.25 no.3
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• pp.720-732
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• 1995

The use of transforming growth $factor-{\beta}1$ which functions as a potent biologic mediator regulating numerous activities of wound healing has been suggested for the promotion of periodontal regeneration. The mitogenic effects of transforming growth $factor-{\beta}1$ on human periodontal ligament cells and human gingival fibroblasts were evaluated by determining the incorporation of $[^3H]-thymidine$ into DNA of the cells dose-dependently. Cells were prepared with primary cultured fibroblasts and periodontal ligament cells from humans, and used in experiments were the fourth or sixth subpassage. Cells were seeded with serum free Dulbecco's modified Eagle medium containing 0.1% bovine serum albumine. The added concentrations of transforming growth $factor-{\beta}1$ were 0.25, 0.5, 1, 2.5, 5ng/ml and transforming growth $factor-{\beta}1$ were added to the quiescent cells for 24hours, 48hours, 72hours. They were labeled with lnCi/ml $[^3H]$ thymidine for the last 24hour of the each culture. The results were presented as the mean counts per minute (CPM) per well and S.D. of four determinations. The results were as follows. : The DNA synthetic activity of human gingival fibroblasts was increased dose-dependently by transforming growth $factor-{\beta}1$ at 24 hours, 48 hours and 72 hours. The maximum mitogenic effects were at the 48 hour application of transforming growth $factor-{\beta}1$. The DNA synthetic activity was generally more decreased at the 72 hour application than at the 48 hour the application of transforming growth $factor-{\beta}1$. The DNA synthetic activity of human periodontal ligament cells was increased dose-dependently by transforming growth $factor-{\beta}1$ at 24 hours and 48 hours. But the DNA synthetic activity was decreased at 5ng/ml of the 72 hour application. The maximum mitogenic effects were also at the 48 hour application of transforming growth $factor-{\beta}1$. The DNA synthetic activity of human periodontal ligament cells was generally more decreased at the 72 hour application than at the 48 hour application of transforming growth $factor-{\beta}1$. In the comparision of DNA synthetic activity between the human gingival fibroblasts and human periodontal ligament cells, the human gingival fibroblasts had more activity than the human periodontal ligament cells at all time application with the concentration of transforming growth $factor-{\beta}1$. In conclusion, transforming growth $factor-{\beta}1$ has an important roles in the stimulation of DNA synthesis in human periodontal ligament cells and human gingival fibroblasts, which means an increase in collagen synthesizing cells and thus, may be useful for clinical application in periodontal regenerative procedures.

• Radiation Oncology Journal
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• v.15 no.2
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• pp.79-95
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• 1997

Purpose : Phospholipase C(PLC) isozymes play significant roles in signal transduction mechanism. $PLC-\gamma$ 1 is one of the key regulatory enzymes in signal transduction for cellular proliferation and differentiation. Ras oncoprotein, EGFR, and PKC are also known to be involved in cell growth. The exact mechanisms of these signal transduction following irradiation, however, were not clearly documented Thus, this study was Planned to determine the biological significance of PLC, ras oncoprotein, EGFR, and PKC in damage and regeneration of rat intestinal mucosa following irradiation. Material and Method : Sixty Sprague-Dawley rats were irradiated to entire body with a single dose of 8Gy. The rats were divided into S groups according to the sacrifice days after irradiation. The expression of PLC, ras oncoprotein, EGFR and PKC in each group were examined by the immunoblotting and immunohistochemistry. The histopathologic findings were observed using H&I stain, and the mitoses for the evidence of regeneration were counted using the light microscopy & PCNA kit. The Phosphoinositide(PI) hydrolyzing activity assay was also done for the indirect evaluation of $PLC-\gamma$ 1 activity. Results: In the immunohistochemistry , the expression of $PLC-{\beta}$ was negative for all grøups. The expression of $PLC-{\gamma}1$ was highest in the group III followed by group II in the proliferative zone of mucosa. The expression of $PKC-{\delta}1$ was strongly positive in group 1 followed by group II in the damaged surface epithelium. The above findings were also confirttled in the immunoblotting study. In the immunoblotting study, the expressions of $PLC-{\beta}$, $PLC-{\gamma}1$, and $PKC-{\delta}1$ were the same as the results of immunohis-tochemistry. The expression of ras oncoprctein was weakly positive in groups II, III and IV. The of EGFR was the highest in the group II, III, follwed by group IV and the expression of PKC was weakly positive in the group II and III. Conclusion: $PLC-{\gamma}1$ mediated signal transduction including ras oncoprotein, EGFR, and PKC play a significant role in mucosal regeneration after irradiation. $PLC-{\delta}1$ mediated signal transduction might have an important role in mucosal damage after irradiation. Further studies will be necessary to confirm the signal transduction mediating the $PKC-{\delta}1$.

