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Yu, Kim, Ko, Joo, Yang, and Lim: Evaluation of the adjuvants effect on the immunoglobulin class switching in generation of monoclonal antibodies against Paenibacillus larvae


American foulbrood (AFB) is caused by the bacterium Paenibacillus larvae, which is highly contagious and often lethal to honeybee broods. To control AFB, rapid diagnostic tools including those based on immunological methods are required. We produced several specific mouse monoclonal antibodies (MAbs) against P. larvae. Interestingly, a few of the MAbs were revealed to be an IgM-type antibody. To ascertain the effects of adjuvants on immunoglobulin isotype switching, BALB/c mice were immunized with various adjuvants, i.e., Freund's adjuvant (FA), Alum adjuvant, and AddaVaxโ„ข followed by the generation of hybridoma that secreted monoclonal antibodies to P. larvae. In the case of AddaVaxโ„ข, all screened hybridoma clones secreted IgG-type MAbs, whereas hybridomas generated by Alum and FA secreted 91.25% (7/80) and 66.67% (11/33) respectively, IgG-type MAbs. Although the mechanism of incomplete immunoglobulin isotype switching associated with the P. larvae antigen needs further study, our results indicate that the applied adjuvants can have a significant effect on immunoglobulin isotype switching results.

์„œ ๋ก 

๋ฏธ๊ตญ๋ถ€์ €๋ณ‘(American foul brood, AFB)์€ Paenibacillus larvae(P. larvae)์˜ ๊ฐ์—ผ์— ์˜ํ•œ ๊ฟ€๋ฒŒ์˜ ์น˜๋ช…์ ์ธ ์งˆ๋ณ‘์œผ๋กœ์„œ ์œ ์ถฉ์— ๊ฐ์—ผํ•˜์—ฌ ์œ ์ถฉ์„ ์ฉ๊ฒŒ ๋งŒ๋“œ๋Š” ๊ฒƒ์ด ํŠน์ง•์ด๋‹ค [5]. ๋ฏธ๊ตญ๋ถ€์ €๋ณ‘์€ ๋น ๋ฅธ ๊ฐ์—ผ์†๋„์™€ ๋†’์€ ์น˜์‚ฌ์œจ์„ ๋ณด์ด๋ฏ€๋กœ [3] ์งˆ๋ณ‘์˜ ์ œ์–ด๋ฅผ ์œ„ํ•ด์„œ๋Š” ์‹ ์†ํ•˜๊ณ  ์ •ํ™•ํ•œ ์ง„๋‹จ์ด ํ•„์š”ํ•˜๋ฉฐ ๋ฉด์—ญํ•™์  ์ง„๋‹จ๋ฒ•์€ ์ด๋Ÿฌํ•œ ๋ชฉ์ ์— ๋ถ€ํ•ฉํ•œ๋‹ค [7].
๋ฉด์—ญํ•™์  ์ง„๋‹จ๋ฒ•์˜ ๊ฐœ๋ฐœ์„ ์œ„ํ•ด์„œ๋Š” ๋ณ‘์›์ฒด์— ํŠน์ด์ ์ธ ํ•ญ์ฒด๊ฐ€ ์š”๊ตฌ๋œ๋‹ค. ์ด๋ฅผ ์œ„ํ•˜์—ฌ ์„ ํ–‰ ์—ฐ๊ตฌ์—์„œ๋Š” [8] ์„ธํฌ์œตํ•ฉ๊ธฐ๋ฒ•์„ ํ†ตํ•˜์—ฌ P. larvae๊ท ์ฒด ํ•ญ์›์— ํŠน์ด์ ์ธ ๋‹จํด๋ก ํ•ญ์ฒด(monoclonal antibodies, MAbs)๋ฅผ ๋ถ„๋น„ํ•˜๋Š” ์žก์ข…์„ธํฌ(hybridoma)๋“ค์„ ์–ป์–ด๋‚ธ๋ฐ” ์žˆ๋‹ค. ํฅ๋ฏธ๋กญ๊ฒŒ๋„, ์ด๋•Œ ํ™•๋ฆฝ๋œ ์žก์ข…์„ธํฌ๋“ค์—์„œ ๋ชจ๋‘ IgM type์˜ ๋‹จํด๋ก ํ•ญ์ฒด๊ฐ€ ๋ถ„๋น„๋˜์—ˆ๋‹ค. ์ผ๋ฐ˜์ ์œผ๋กœ ๊ณผ๋ฉด์—ญ์‹œ์ผœ ํ™œ์„ฑํ™”๋œ B cell์—์„œ๋Š” ์ดˆ๊ธฐํ•ญ์ฒด IgM์„ ๋ฐœํ˜„์‹œํ‚ค๋Š” IgM+ B cell์—์„œ IgG+ B cell๋กœ์˜ Immunoglobulin isotype switching [15]์ด ์ผ์–ด๋‚˜๋Š” ๊ฒƒ์— ๋น„์ถ”์–ด๋ณด๋ฉด ์ด๋Š” ์ด๋ก€์ ์ธ ํ˜„์ƒ์ด์—ˆ๋‹ค.
