무독화 논문 번역(한국어 원본)화학적 무독화처리에 따라 불활성 된 백일해독소의 residual PTx activity와 reversion to toxicity를 확인하기 위해서 Tohama I strain에서 pertussis toxin을 생산하고, 이를 분리정제하였다. 정제된 백일해독소는 단백질정량분석을 통해 독소량을 측정하였으며, 희석하여 100 μg/ml의 농도의 조정하여 포름알데히드, 글루탈알데히드, 글루탈알데히드와 포름알데히드 혼합액에 각각 무독화처리를 하였다. 무독화처리 이후에 centricon을 이용하여 잔존하는 aldehyde를 충분히 제거한 후, 다시 100 μg/ml의 농도로 맞추었다. Reversion to toxicity of detoxifying agent treated PTx samples were carried out incubating the samples at 37℃ for 4 weeks. 대조군으로서 native PTx를 각각 4도와 37도에 4주간 같이 보관하였다.
무독화 처리한 각각의 sample을 이용하여 5,000 ng/ml 부터 2 ng/ml까지 단계희석하여 carbohydrate binding assay as previously described by Gomez et al.14 을 수행하였다. Native PTx의 경우 그림 1(A)와 같이 PTx activity 범위를 보였으며, 37도에서 4주간 보관한 경우, activity가 약간 감소하는 결과가 관찰되었다. 포름알데히드처리한 경우 백일해독소가 dramatically하게 감소하는 경향이 보였으며, reversion to toxicity가 native PTx 수준가까이로 회복되는 결과를 보였다 (Fig.1(B)). 글루탈알데히드처리한 경우 백일해독소가 급격히 감소하는 경향을 보였으나, reversion to toxicity는 오히려 4도 4주간 보관한 PTx activity보다 PTx activity가 떨어지는 결과를 관찰되었다 (Fig.1(C)). 글루탈알데히드-포름알데히드 혼합액을 처리한 경우 백일해독소가 급격히 감소하는 경향이 관찰되었고, reversion to toxicity는 비록 적은 수준이지만 독성회복양상을 보였다 (Fig.1.(D)).
또한 무독화 처리한 각각의 sample을 이용하여 8,000 ng/ml 부터 61.5 ng/ml까지 단계희석하여 enzymatic HPLC assay as previously described by Yuen et al.13을 수행하였다. Native PTx의 경우 그림 2(A)와 같이 PTx activity 범위를 보였으며, 37도에서 4주간 보관한 경우, 역시 activity가 약간 감소하는 결과가 관찰되었다. 포름알데히드처리한 경우 carbohydrate binding assay 결과와 같이 백일해독소가 dramatically하게 감소하는 경향이 보였고, reversion to toxicity가 native PTx 수준가까이로 회복되는 결과를 보였다 (Fig.2(B)). 그러나 흥미롭게도, 글루탈알데히드처리한 경우 백일해독소가 감소하는 결과가 관찰되지 않았으며, 4도에서 4주간 보관한 PTx activity와 37도 4주간 보관한 PT activity사이에 큰 변화가 없는 것으로 관찰되었다 (Fig.2(C)). 글루탈알데히드-포름알데히드 혼합액을 처리한 경우 백일해독소가 급격히 감소하는 경향이 관찰되었고, reversion to toxicity는 비록 적은 수준이지만 독성회복양상을 보였다 (Fig.2.(D)).
8주령 수컷 CD1 mice 10마리를 그룹으로하여 dose당 200ng에서 25ng을 복강주사하고, 4일후에 2mg의 히스타민주사를 한 후 1시간동안의 치사율을 확인하였다 (Table 1). Native PTx의 경우 농도에 비례하여 높은 사망률을 나타냈음에 반해, 포름알데히드, 글루탈알데히드, 글루탈알데히드-포름알데히드 혼합액을 처리한 백일해독소 어느 희석구간에서도 죽은 마우스는 관찰되지 않았다. 37도에서 4주간 보관조건에서 포름알데히드에 무독화된 PTx, 글루탈알데히드-포름알데히드 혼합액을 처리한 PTx순으로 reversion to toxicity 현상이 관찰되었다. 그러나 글루탈알데히드를 처리한 detoxified PTx의 경우에는 어느 희석구간에서도 죽은 마우스는 관찰되지 않았다. |
무독화 논문 번역(영어 번역본)In order to measure residual PTx activity and reversion to toxicity in PTx detoxified by chemical treatment, PTx was produced from Tohama I strain and purified. Obtained PTx was quantified by Lowry assay then diluted to concentration of 100 μg/ml before being detoxified by formaldehyde, glutaraldehyde, or glutaraldehyde-formaldehyde mixture. Afterwards, Centricon was used to sufficiently remove remaining aldehyde. Concentration of the samples was set again at 100 μg/ml. PTx samples were incubated at 37℃ for 4 weeks. As a control, native PTx samples were incubated together at 4℃ or 37℃ for 4 weeks.
Each sample that has been detoxified was serially diluted from 5,000 ng/ml to 2 ng/ml and underwent carbohydrate binding assay as previously described by Gomez et al.14 Native PTx had activity range as shown in Fig. 1 (A), and the activity slightly decreased when stored at 37℃ for 4 weeks. When treated with formaldehyde, PTx activity dramatically declined at first but reversion to toxicity was comparable to that of native PTx after storage at 37℃ for 4 weeks (Fig. 1(B)). For samples treated with glutaraldehyde, PTx activity rapidly declined at first but reversion to toxicity was absent in PTx stored at 37℃ for 4 weeks and its activity was actually lower than that of PTx stored at 4℃ for 4 weeks (Fig. 1(C)). When samples were treated with glutaraldehyde-formaldehyde mixture, PTx activity rapidly declined and some reversion to toxicity was observed (Fig. 1 (D)).
Additionally, each detoxified sample was serially diluted from 8,000 ng/ml to 61.5 ng/ml and underwent enzymatic HPLC assay as previously described by Yuen et al.13 Native PTx had activity range as shown in Fig. 2 (A), and the activity slightly decreased when stored at 37℃ for 4 weeks. Similar to result from carbohydrate binding assay, formaldehyde-treated samples showed dramatic decline in PTx activity and its reversion to toxicity after storage at 37℃ for 4 weeks was comparable to that of native PTx (Fig. 1(B)). Interestingly for samples treated with glutaraldehyde, no decrease in PTx activity was observed, and there was no significant difference in activity between PTx stored at 4℃ for 4 weeks and PTx stored at 37℃ for 4 weeks (Fig. 2(C)). When samples were treated with glutaraldehyde-formaldehyde mixture, PTx activity rapidly declined and some reversion to toxicity, although to a very small extent, was observed (Fig. 1 (D)).
8-week-old male CD1 mice in groups of 10 were i.p. injected with 200ng to 25ng of the samples for each dose. After 4 days, 2mg of histamine was injected and the number of deceased animals within 1 hour was counted (Table 1). Native PTx showed high rate of mortality proportionate to the concentration, but no mouse was deceased for any dilutions of PTx treated with formaldehyde, glutaraldehyde, or glutaraldehyde-formaldehyde mixture when the samples were not stored for extended period of time. Reversion to toxicity was observed for PTx detoxified by formaldehyde and by glutaraldehyde-formaldehyde mixture then stored at 37℃ for 4 weeks, with reversion being greater in formaldehyde-detoxified PTx. However, no mouse was deceased for any concentration of PTx detoxified by glutaraldehyde. |