• Journal of Aquaculture
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• v.11 no.4
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• pp.529-546
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• 1998

Gonadal part that developed by indifferentiation period for 6 months after hatching is made as gonad and fat body. These gonad are thin semi-transparant and undistinguished germ cell. Germinal epithelium is distinguished by development of gonad epithelial tissue from 7 months after hatching. Sex differentiation is begun by oogonia develoment at 8 months after hatching. Primary oocytes grow over germinal epithelium of gonadal cavity, at 9 months after hatching, gonadal cavity become ovarian cavity as they increasing. As soon as oocytes at 13 months after hatching are filled with the whole part of gonad, degeneration of oocyte is begun. And then, gonad has cavity tissue, a small number of oocyte are located in gonadal cavity. At 15 months after hatching, new primary oocyte develop and cavity of ovarian tissue in the central of ovarian cavity. Spermatogonia multiplicate and cavity tissue consist of testicular tissue. These gonad become hermaphrodite and then ditermine the sex of female and male. These results show the red sea bream is juvenile hermaphrodite and undif-ferentiated gonochoristic teleost. Male and female differentiation type of gonad is divided in undifferentiation stage, oogonia-like stage, ovary-like stage, ovary development stage, hermaphroditic testis stage, hermaphroditic ovary stage, and testis development stage. Undifferentiation stage is continued total lenth 18cm at 13 months after hatching. ovary-like stage is continued total length 11~18cm at 13 months after hatching. Ovary-like stage is continued total length 14~26cm at 10~14 months after hatching. Ovary development stage begins from total length 20cm, 14 months after hatching. At 20 months after hatching, 44 percent of total sampled individuals had ovary. Hermaphroditic ovary stage first begins total length 19~20 cm at 15 months after hatching, but it is not observed total length 28~29cm at 20months after hatching. Hermaphroditic testis stage first begins total length 21~22cm at 20months after hatching and is continued for 20months. Testis development stage first begins total length 20~21cm at 20 months after hatching, and is occupied 33 percent total length 28~29cm at 20 months. The beginning of sex differentiation more than 50 percent is from total length 16cm at 11 months after hatching. Sex determination begins total length 20cm, 14months after hatching in female and total length 20cm, 15 months after hatching in male. Sex determination more than 50 percent begins total length 23cm,, 17 months after hatching. Undifferentiated gonadal part of red sea bream consist gonad and fat body. As differentiation is going on and gonad is growing, fat body shrinks. This appearence is showed the same tendency in 3-year old red sea bream. 1.9mm larvae after hatching grow about 19mm larvae for 47 days. The relationship between the total length and body weight of larvae and juveniles in $BW=4.45{\times}10^{-6}TL^{3.4718}$ r=0.9820. Fishes in cage culture grow to maximum total length 28.4cm. The relationship between the total length and body weight of these fishes is $BW=2.36{\times}10^{-2}TL^{2.9180}$, r=0.9971. Undifferentiated gonadal part of red sea bream consist gonad and fat body. As differentiation is going on and gonad is growing, fat body shrinks.

• Tuberculosis and Respiratory Diseases
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• v.53 no.3
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• pp.294-308
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• 2002