ํ•œํŽธ, ELISA ํ˜น์€ Immunochromatography๋ฒ• ๋“ฑ์— ์ ์šฉํ•  ๋ฉด์—ญํ•™์  ์ง„๋‹จ์†Œ์žฌ๋กœ์„œ๋Š” avidity์˜ ๊ด€์ ์ด๋‚˜ [16] ์ˆœ์ˆ˜๋ถ„๋ฆฌ์˜ ํŽธ๋ฆฌ์„ฑ ๊ด€์ ์—์„œ [12] isotype switching ์™„๋ฃŒ๋œ IgG type์˜ ํ•ญ์ฒด๊ฐ€ ์œ ๋ฆฌํ•œ ์ ์ด ๋งŽ๋‹ค. ๋ณธ ์—ฐ๊ตฌ๋Š” P. larvae์— ํŠน์ด์ ์ธ IgG type์˜ ๋‹จํด๋ก ํ•ญ์ฒด๋ฅผ ๋ถ„๋น„ํ•˜๋Š” ์žก์ข…์„ธํฌ๋ฅผ ์ˆ˜๋ฆฝํ•˜๊ธฐ ์œ„ํ•˜์—ฌ ์ˆ˜ํ–‰๋˜์—ˆ๋‹ค.
Isotype switching์„ ์œ„ํ•ด์„œ๋Š” ๊ฐ•ํ•œ ๋ฉด์—ญ์ž๊ทน์ด ํ•„์š”ํ•˜๋ฉฐ, ๋ฉด์—ญ๋ณด๊ฐ•์ œ(adjuvant)๋ฅผ ์ด์šฉํ•˜์—ฌ ๋”์šฑ ๊ฐ•ํ•œ ํ•ญ์›์ž๊ทน์„ ์ผ์œผํ‚ฌ ํ•„์š”๊ฐ€ ์žˆ๋‹ค. ํ•ญ์› ๋ฉด์—ญ์‹œ ํ•จ๊ป˜ ์‚ฌ์šฉ๋˜๋Š” adjuvant๋“ค์€ ๊ฐ๊ธฐ ๊ทธ ํ˜•ํƒœ์™€ ์ž‘์šฉ๊ธฐ์ „, ๋ฉด์—ญ์œ ๋„ ํšจ๊ณผ๊ฐ€ ๋‹ค์–‘ํ•œ ๊ฒƒ์œผ๋กœ ์•Œ๋ ค์ ธ ์žˆ๋Š”๋ฐ” [13], ๋น„๋ก ์„ ํ–‰์—ฐ๊ตฌ์—์„œ์ฒ˜๋Ÿผ P. larvae ํ•ญ์›์„ Freundโ€™s adjuvant๋กœ ๋ฉด์—ญ์‹œ์ผฐ์„ ๋•Œ IgM type์˜ ๋‹จํด๋ก ํ•ญ์ฒด๋“ค์ด ์ž‘์„ฑ๋˜์—ˆ์œผ๋‚˜, ๋‹ค์–‘ํ•œ adjuvant์˜ ์ ์šฉ์„ ํ†ตํ•˜์—ฌ isotype switching์ด ์™„๋ฃŒ๋œ ๋‹จํด๋ก ํ•ญ์ฒด๋ฅผ ์ž‘์ถœํ•  ์ˆ˜ ์žˆ์„ ๊ฒƒ์œผ๋กœ ์‚ฌ๋ฃŒ๋˜์—ˆ๋‹ค.
์ด๋ฅผ ํ™•์ธํ•˜๊ธฐ ์œ„ํ•˜์—ฌ, ๋ณธ ์—ฐ๊ตฌ์—์„œ๋Š” 3์ข…์˜ adjuvant ์ฆ‰, Water in Oil์˜ ํ˜•ํƒœ์˜ Freundโ€™s adjuvant(FA), ๊ธˆ์†์—ผ์ธ Alum adjuvant ๋ฐ Oil in water ํ˜•ํƒœ์ธ AddaVaxโ„ข(adjuvant MF59 like squalene adjuvant) [2]๋ฅผ ์‚ฌ์šฉํ•˜์—ฌ P. larvae์˜ ํ•ญ์›์„ BALB/c mouse์— ๋ฉด์—ญ์‹œ์ผœ ์žก์ข…์„ธํฌ clone์„ ์œ ๋„ํ•˜๊ณ  ๊ฐ ์žก์ข…์„ธํฌ๊ฐ€ ๋ถ„๋น„ํ•˜๋Š” ํŠน์ด ๋‹จํด๋ก ํ•ญ์ฒด์˜ isotype์„ ๋น„๊ตํ•˜์—ฌ ๋ณด์•˜๋‹ค.

์žฌ๋ฃŒ ๋ฐ ๋ฐฉ๋ฒ•

P. larvae ๋ฉด์—ญ์›

P. larvae ์ข…๊ท (ATCC9545)์„ Brain Heart Infusion(BHI) ๋ฐฐ์ง€์— ์ ‘์ข…ํ•˜์—ฌ 37โ„ƒ์— 24์‹œ๊ฐ„ ๋ฐฐ์–‘ํ•œ ํ›„ ์ˆ˜ํ™•ํ•˜์˜€๋‹ค. ๊ท ์ฒด๋Š” ์›์‹ฌ๋ถ„๋ฆฌ(3,735xg, 15๋ถ„, 3ํšŒ)ํ•˜์—ฌ PBS๋กœ ์„ธ์ฒ™ํ•˜์˜€๋‹ค. ์„ธ์ฒ™ํ•œ ๊ท ์ฒด๋Š” Homogenizer(WiseTis HG-15A, Witeg, Germany)๋กœ ๋ถ„์‡„ํ•œ ํ›„(27,000rpm x 2min, 1๋ถ„ ๊ฐ„๊ฒฉ์œผ๋กœ ์ด 5ํšŒ) -80โ„ƒ์— ๋ณด๊ด€ํ•˜๋ฉฐ ๋ฉด์—ญ์›์œผ๋กœ ์‚ฌ์šฉํ•˜์˜€๋‹ค. ๋ถ„์‡„ํ•œ ๊ท ์ฒด์•ก์˜ ๋‹จ๋ฐฑ๋Ÿ‰์€ Bio-rad protein assay(Bio-rad, USA) ์‹œ์•ฝ์„ ์‚ฌ์šฉํ•˜์—ฌ ์ •๋Ÿ‰ํ•˜์˜€๋‹ค.

