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125I-Labelling of human thyrotropin (h-TSH) Was performed using a small amount of chloramine T(CT) as an oxidant at room temperature. When compared with the conventional method that uses a large amount of CT, this method facilitated uniform labelling and reduced the damage of h-TSH by CT, a strong oxidizing reagent. h-TSH* prepared by the new method was comparable to the commercial product of Daiichi Company, in terms of affinity to antibody and effective period.
최근 단일클론항체의 상업적 이용가치가 증대하면서 그 수요량 또한 급격히 증가, 이를 뒷받침할 수 있는 대량생산 기술이 요구되고 있다. 본 연구에서는 쥐 하이브리도마 Alps 25-3의 대량배양을 위해 우선적으로 무혈청 배지(serum-free media)의 개발을 시도하였다. 동물세포 배양액 IMDM과 Ham's F-12를 1:1로 혼합한 배지를 기본으로하여 ITES용액(insulin 10 ㎍/㎖, transferrin 10 ㎍/㎖, ethanolamine 10 μM and selenium 30 nM)을 첨가하여 일차적으로 결정한 EBM(enriched basal medium)에 steroid hormones, vitamins, lipid, mineral 및 여러 성분들을 각각 첨가하여 혈청과 대치될 수 있는 성분들의 최적농도를 조사하였다. 각 성분들의 개별 효과와 함께 그들을 혼합하였을 때의 상호효과를 통해 혈청배지와 거의 동일한 성장을 보이는 무혈청배지 KM3(EBM with BSA 100 ㎍/㎖, mineral solution and 0.05% PEG)를 결정할 수 있었으며 이 배지에서의 최고 항체 생성량은 혈청배지의 약 80%로 나타났다. 또한 무혈청 배지에서 여러 cell line의 성장과 그들의 항체 생성을 조사함으로써 다른 쥐 하이브리도마 세포배양에도 적용시킬 수 있음을 확인할 수 있었다. A serum-free medium that could be used for the large-scale culture of mouse hybridoma to produce monoclonal antibodies was developed. The medium was based on a 1:1 mixture of Iscove's Modified Dulbecco's Medium and Ham's F-12, supplemented with insulin 10 ㎍/㎖, transferrin 10 ㎍/㎖, ethanolamine 10 μM and selenium 30 nM(designated EBM (enriched basal medium) with the supplements). The effect of various supplements of steroid hormones, vitamins, lipid and mineral salts was investigated and their optimal concentration was determined to replace fetal calf serum (FCS). These components were added respectively and then added by way of two or three combination to discern of which component combination was effective to the culture of hybridoma. As a result, serum-free medium KM3(EBM with BSA 100 ㎍/㎖, mineral cocktail and 0.05% PEG) was determined. The hybridoma Alps 25-3 cultured in this medium showed almost the same growth rate as in medium added with 2% fetal bovine serum. However, the antibody concentration from KM3 cultures was 80% of that obtained from culture with FCS. KM3 was also examined for the culture of other mouse hybridomas, KW, A4W & HCGK, and it was confirmed that it could support the growth of these hybridomas and the production of monoclonal antibodies.