Background : The cell-mediated immune response plays an important role in tuberculosis. After being activated by mycobacterial antigens, T lymphocytes express a high affinity receptor (IL-2R) for interleukin-2 (IL-2) on their own surface and release a soluble fraction of the IL-2 receptor (sIL-2R) from the cell membrane into the circulation. Neopterin is a metabolite of guanosine-triphosphate, which is produced by stimulated macrophages under the influence of IFN-$\gamma$ with a T lymphocyte origin. Therefore, the utility of sIL-2R, IFN-$\gamma$ and the neopterin levels as immunologic indices of the cell-mediated immune response and severity of disease in patients with pulmonary tuberculosis was assessed. Methods : The serum sIL-2R, IFN-$\gamma$ and neopterin levels were measured in 39 patients with pulmonary tuberculosis, 6 patients with tuberculous lymphadenitis prior to treatment and 10 healthy subjects. The serum and pleural sIL-2R, neopterin and ADA levels were measured in 22 patients with tuberculous pleurisy. The patients with pulmonary tuberculosis were divided into a mild, moderate and severe group according to the severity by ATS guidelines. To compare the results from these patients with those of the pretreatment levels, the sIL-2R, IFN-$\gamma$ and neopterin levels were measured in 36 of the 39 patients(1 patient, expired; 2 patients were referred to a sanitarium) with pulmonary tuberculosis after 2 months of treatment. Results : 1) the serum sIL-2R and IFN-$\gamma$ levels were elevated in patients with tuberculosis when compared to those of healthy subjects (p>0.05). The neopterin concentration in the serum was significantly lower in patients with pulmonary tuberculosis($2967{\pm}2132.8$ pg/ml) than in healthy controls($4949{\pm}1242.1$ pg/ml)(p<0.05). 2) In the pulmonary tuberculosis group, the serum sIL-2R and IFN-$\gamma$ levels were higher in patients with severe disease than those in patients with mild and moderate disease. However, the neopterin levels declined as the pulmonary tuberculosis became more severe (p<0.01). 3) The mean serum sIL-2R and IFN-$\gamma$ levels declined from $1071{\pm}1139.4$ U/ml to $1023{\pm}1920.9$ U/ml(p>0.05), $41{\pm}52.8$ pg/ml to $22{\pm}23.9$ gm/ml(p<0.05), respectively, after 2 month of treatment. The mean serum neopterin levels increased from $3158{\pm}2272.6$ pg/ml to $3737{\pm}2307.5$ pg/ml(p>0.05) after a 2 month of treatment. These findings were remarkable in the severe group of pulmonary tuberculosis with a clinical correlation. 4) In the patients with tuberculous pleurisy, the serum sIL-2R and ADA were significantly higher than those in the pleural fluid, However, the neopterin levels in the sera and pleural effusion were similar. Conclusion : On the basis of this study, sIL-2R, IFN-$\gamma$ and neopterin measurements may not only provide an insight into the present state of the cell-mediated immune response, but also serve as parameters monitoring of the prognosis of the disease, particularly in patients with severe pulmonary tuberculosis. In addition, an assay of the pleural sIL-2R levels might signal a stimulated local immunity including T cell activation in the tuberculous pleural effusion.

• Tuberculosis and Respiratory Diseases
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• v.55 no.5
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• pp.449-458
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• 2003

Background : The poor prognostic factors of far-advanced pulmonary tuberculosis(FAPTB) are lymphocytopenia in the peripheral blood(PB)(< $1,000/mm^3$) and $T_4$-cell count ${\leq}500/mm^3$. However, the cause of PB lymphocytopenia in FAPTB is unclear. The aim of this study was to analyze the lymphocyte proportion and cytokines of the bone marrow(BM) in FAPTB patients with peripheral lymphocytopenia in order to clarify whether the limiting step of the lymphocytopenia occurs in production, differentiation, or circulation. Methods : This study included patients with FAPTB between August 1999 and August 2002 who visited Gachon Medical School Gil Medical Center. The exclusion criteria were old age(${\geq}65years$), cachexia or a low body weight, shock, hematologic diseases, or BM involvement of tuberculosis. The distributions of cells in PB and BM were analyzed and compared to the control group. The interleukin(IL)-2, IL-7, IL-10, TNF-${\alpha}$, Interferon-${\gamma}$, and TGF-${\beta}$ levels in the BM were measured by ELISA. Result : Thirteen patients(male : female=9:4) were included and the mean age was $42{\pm}12$years. The proportion and count of the lymphocytes in the PB were significantly lower in the FAPTB group ($7.4{\pm}3.0%$, $694{\pm}255/mm^3$ vs. $17.5{\pm}5.8%$, $1,377{\pm}436/mm^3$, each p=0.0001 and 0.002). The proportion of immature lymphocyte in the BM showed a decreasing trend in the FAPTB group($9{\pm}4%$ vs. $12{\pm}3%$, p=0.138). The IL-2($26.0{\pm}29.1$ vs. $112.2{\pm}42.4pg/mL$, p=0.001) and IL-10($3.4{\pm}4.7$ vs. $12.0{\pm}8.0pg/mL$, p=0.031) levels in the BM were significantly lower in the FAPTB group than those in control. The levels of the other cytokines in FAPTB group and control were similar. Conclusion : These results suggest that the cause of lymphocytopenia in PB is associated with a abnormality IL-2 and IL-10 production in the BM. More study will be needed to define the mechanism of a decreased reservoir in BM.