Balb/c Mouse ๋ฉด์—ญ

P. larvae ๋ฉด์—ญ์›(400ใŽ protein/50ใŽ•)์„ 7์ฃผ๋ น์˜ ์•”์ปท BALB/c ๋งˆ์šฐ์Šค์— 2์ฃผ ๊ฐ„๊ฒฉ์œผ๋กœ 3ํšŒ ํ”ผํ•˜๋กœ ์ ‘์ข…ํ•˜์˜€๋‹ค. ๊ฐ๊ฐ ์‚ฌ์šฉ๋œ adjuvant๋Š” FA(Sigma-Aldrich, USA), Alum adjuvant(1.0% Aluminum Hydroxide Gel, Sigma-Aldrich, USA), AddaVaxโ„ข(Invivogen, USA)์œผ๋กœ์„œ, ๋ฉด์—ญ์›๊ณผ ๋™๋Ÿ‰์„ ์„ž์–ด ์‚ฌ์šฉํ•˜์˜€๋‹ค. ์ตœ์ข…๋ฉด์—ญ 24์‹œ๊ฐ„ ํ›„ ์„ธํฌ์œตํ•ฉ์„ ์ง„ํ–‰ํ•˜์—ฌ ํŠน์ดํ•ญ์ฒด๋ฅผ ๋ถ„๋น„ํ•˜๋Š” ์žก์ข…์„ธํฌ๋ฅผ ์„ ํƒํ•˜์˜€๋‹ค(์ œ์ฃผ๋Œ€ํ•™๊ต ๋™๋ฌผ์‹คํ—˜์œค๋ฆฌ์œ„์›ํšŒ, ์Šน์ธ๋ฒˆํ˜ธ:2017-0032).

Immunoglobulin isotype ํ™•์ธ ELISA

๊ฐ adjuvand๋ณ„๋กœ ๊ฐ๊ฐ ๊ณผ๋ฉด์—ญ์‹œํ‚จ BALB/c mice ํ˜ˆ์ฒญ ์ค‘์˜ ํŠน์ด ํ•ญ์ฒด๊ฐ€๋ฅผ ๋น„๊ตํ•˜๊ธฐ ์œ„ํ•˜์—ฌ, ์ œ 3์ฐจ ๋ฉด์—ญ ํ›„ 7์ผ์— ๋ฏธ์ •๋งฅ์—์„œ ์ฑ„ํ˜ˆํ•œ ํ˜ˆ์•ก๊ณผ, cell fusion ์ง์ „ mice์˜ ์‹ฌ์žฅ์—์„œ ์ฑ„ํ˜ˆํ•œ ํ˜ˆ์•ก์„ ์›์‹ฌ๋ถ„๋ฆฌํ•˜์—ฌ ELISA๋ฅผ ์‹ค์‹œํ•˜์˜€๋‹ค. Isotype ๋ณ„ ํ•ญ์ฒด๊ฐ€๋ฅผ ์ธก์ •ํ•˜๊ธฐ ์œ„ํ•œ ELISA๋Š” ๋‹ค์Œ๊ณผ ๊ฐ™์ด ์‹ค์‹œํ•˜์˜€๋‹ค. ์ฆ‰, P. larvae ๋ถ„์‡„ํ•ญ์›์ด ํก์ฐฉ๋œ 96 well micro ELISA plate์— ํฌ์„๋œ ํ˜ˆ์ฒญ ํ˜น์€ ๋ฐฐ์–‘ ์ƒ์ธต์•ก์„ ๊ฐ€ํ•˜์—ฌ ๋ฐ˜์‘์‹œํ‚จ ํ›„, 2์ฐจํ•ญ์ฒด ์ ‘ํ•ฉ์ฒด๋กœ๋Š” horseradish peroxidase (HRP)๊ฐ€ ํ‘œ์ง€๋œ goat, anti-mouse IgM(ฮผ-chain Specific, Sigma- Aldrich, USA), goat, anti-mouse IgG(ฮณ-chain specific, Sigma- Aldrich, USA) ๋ฐ goat, anti-mouse IgG(H+L)(kpl, USA)๋ฅผ ์‚ฌ์šฉํ•˜์—ฌ Immunoglobulin isotype๋ณ„์˜ ํ•ญ์ฒด๊ฐ€๋ฅผ ํ™•์ธํ•˜์˜€๋‹ค. ๋ฐœ์ƒ‰์ œ๋กœ๋Š” 2,2โ€ฒ-Azino-bis (3-ethylbenzothiazoline-6-sulfonic acid(ABTS, Sigma-Aldrich, USA)๋ฅผ ์‚ฌ์šฉํ•˜์˜€์œผ๋ฉฐ, 405nm(reference 492nm)์—์„œ ํก๊ด‘๋„๋ฅผ ์ธก์ •ํ•˜์˜€๋‹ค.