To evaluate mechanisms of action of blocking type-TSH receptor antibody in patients with prmary nongoitrous hypothyroidism, we studied the effects of IgGs form 17 patients with primary nongoitrous hypothyroidis, on 3H-thymidine incorporation into rat thyroid cells, FRTL-5, induced by TSH, Graves' IgG, dibutyl cAMP (dBcAMP), forskolin, and insulin-like growth factor I (IGF-I). IgGs from 11 out of 12 patients with primary nongoitrous hypothyroidism who had TBII activities inhibited both TSH- and Graves' IgG-stimlated 3H-thymidine incorporation in a dose-dependent manner. dBcAMP, forskolin, and IGF-I stimulated 3H-thymidine incorporation into FRTL-5 cells dose-dependently. However, all IgGs from 17 patients with primary nongoitrous hypothyroidism did not inhibit 3H-thymidine incorporation induced by dBcAMP (10-4M), forskolin (10-4M), and IGF-I (10ng/ml)in the range of 0.5-10 mg/ml IgG. The synergistic effect of coincubation with IGF-I (10 ng/ml) and TSH (1 mU/ml) or Gravrs' IgG (5 mg/ml) on 3H-thymidine incorporation into FRTL-5 cells disappeared by IgGs from patients with primary nongoitrous hypothyroidism. However, the proportion of 3H-thymidine incorporation induced by IGF-I itself was not inhibited by these IgGs. These frsults suggest that blocking type-TSH receptor antibody dose not affect post-receptor signal transduction and action of IGF-I. Therefore, the inhibitory action of blocking type-TSH receptor antibody may be on the-TSH receptor level through the inhibition of TSH binding to the TSH recptor. (J Kor Soc Endocrinol 6:8 16, 1991)
It is well Known that antithyroid drug treatment of Graves' disease suppresses excessive thyroid hormone synthesis and causes a parallel decrease in serum thyroid autoantibody levels including thyroid stimulating antibodies(TSAb) in most patients suggesting the immunosuppressive or immunomodulating effects of antithyroid drugs. In the context of view that thyrotropin receptor blocking antibody may play an important pathogenetic role at least in some patients with primary myxedema(chronic atrophic autoimmune thyroiditis), antithyroid drug treatment in these patients might be beneficial to disease course. To evaluate the effect of antithyroid drug on the thyrotropin receptor blocking antibody levels, we serially measured thyrotropin-binding inhibitor immunoglobulins(TBII) and thyroid stimulation blocking antibodies(TSBAb) using FRTL-5 cells, antimicrosomal- and antithyroglobulin antibody activities in 7 patients with primary myedema who have blocking TSH receptor antibodies during 6 months of methimazole(MMI, 40mg/day) administration. TBII and TSBAb activities did not change after MMI, but one of them showed stepwise decrease and disappearance of TBII and TSBAb activities. Antimicrosomal- and antithyroglobulin antibody activities decreased significantly after 3 months of MMI administration in those patients. These results suggest a minimal effect of antithyroid drug treatment on the level of thyrotropin receptor blocking antibodies. Persistence of thyrotropin receptor blocking antibodies despite of the decrease in antimicrosomal and antithyroglobulin antibodies might suggest that blocking TSH receptor antibodies of primary myxedema is produced mainly in extrathyroidal tissue in contrast to the thyroid stimulating antibodies of Graves' disease. One patient, whose blocking antibody have disappeared after MMI treatment, is under observation to see if she will remain in remission of hypothyroidism(J Kor Soc Endocrinol 10: 229-241, 1995).
We previously reported that IgGl was the most important subclass containing TSH receptor blocking anitbody activity but clearaly detectable blocking activity was found in the IgG2, IgG4, We tried to confirm the IgG subclass distribution of TSH receptor blocking antibody activity by measuring the TSH binding inhibitory immunoglobulin in the purified IgG subclass fractions and by converting the TSH receptor bound blocking type IgG subclass to the stimulating type by antihuman IgG antibody (Fab specific) in rat thyroid (FRTL-5) cells. From four patients with primary nongoitrous hypothyroidism, sera containing TSH receptor blocking activity were separated into four IgG subclasses and non IgG fraction by deletion affinity chromatography using affinity columns of subclass specific monoclonal antibodies. The TBII activities were dominant in IgG1 fractions in all four patients, but IgG2 and IgG4, fractions also showed significant TBII activites compared to non-IgG fractions in 2 patients. The significant amount of cAMP production after the addition of gaoat antihuman IgG antibody (Fab specific) to the IgG subclass (TSH receptor blocking antibody) bound FRTL-5 cells was noted in IgG1, IgG2, IgG4 frations in cases 1, in IgG1, IgG2, fractions in case 2, in IgG1 fraction in case 3, in IgG1, IgG4 fractions in case 4. These findings support the previous our study on the unrestricted IgG subclass distribution of TSH receptor blocking antibldies in primary nongoitrous hypothyroidism. (J Kor Soc Endocrinol 8:164~170, 1993)