• Journal of Periodontal and Implant Science
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• v.23 no.3
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• pp.535-563
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• 1993

New techniques for regenerating the destructed periodontal tissue have been studied for many years. Current acceptable methods of promoting periodontal regeneration alre basis of removal of diseased soft tissue, root treatment, guided tissue regeneration, graft materials, biological mediators. Platelet-derived growth factor (PDGF) is one of polypeptide growth factor. PDGF have been reported as a biological mediator which regulate activities of wound healing progress including cell proliferation, migration, and metabolism. The purposes of this study is to evaluate the possibility of using the PDGF as a regeneration promoting agent for furcation involvement defect. Eight adult mongrel dogs were used in this experiment. The dogs were anesthetized with Pentobarbital Sodium (25-30 mg/kg of body weight, Tokyo chemical Co., Japan) and conventional periodontal prophylaxis were performed with ultrasonic scaler. With intrasulcular and crestal incision, mucoperiosteal flap was elevated. Following decortication with 1/2 high speed round bur, degree III furcation defect was made on mandibular second(P2) and fourth(P4) premolar. For the basic treatment of root surface, fully saturated citric acid was applied on the exposed root surface for 3 minutes. On the right P4 20ug of human recombinant PDGF-BB dissolved in acetic acid was applied with polypropylene autopipette. On the left P2 and right P2 PDGF-BB was applied after insertion of ${\beta}-Tricalcium$ phosphate(TCP) and collagen (Collatape) respectively. Left mandibular P4 was used as control. Systemic antibiotics (Penicillin-G benzathine and penicillin-G procaine, 1 ml per 10-25 1bs body weight) were administrated intramuscular for 2 weeks after surgery. Irrigation with 0.1% Chlorhexidine Gluconate around operated sites was performed during the whole experimental period except one day immediate after surgery. Soft diets were fed through the whole experiment period. After 2, 4, 8, 12 weeks, the animals were sacrificed by perfusion technique. Tissue block was excised including the tooth and prepared for light microscope with H-E staining. At 2 weeks after surgery, therer were rapid osteogenesis phenomenon on the defected area of the PDGF only treated group and early trabeculation pattern was made with new osteoid tissue produced by activated osteoblast. Bone formation was almost completed to the fornix of furcation by 8 weeks after surgery. New cementum fromation was observed from 2 weeks after surgery, and the thickness was increased until 8 weeks with typical Sharpey’s fibers reembedded into new bone and cementum. In both PDGF-BB with TCP group and PDGF-BB with Collagen group, regeneration process including new bone and new cementum formation and the group especially in the early weeks. It might be thought that the migration of actively proliferating cells was prohibited by the graft materials. In conclusion, platelet-derived growth factor can promote rapid osteogenesis during early stage of periodontal tissue regeneration.

• Korean Journal of Agricultural Science
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• v.19 no.1
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• pp.51-64
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• 1992

This experiment was carried out to elucidate the effects of the thyroid function on lactation in female rats. One hundred and five female rats, whose body weight was approximately 250g with normal parturition, were divided into 3.5 THY, 35 PTU, and 35 CON. The $30{\mu}g$ L-thyroxine per rat was administered subcutaneously for the THY group with 3-days intervals arid 0.03% propylthiouracil solution was drunk for the PTU group. After the treatments body weight, thyroid weights and prolactin levels in serum, and histological changes in thyroid and mammary gland were investigated for 3 weeks with 3-days interval. The results obtained were as follows 1. The body weights of PTU group were lower than those of CON arid THY groups. The changes in body weights were significant between 3 and 6 days and between 15 and 18 days. 2. Differences in thyroid weight among the groups were significant after 9 days, The thyroid weights of PTU group were much higher than those of CON and THY group. 3. The follicular epithelia of PTU group after 6 days showed cuboidal phenomena which were accompanied by hypertrophy and hyperplasia, and this phenomena continued until post weaning period. Those of THY group after 9 days showed a squamous degeneration together with pyknosis. 4. The prolactin concentrations of THY group were higher than the other groups after 12 days. and those of CON group were higher than the others after 18 days. However, those of PTU group were lower all through the period. 5. The secretary epithelial cells of THY and CON groups became cuboidal after 12 days, but after 15 days the differentiation of mammary tissue was progressing in THY group faster than CON group. The degeneration of mammary tissue were observed in PTU group as time lapses, so after 15 days the exfoliation of secretory epithelium and atrophy of alveolus were recognized. 6. The body weights of offspring for all experimental groups were increasing as time lapses, but the values for PTU group were markedly lower than the others.