๋‹จํด๋ก  ํ•ญ์ฒด์˜ Immunoglobulin isotype ๊ฒ€์‚ฌ

์žก์ข…์„ธํฌ์˜ ๋ฐฐ์–‘ ์ƒ์ธต์•ก์„ ์ทจํ•˜์—ฌ ์ „์ˆ ํ•œ ELISA ๋ฐฉ๋ฒ•์œผ๋กœ ๋ถ„์„ํ•˜์˜€๋‹ค.

๊ฒฐ ๊ณผ

Stock Mab clones์˜ Isotype

๊ฐ๊ฐ ๊ฐœ๋ณ„์ ์œผ๋กœ ์ˆ˜ํ–‰๋œ ์„ ํ–‰์—ฐ๊ตฌ์—์„œ ์ž‘์„ฑ๋œ ์œ ํšจํด๋ก  ์ค‘, P. larvae flagella ํ•ญ์›์œผ๋กœ ๋ฉด์—ญ์‹œ์ผœ ์ž‘์„ฑํ•˜์˜€์œผ๋ฉฐ, ์ง€์†์ ์œผ๋กœ ํŠน์ดํ•ญ์ฒด๋ฅผ ๋ถ„๋น„ํ•˜๋Š” 4์ข… ์žก์ข…์„ธํฌ์˜ ๋‹จํด๋ก ํ•ญ์ฒด isotype ๋ฐœํ˜„์–‘์ƒ์„ Table 1์— ์ •๋ฆฌํ•˜์˜€๋‹ค. ํ•ด๋‹น ํด๋ก ๋“ค์€ FA๋ฅผ adjuvant๋กœ ์‚ฌ์šฉํ•œ P. larvae Flagella๋ฅผ ๋ฉด์—ญ์‹œ์ผœ ๊ฐœ๋ฐœ๋œ ๊ฒƒ๋“ค๋กœ์„œ, 6ํšŒ์˜ ์ถ”๊ฐ€์ ‘์ข…์œผ๋กœ ์ธํ•œ ๊ณผ๋ฉด์—ญ์—๋„ ๋ถˆ๊ตฌํ•˜๊ณ  isotype์€ ๋ชจ๋‘ IgM์œผ๋กœ ํ™•์ธ๋˜์—ˆ๋‹ค.
๋˜ํ•œ, P. larvae๊ท ์ฒด ํŒŒ์‡„ ํ•ญ์›์„ ๋ฉด์—ญ์‹œ์ผœ ์–ป์€ ์œ ํšจํด๋ก ์— ์˜ํ•˜์—ฌ ๋ถ„๋น„๋œ ๋‹จํด๋ก ํ•ญ์ฒด์˜ isotype์€ Table 2์— ์ •๋ฆฌํ•˜์˜€๋‹ค. FA์™€ Alum์€ ๊ฐ๊ฐ 66%(2 clones) ๋ฐ 25%(1 clones)์˜ IgG type์˜ ๋ฐœํ˜„์œจ์„ ๋ณด์ธ ๊ฒƒ์— ๋ฐ˜ํ•ด AddaVaxโ„ข adjuvant๋กœ ์ ‘์ข…ํ•œ ๊ฒฝ์šฐ์—์„œ 100%(5 clones) IgG๋กœ์˜ class switching์„ ๋ณด์˜€๋‹ค.

Adjuvant์— ๋”ฐ๋ฅธ ๋ฉด์—ญ๋งˆ์šฐ์Šค ํ˜ˆ์ฒญ Immunoglobulin์˜ ํ•ญ์ฒด๊ฐ€ ๋น„๊ต

๋™์‹œ์— ๋™์ผํ•œ ์šฉ๋Ÿ‰์˜ ํ•ญ์›(400 ใŽ/mouse)์„ ๋™์ผํ•œ ์—ฐ๋ น์˜(7์ฃผ๋ น) BALB/c mice์— ๋ฉด์—ญ์‹œํ‚ค๊ณ  ์žก์ข…์„ธํฌ ์œตํ•ฉ๊ณผ์ •์„ ์‹ค์‹œํ•˜์—ฌ adjuvant๋ณ„์˜ ๋ฉด์—ญ๊ฐ•๋„์™€ IgM ๋ฐ IgG type๋ณ„์˜ ํ•ญ์ฒด๊ฐ€๋ฅผ ๋น„๊ตํ•˜์˜€๋‹ค. ์ œ 3์ฐจ ๋ฉด์—ญ ํ›„ 7์ผ์งธ์— ๋ฏธ์ •๋งฅ์„ ํ†ตํ•˜์—ฌ ์ฑ„ํ˜ˆํ•œ BALB/c miceํ˜ˆ์ค‘์˜ ํ•ญ์ฒด๊ฐ€์— ๋น„ํ•˜์—ฌ ์ตœ์ข…๋ฉด์—ญ 24์‹œ๊ฐ„ํ›„ ์ฆ‰, cell fusion ์ง์ „์— ์‹ฌ์žฅ์„ ํ†ตํ•˜์—ฌ ์ฑ„ํ˜ˆํ•œ ํ˜ˆ์ค‘ ํ•ญ์ฒด๊ฐ€ ๋ฐ IgG type์˜ ํ•ญ์ฒด๊ฐ€๋Š” ๊ฐ adjuvant ๋ณ„๋กœ ์ฃผ๋ชฉํ•  ๋งŒํ•œ ์ฐจ์ด๋Š” ์ธ์ •๋˜์ง€ ์•Š์•˜๋‹ค(๋ชจ๋‘ 105 titer ์ด์ƒ). IgM type์˜ ํ•ญ์ฒด๊ฐ€์˜ ์ฆ๊ฐ€๋Š” 2๊ฐœ ๋ฉด์—ญ ์‹œ์  ๋ฐ adjuvant๋ณ„์˜ ๊ฐ ๊ฒฝ์šฐ์— ๋ชจ๋‘ ๊ฐ์ง€๋˜์ง€ ์•Š์•˜๋‹ค(๋ชจ๋‘ 103 titer ๋ฏธ๋งŒ). ํ•œํŽธ, Cell fusion ์‹œ์ ์—์„œ AddaVaxโ„ข adjuvant๋กœ ์ ‘์ข…ํ•œ ๋งˆ์šฐ์Šค์—์„œ์˜ IgG type์˜ ํ•ญ์ฒด๊ฐ€๋Š” ๋‹ค๋ฅธ ์ ‘์ข…๊ตฐ์— ๋น„ํ•˜์—ฌ ๋‹ค์†Œ ๋‚ฎ์€ ๊ฒฝํ–ฅ์„ ๋ณด์˜€๋‹ค(Fig. 1).