• Tuberculosis and Respiratory Diseases
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• v.45 no.5
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• pp.962-974
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• 1998

Background: Alteration of p53 tumor suppressor genes is most frequently identified in human neoplasms, including lung carcinoma. It is well known that bcl-2 oncoprotein protects cells from apoptosis. Recent studies have demonstrated that bcl-2 expression is associated with favorable prognosis for patients with non-small cell lung carcinoma. However, the precise biologic role of bcl-2 in the development of these tumors is still obscure. p53 and bcl-2 have important regulatory influence in the apoptotic pathway and thus their relationship is of interest in tumorigenesis, especially lung cancer. Purpose: The author investigated to know the prognostic significance of the expression of p53 and bcl-2 in radically resected non-small cell lung cancer. Method: 84 cases of formalin-fixed paraffin-embedded blocks from resected primary non-small cell lung cancer from 1980 to 1994 at Hanyang University Hospital were available for both clinical follow-up and immunohistochemical staining using monoclonal antibodies for p53 and bcl-2. Results : The histologic classification of the tumor was based on WHO criteria., and the specimens included 45 squamous cell carcinomas(53.6%), 28 adeonocarcinomas(33.3%) and 11 large cell carcinomas(13.1 %). p53 immunoreactivity was noted in 47 cases of 84 cases(56.0%). bcl-2 immunoreactivity was noted in 15 cases of 84 cases(17.9%). The mean survival duration was $64.23{\pm}10.73$ months in bcl-2 positive group and $35.28{\pm}4$. 39 months in bcl-2 negative group. The bcl-2 expression was significantly correlated with survival in radically resected non-small cell lung cancer patients(p=0.03). The mean survival duration was $34.71{\pm}6.12$ months in p53 positive group and $45.35{\pm}6.30$ months in p53 negative group(p=0.21). The p53 expression was not predictive for survival. There was no correlation between combination of the different status of p53 and bcl-2 expression in our study. Conclusions : The interaction and the regulation of new biologic markers, such as those involved in the apoptotic pathway, are complex. bcl-2 overexpression is a good prognostic factor in non-small cell lung cancer and p53 expression is not significantly associated with the prognostic factor in non-small cell lung cancer.

• Korean Journal of Veterinary Research
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• v.36 no.4
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• pp.783-794
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• 1996

It has been suggested that ion transport systems are intimately involved in mediating the effects of growth regulatory factors on the growth of a number of different types of animal cells in vivo. The functional importance of the apical membrane $Na^+/H^+$ antiporter in the renal proximal tubule is evidenced by estimates that this transporter mediates the reabsorption of approximately one third of the filtered load of sodium and the bulk of the secretion of hydrogen ions. This study was designed to investigate the pathway utilized by IGF-I in regulating sodium transport in primary cultured renal proximal tubule cells. Results were as follows : 1. $Na^+$ was observed to accumulate in the primary cells as a function of time. Raising the concentration of extracellular NaCl induced an decrease in $Na^+$ uptake compared with control cells in a dose dependent manner. The rate of $Na^+$ uptake into the primary cells was about two times higher in the absence of NaCl($40.11{\pm}1.76pmole\;Na^+/mg\;protein/min$) than in the presence of 140mM NaCl($17.82{\pm}0.94pmole\;Na^+/mg\;protein/min$) at the 30 minute uptake. 2. $Na^+$ uptake was inhibited by IAA($1{\times}10^{-4}M$) or valinomycin($5{\times}10^{-6}M$) treatment($50.51{\pm}4.04$ and $57.65{\pm}2.27$ of that of control, respectively). $Na^+$ uptake by the primary proximal tubule cells was significantly increased by ouabain($5{\times}10^{-5}M$) treatment($140.23{\pm}3.37%$ of that of control). When actinomycin D($1{\times}10^{-7}M$) or cycloheximide($4{\times}10^{-5}M$) was applied, $Na^+$ uptake was decreased to $90.21{\pm}2.39%$ or $89.64{\pm}3.69%$ of control in IGF-I($1{\times}10^{-5}M$) treated cells, respectively. 3. Extracellular cAMP decreased $Na^+$ uptake in a dose-dependent manner($10^{-8}-10^{-4}M$). IBMX($5{\times}10^{-5}M$) also inhibited $Na^+$ uptake. Treatment of cells with pertussis toxin(50pg/ml) or cholera toxin($1{\mu}g/ml$) inhibited $Na^+$ uptake. Extracellular PMA decreased $Na^+$ uptake in a dose-dependent manner(1-100ng/ml). 100 ng/ml PMA concentration significantly inhibited $Na^+$ uptake in IGF-I treated cells. However, staurosporine($1{\times}10^{-7}M$) had no effect on $Na^+$ uptake. When PMA and staurosporine were added together, the inhibition of $Na^+$ uptake was not observed. In conclusion, sodium uptake in primary cultured rabbit renal proximal tubule cells was dependent on membrane potentials and intracellular energy levels. IGF-I stimulates sodium uptake through mechanisms that involve some degree of de novo protein and/or RNA synthesis, and cAMP and/or PKC pathway mediating the action mechanisms of IGF-I.

• The Korean Journal of Nuclear Medicine Technology
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• v.21 no.1
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• pp.39-43
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• 2017

Purpose Thyroglobulin (Tg) is a biologic marker of differentiated thyroid carcinoma (DTC), produced by normal thyroid tissue or thyroid cancer tissue. Therefore, the Tg values of DTC patients is the most specific indicator for judging whether recurrence occur or whether the remaining thyroid cancer is present. Thyroid cancer is currently the most common cancer in Korea, of which 90% is differentiated thyroid cancer. The number of patients with thyroid disease of this application also increased, and an accurate and prompt results are required. However, the incubation time of the Tg commonly takes about 24 hours in our hospital, and the result reporting time is delayed, and We could not satisfied with the requirements of clinical departments and patients. In order to fulfill these requirements, experiments were conducted by shortening the incubation time between company B's Kit currently in use and company C's Kit used in other hospitals. Through these experiments, we could perform the correlation with the original method and shortening method, and could find the optimum reaction time to satisfy the needs of the departments and the patients, and we will improve the competitiveness with the EIA examination. Materials and Methods In September 2016, we tested 65 patients company B's kit and company C's kit by three incubation ways. First method $37^{\circ}C$ shaking 2hr/2hr, Second method RT shaking 3hr/2hr, Third method 1hr/1hr shaking at $37^{\circ}C$. Fourth method RT shaking 3hr method which is the original method of Company C's Kit. Fifth method, the incubation time was shortened under room temperature shaking 2hr, Sixth method $37^{\circ}C$ shaking 2hr. And we performed and compared the correlation and coefficient of each methods. Results As a result of performing shortening method on company B currently in use, when comparing the Original method of company B kit, First method $37^{\circ}C$ shaking 2hr/2hr was less than Tg 1.0 ng/mL and the ratio of $R^2=0.5906$, above 1.0 ng/mL In the value, $R^2=0.9597$. Second method RT shaking 3hr/2hr was $R^2=0.7262$ less than value of 1.0 ng/mL, $R^2=0.9566$ above than value of 1.0 ng/mL. Third method $37^{\circ}C$ shaking 1hr/1hr was $R^2=0.7728$ less than value of 1.0 ng/mL, $R^2=0.8904$ above than value of 1.0 ng/mL. Forth, Company C's The original method, RT shaking 3hr was $R^2=0.7542$ less than value of 1.0 ng/mL, and $R^2=0.9711$ above than value of 1.0 ng/mL. Fifth method RT shaking 2hr was $R^2=0.5477$ less than value of 1.0 ng/mL, $R^2=0.9231$ above than value of 1.0 ng/mL. Sixth method $37^{\circ}C$ shaking 2hr showed $R^2=0.2848$ less than value of 1.0 ng/mL, $R^2=0.9028$ above than value of 1.0 ng/mL. Conclusion Samples with both values of 1.0 ng/mL or higher in both of the six methods showed relatively high correlation, but the correlation was relatively low less than value of 1.0 ng/mL. Especially, the $37^{\circ}C$ shaking 2hr method of company C showed a sharp fluctuation from the low concentration value of 1.0 ng/mL or less. Therefore, we are planning to continuously test the time, equipment, incubation temperature and so on for the room temperature shaking 2hr method and $37^{\circ}C$ shaking 1hr/1hr of company C which showed a relatively high correlation. After that, we can search for an appropriate shortening method through additional experiments such as recovery test, dilution test, sensitivity test, and provide more accurate and prompt results to the department of medical treatment, It is competitive with EIA test.