Adjuvant๋ณ„๋กœ ๋น„๊ตํ•œ MAb์˜ Isotype

Adjuvant๊ฐ€ Ig isotype switching์— ๋ฏธ์น˜๋Š” ์˜ํ–ฅ์„ ํ™•์ธํ•˜๊ณ ์ž, ๋™์ผํ•œ ์กฐ๊ฑด์œผ๋กœ adjuvant๋ณ„๋กœ ๊ตฌ๋ถ„ํ•˜์—ฌ ๋ฉด์—ญ์‹œํ‚จ ํ›„ ์–ป์–ด์ง„ ๋‹จํด๋ก ํ•ญ์ฒด์˜ isotype์„ ๋น„๊ตํ•˜์˜€๋‹ค(Table 3). FA๋ฅผ adjuvnat๋กœ ์‚ฌ์šฉํ•œ MAb clone์˜ isotype์€ IgG type๊ณผ IgM type ์ด ๋ชจ๋‘ ๋ฐœํ˜„๋˜์—ˆ์œผ๋ฉฐ IgG type์ด 67%, IgM type์ด 33%๋กœ ๋‚˜ํƒ€๋‚ฌ๋‹ค. Alum adjuvant๋ฅผ ์‚ฌ์šฉํ•œ ๊ฒฝ์šฐ์—๋„ IgG type๊ณผ IgM type์˜ MAb clone์ด ๋ชจ๋‘ ๋ฐœํ˜„๋˜์—ˆ์œผ๋ฉฐ, IgG type์ด 91%, IgM type์ด 9%๋กœ ๋‚˜ํƒ€๋‚ฌ๋‹ค. ํ•œํŽธ, AddaVaxโ„ข์„ Adjuvant๋กœ ์‚ฌ์šฉํ•˜์˜€์„ ๊ฒฝ์šฐ์—๋Š” ์„ ํ–‰์—ฐ๊ตฌ์˜ ๊ฒฐ๊ณผ์™€ ๋งˆ์ฐฌ๊ฐ€์ง€๋กœ ์žก์ข…์„ธํฌ์˜ 100%์—์„œ Isotype switching์ด ์™„์„ฑ๋œ IgG type์˜ ํ•ญ์ฒด๋ฅผ ๋ถ„๋น„ํ•˜์˜€๋‹ค.

๊ณ  ์ฐฐ

๊ฟ€๋ฒŒ์€ ๋‹ค์–‘ํ•œ ์–‘๋ด‰์‚ฐ๋ฌผ์„ ์ƒ์‚ฐํ•  ๋ฟ ์•„๋‹ˆ๋ผ ์‹๋ฌผ์˜ ์ˆ˜๋ถ„ํ™œ๋™์„ ์ฃผ๋„ํ•˜๋Š” ์ค‘์š”ํ•œ ์—ญํ• ์„ ํ•œ๋‹ค [4, 11]. AFB๋Š” ๊ฟ€๋ฒŒ์˜ ์ƒ์œก์„ ์œ„ํ˜‘ํ•˜๋Š” ์ค‘์š”ํ•œ ๊ฐ์—ผ๋ณ‘์ด๋‹ค. ์ €์ž๋“ค์€ ๋ฏธ๊ตญ๋ถ€์ €๋ณ‘์— ๋Œ€ํ•œ ๋ฉด์—ญํ•™์  ์ง„๋‹จ์†Œ์žฌ ๊ฐœ๋ฐœ์„ ์œ„ํ•˜์—ฌ P. larvae์— ๋Œ€ํ•œ Mab๋ฅผ ๊ฐœ๋ฐœํ•œ ๋ฐ” ์žˆ๋‹ค. ์ด ๊ณผ์ • ์ค‘ ํฅ๋ฏธ๋กญ๊ฒŒ๋„ IgM type์˜ ๋‹จํด๋ก ํ•ญ์ฒด๋ฅผ ๋ถ„๋น„ํ•˜๋Š” ์žก์ข…์„ธํฌ์ฃผ๊ฐ€ ๋‹ค์ˆ˜ ์ˆ˜๋ฆฝ๋˜์—ˆ๋‹ค [8]. ๋น„๋ก ๋‹จํด๋ก ํ•ญ์ฒด์˜ ๊ฐ isotype๋“ค์€ ๋ถ„์ž์ ์ธ ํŠน์„ฑ์— ์˜ํ•˜์—ฌ ๊ฐ๊ฐ์˜ ์ ํ•ฉํ•œ ์šฉ๋„๋กœ ํ™œ์šฉ๋  ์ˆ˜ ์žˆ์„ ๊ฒƒ์ด๋‚˜, ์ด๋Ÿฌํ•œ isotype์˜ ๊ฒฐ์ •์€ ์—ฐ๊ตฌ์ž์˜ ์˜๋„์™€๋Š” ์ƒ๊ด€์—†์ด ์ด๋ฃจ์–ด์ง„๋‹ค.
Apiratmateekul ๋“ฑ [1]์€ Hemoglobin F์— ๋Œ€ํ•œ IgM type์˜ ๋‹จํด๋ก ํ•ญ์ฒด๋ฅผ ์–ป๊ธฐ ์œ„ํ•˜์—ฌ IgM+ Spleen cell๋งŒ์„ ๋”ฐ๋กœ ์„ ๋ณ„ํ•œ ํ›„ ์„ธํฌ์œตํ•ฉ์„ ์‹ค์‹œํ•˜์—ฌ IgM type์˜ ๋‹จํด๋ก ํ•ญ์ฒด๋ฅผ ๋ถ„๋น„ํ•˜๋Š” ์žก์ข…์„ธํฌ์˜ ์ถœํ˜„์ด ์šฐ์„ธํ•˜๋„๋ก ์ธ์œ„์ ์ธ ์ฒ˜์น˜๋ฅผ ํ•œ ๊ฒฐ๊ณผ๋ฅผ ๋ณด๊ณ ํ•˜๊ธฐ๋„ ํ•˜์˜€๋‹ค.
ํ•œํŽธ, Soukhtanloo ๋“ฑ [14]์ด chitinase์— ๋Œ€ํ•œ IgM type์˜ Mab์— ๋Œ€ํ•œ ๋ณด๊ณ ๋‚˜, Lee ๋“ฑ [10]์ด Ebola virus glycoprotein์ด encoding๋œ DNA ๋ฐฑ์‹ ์œผ๋กœ ์–ป์–ด๋‚ธ IgM type์˜ MAb clone์— ๋Œ€ํ•œ ๋ณด๊ณ ๋Š” immunoglobulin isotype switching์˜ ์™„์„ฑ์—ฌ๋ถ€๊ฐ€ ๊ฐœ๋ณ„ ๋ฉด์—ญํ•ญ์›์˜ ํŠน์„ฑ์— ์˜ํ•˜์—ฌ ์ด๋ฃจ์–ด์ง์„ ์‹œ์‚ฌํ•˜๊ณ  ์žˆ๋‹ค.
๋ณธ ์—ฐ๊ตฌ์—์„œ์˜ P. larvae ํ•ญ์›์— ์˜ํ•œ ์œ ๋„๋˜๋Š” MAb์— ๊ด€ํ•œ ์—ฐ๊ตฌ๊ฒฐ๊ณผ๋Š” ๋ณด๊ณ ๋œ ๋ฐ”๊ฐ€ ๋งŽ์ง€ ์•Š๊ธฐ ๋•Œ๋ฌธ์— [7, 8], immunoglobulin isotype switching์„ ๋ถˆ์™„์ „ํ•˜๊ฒŒ ์œ ๋„ํ•˜๋Š” ํ•ญ์›์ ์ธ ํŠน์„ฑ์— ๊ด€ํ•œ ์ž๋ฃŒ๋Š” ์•„์ง ์•Œ๋ ค์ ธ ์žˆ์ง€ ์•Š๋‹ค. ๋˜ํ•œ ๋ฉด์—ญ๋ณด๊ฐ•์„ ์œ„ํ•˜์—ฌ ํ˜ผํ•ฉํ•˜์—ฌ ์‚ฌ์šฉ๋˜๋Š” adjuvant์˜ ์ ์šฉ์— ๋”ฐ๋ผ ๋„์ถœ๋˜๋Š” ๊ฒฐ๊ณผ์˜ ์ฐจ์ด๋ฅผ ์‚ดํŽด๋ณด๋Š” ๊ฒƒ๋„ ํฅ๋ฏธ๋กœ์šด ์‚ฌํ•ญ์ด๋‹ค.
์ €์ž๋“ค์€ P. larvae์— ๋Œ€ํ•œ Mab๋ฅผ ๋ถ„๋น„ํ•˜๋Š” ์žก์ข…์„ธํฌ์˜ Isotype switching์˜ ์ •๋„๊ฐ€ adjuvant์— ๋”ฐ๋ผ ์ฐจ์ด๊ฐ€ ๋‚˜๋Š”์ง€ ์•Œ์•„๋ณด๊ณ ์ž, P. larvae๊ท ์ฒดํ•ญ์›์„ 3์ข…์˜ adjuvant์™€ ํ˜ผํ•ฉํ•˜์—ฌ ๊ฐ๊ฐ BALB/c ๋งˆ์šฐ์Šค์— ๋ฉด์—ญ์‹œํ‚จ ํ›„, ์„ธํฌ์œตํ•ฉ์— ์˜ํ•˜์—ฌ ์ œ์ž‘๋œ ์žก์ข…์„ธํฌ๋“ค์ด ๋ถ„๋น„ํ•˜๋Š” ๋‹จํด๋ก ํ•ญ์ฒด์˜ isotype ์ƒ์„ฑ์„ ๋น„๊ตํ•˜์˜€๋‹ค.
Isotype switching์—๋Š” CD40 signaling๊ณผ NF-ฮบB์˜ ํ™œ์„ฑ์„ ํ•„์š”๋กœ ํ•˜๋Š”๋ฐ [15] NF-ฮบB์˜ ํ™œ์„ฑํ™”์—๋Š” MyD88์ด ์˜ํ–ฅ์„ ์ฃผ๋Š” ๊ฒƒ์œผ๋กœ ์•Œ๋ ค์ ธ ์žˆ๋‹ค. Garg ๋“ฑ [6]์€ Salmonella Typhi Vi ํ•ญ์›์œผ๋กœ ๋งˆ์šฐ์Šค๋ฅผ ๋ฉด์—ญ์‹œ์ผฐ์„ ๋•Œ, B cell์—์„œ ์ƒ์„ฑ๋˜๋Š” immunoglobulin์ด IgG๋กœ isotype switching ๋˜๋Š” ๊ณผ์ •์— MyD88์ด ํ•ต์‹ฌ์ ์ธ ์˜ํ–ฅ์„ ์คŒ์„ ๋ณด๊ณ ํ•˜์˜€๋‹ค.
์ด๋Ÿฌํ•œ ๊ด€์ ์—์„œ ๋ณธ๋‹ค๋ฉด, ๋ณธ ์—ฐ๊ตฌ์—์„œ ์‚ฌ์šฉํ•œ P. larvae ํ•ญ์›์ด MyD88์˜ ํ™œ์„ฑํ™”์— ์ง€์žฅ์„ ์ฃผ์–ด ๊ทธ ๊ฒฐ๊ณผ๋กœ isotype switching์ด ์™„์„ฑ๋˜์ง€ ์•Š์€ IgM type์˜ clone์ด ๋‹ค์ˆ˜ ์œ ๋„๋œ ๊ฒƒ์œผ๋กœ ์ถ”๋ก ํ• ์ˆ˜๋„ ์žˆ์„ ๊ฒƒ์ด๋ฉฐ, ์ด์— ๋Œ€ํ•œ ์ถ”๊ฐ€์ ์ธ ์—ฐ๊ตฌ๊ฐ€ ์ˆ˜ํ–‰๋˜์–ด์•ผ ํ•  ๊ฒƒ์ด๋‹ค.
๊ณผ๋ฉด์—ญ์„ ์‹ค์‹œํ•œ 3ํšŒ์˜ ๋ฉด์—ญ ์ดํ›„์˜ ์‹œ์  ๋ฐ B cell๊ณผ myeloma cell์˜ fusion ์‹œ์ ์—์„œ, 3์ข…์˜ adjuvant๋กœ ๋ฉด์—ญ์‹œํ‚จ ๊ฐ๊ฐ์˜ ๋งˆ์šฐ์Šค ๋ชจ๋‘์—์„œ๋Š” ์ด๋ฏธ ์ถฉ๋ถ„ํ•œ ์ •๋„์˜ ํ˜ˆ์ค‘ IgG ํ•ญ์ฒด๊ฐ€์˜ ์ƒ์Šน์„ ๋ณด์˜€๋‹ค(x105<). ๊ทธ๋Ÿฌ๋‚˜ IgM ํ•ญ์ฒด๊ฐ€๋Š” ํ™•์ธ๋˜์ง€ ์•Š์•˜๋‹ค(x103>), ์ด๋Š” cell fusion ์‹œ์ ์—์„œ ์ด๋ฏธ ๋ฉด์—ญ์›์— ๋Œ€ํ•œ ํ•ญ์ฒด๋ถ„๋น„์„ธํฌ์—์„œ isotype switching์ด ์ถฉ๋ถ„ํžˆ ์ผ์–ด๋‚ฌ๊ธฐ ๋•Œ๋ฌธ์ด๋ผ๊ณ  ์ถ”๋ก ํ•  ์ˆ˜ ์žˆ์—ˆ๋‹ค. ๊ทธ๋Ÿผ์—๋„ ๋ถˆ๊ตฌํ•˜๊ณ  ๋‹จํด๋ก ํ•ญ์ฒด๋ฅผ ๋ถ„๋น„ํ•˜๋Š” ์žก์ข…์„ธํฌ ์ค‘์—๋Š” IgM type์˜ Mab๋ฅผ ๋ถ„๋น„ํ•˜๋Š” clone๋„ ๋‹ค์ˆ˜ ๋ฐœํ˜„๋œ ๊ฒฐ๊ณผ๋ฅผ ๋ฏธ๋ฃจ์–ด ๋ณผ ๋•Œ, ๋น„์žฅ์„ธํฌ ์ค‘์—๋Š” IgM+ Spleen cell๋“ค์ด ์—ฌ์ „ํžˆ ์ƒ๋‹นํ•œ ์ •๋„๋กœ ์กด์žฌํ•˜๊ณ  ์žˆ์Œ์„ ์‹œ์‚ฌํ•œ๋‹ค.
๊ฒฐ๋ก ์ ์œผ๋กœ, ํ˜ˆ์ค‘ ํ•ญ์ฒด๊ฐ€๋กœ ๋น„๊ตํ•œ adjuvants ๊ฐ„์˜ ๋ฉด์—ญ์ฆ๊ฐ•ํšจ๊ณผ๋Š” ์ด๋Ÿ‰์ ์œผ๋กœ๋‚˜ isotype๋ณ„ ์ฆ‰, IgG ๋ฐ IgM์˜ ๊ฐœ๋ณ„์ ์ธ ๊ฐœ๋ณ„์ ์ธ ํ•ญ์ฒด๊ฐ€์˜ ์ฐจ์ด๋ฅผ ๋ณด์ด์ง€ ์•Š์•˜์œผ๋‚˜, ์žก์ข…์„ธํฌ๊ฐ€ ๋ถ„๋น„ํ•˜๋Š” MAb์˜ isotype switching ์–‘์ƒ์€ ๋šœ๋ ทํ•œ ์ฐจ์ด๊ฐ€ ์žˆ์—ˆ๋‹ค. ์ฆ‰, Freundโ€™s adjuvant๋Š” 66.67% (11/33), Alum adjuvant๋Š” 91.25%(73/80)๊ฐ€ IgG type์œผ๋กœ ๋‚˜ํƒ€๋‚ฌ์œผ๋ฉฐ, AddaVaxโ„ข adjuvant๋Š” 100%(14/14) IgG type์œผ๋กœ์˜ isotype switching์„ ์œ ๋„ํ•˜์˜€๋‹ค. ์ด๋Ÿฌํ•œ ๊ฒฐ๊ณผ๋Š”, isotype switching์ด ๋ถˆ์™„์ „ํ•˜๊ฒŒ ์œ ๋„๋˜๋Š” ํŠน์„ฑ์„ ๋ณด์ด๋Š” P. larvae ํ•ญ์›์œผ๋กœ ๋ฉด์—ญ์‹œ์ผฐ์Œ์—๋„ ๋ถˆ๊ตฌํ•˜๊ณ  ํŠน์ •ํ•œ adjuvant ์ฆ‰, AddaVaxโ„ข์™€ ๊ฐ™์€ ๋ฉด์—ญ๋ณด๊ฐ•์ œ๋ฅผ ์ ์šฉํ•œ๋‹ค๋ฉด isotype class switching์„ ์œ ๋„ํ•˜๋Š” ์ œ๋ฐ˜์š”์ธ๋“ค์„ ๋ณด์™„ํ•ด ์ค„ ์ˆ˜ ์ฃผ๊ณ  ์žˆ์Œ์„ ์‹œ์‚ฌํ•œ๋‹ค.


๋ณธ ์—ฐ๊ตฌ๋Š” ๋†๋ฆผ์ถ•์‚ฐ์‹ํ’ˆ๋ถ€์˜ ์žฌ์›์œผ๋กœ ๋†๋ฆผ์ˆ˜์‚ฐ์‹ํ’ˆ๊ธฐ์ˆ ๊ธฐํšํ‰ ๊ฐ€์›์˜ ์ฒจ๋‹จ์ƒ์‚ฐ๊ธฐ์ˆ ๊ฐœ๋ฐœ์‚ฌ์—…(112042-3, 115058-02, 115102-03 ์˜ ์ง€์›์„ ๋ฐ›์•„ ์ˆ˜ํ–‰๋˜์—ˆ์œผ๋ฉฐ, ์ €์ž ์ผ๋™์€ ์ด์— ๊ฐ์‚ฌํ•˜๋Š” ๋ฐ”์ž…๋‹ˆ๋‹ค.


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Fig.ย 1.
Serum titers of anti-P. larve antibodies in BALB/c mice at the time of 3rd (A) and final (B) immunization. Notable differences of titers were not shown among sera immunized with 3 kinds of adjuvants. While IgG type antibody titers were raised to higher than x 105, no antibody response was observed in IgM type antibodies (โ—‹: Freundโ€™s adjuvant, โ–ก: Alum adjuvant, โ–ณ: AddaVaxโ„ข).
Tableย 1.
Isotype feature of Mab clones against purified flagella of P. larvae with Freundโ€™s adjuvant
Immunogen Adjuvant Immunizing dose Clone Reaction with anti-mouse Ig
IgG (H+L) ฮผ ฮณ ฮฑ
Purified flagella Freundโ€™s adjuvant 15ใŽ prot., 6 times AF01 + + - -
AF02 + + - -
AF03 + + - -
AF04 + + - -
Tableย 2.
Isotype feature of previously cloned Mabs based on the adjuvants applied in immunization
Immunogen Adjuvant Immunizing dose Clone Reaction with anti-mouse Ig
IgG (H+L) ฮผ ฮณ ฮฑ
Whole cell1) Freundโ€™s adjuvant 200ใŽ prot., 3 times AF13 + - + -
AF14 + + - -
AF16 + - + -
Alum adjuvant 200ใŽ prot., 4 times AF20 + + - -
AF21 + - + -
AF22 + + - -
AF23 + + - -
AddaVaxโ„ข 400ใŽ prot., 3 times AF24 + - + -
AF25 + - + -
AF26 + - + -
AF27 + - + -
AF28 + - + -

1) Disrupted & homogenized bacterial antigen.

Tableย 3.
Isotype feature of MAbs according to the adjuvants used in production of hybridoma to P. larvae antigen
Immunogen Adjuvant Immunizing dose Clone number Reaction with anti-mouse Ig
ฮผ ฮณ
Whole cell1) Freundโ€™s adjuvant 400ฮผg prot, 3 times 33 11 (33%) 22 (66%)
Alum adjuvant 80 7 (9%) 73 (91%)
AddaVaxโ„ข 14 0 (0%) 1 (100%)

1) Disrupted & homogenized bacterial antigen.

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