Abstract
The bacterial endotoxin test, which uses amebocyte lysate reagents of horseshoe crab origin, is a sensitive, reproducible and simple assay to measure endotoxin concentration. To develop sustainable raw materials for lysate reagents that do not require horseshoe crabs, three recombinant protease zymogens (factor C, derived from mammalian cells; factor B; and the proclotting enzyme derived from insect cells) were prepared using a genetic engineering technique. Recombinant cascade reagents (RCRs) were then prepared to reconstruct the reaction cascade in the amebocyte lysate reagent. The protease activity of the RCR containing recombinant factor C was much greater than that of recombinant factor C alone, indicating the efficiency of signal amplification in the cascade. Compared with the RCR containing the insect cell-derived factor C, those containing mammalian cell-derived factor C, which features different glycosylation patterns, were less susceptible to interference by the injectable drug components. The standard curve of the RCR containing mammalian cell-derived recombinant factor C had a steeper slope than the curves for those containing natural lysate reagents, suggesting a greater sensitivity to endotoxin. The present study supports the future production of recombinant reagents that do not require the use of natural resources.
| Original language | English |
|---|---|
| Pages (from-to) | 136-146 |
| Number of pages | 11 |
| Journal | Innate Immunity |
| Volume | 23 |
| Issue number | 2 |
| DOIs | |
| Publication status | Published - Jan 1 2017 |
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All Science Journal Classification (ASJC) codes
- Microbiology
- Immunology
- Molecular Biology
- Cell Biology
- Infectious Diseases
Cite this
Genetic engineering approach to develop next-generation reagents for endotoxin quantification. / Mizumura, Hikaru; Ogura, Norihiko; Aketagawa, Jun; Aizawa, Maki; Kobayashi, Yuki; Kawabata, Shun Ichiro; Oda, Toshio.
In: Innate Immunity, Vol. 23, No. 2, 01.01.2017, p. 136-146.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - Genetic engineering approach to develop next-generation reagents for endotoxin quantification
AU - Mizumura, Hikaru
AU - Ogura, Norihiko
AU - Aketagawa, Jun
AU - Aizawa, Maki
AU - Kobayashi, Yuki
AU - Kawabata, Shun Ichiro
AU - Oda, Toshio
PY - 2017/1/1
Y1 - 2017/1/1
N2 - The bacterial endotoxin test, which uses amebocyte lysate reagents of horseshoe crab origin, is a sensitive, reproducible and simple assay to measure endotoxin concentration. To develop sustainable raw materials for lysate reagents that do not require horseshoe crabs, three recombinant protease zymogens (factor C, derived from mammalian cells; factor B; and the proclotting enzyme derived from insect cells) were prepared using a genetic engineering technique. Recombinant cascade reagents (RCRs) were then prepared to reconstruct the reaction cascade in the amebocyte lysate reagent. The protease activity of the RCR containing recombinant factor C was much greater than that of recombinant factor C alone, indicating the efficiency of signal amplification in the cascade. Compared with the RCR containing the insect cell-derived factor C, those containing mammalian cell-derived factor C, which features different glycosylation patterns, were less susceptible to interference by the injectable drug components. The standard curve of the RCR containing mammalian cell-derived recombinant factor C had a steeper slope than the curves for those containing natural lysate reagents, suggesting a greater sensitivity to endotoxin. The present study supports the future production of recombinant reagents that do not require the use of natural resources.
AB - The bacterial endotoxin test, which uses amebocyte lysate reagents of horseshoe crab origin, is a sensitive, reproducible and simple assay to measure endotoxin concentration. To develop sustainable raw materials for lysate reagents that do not require horseshoe crabs, three recombinant protease zymogens (factor C, derived from mammalian cells; factor B; and the proclotting enzyme derived from insect cells) were prepared using a genetic engineering technique. Recombinant cascade reagents (RCRs) were then prepared to reconstruct the reaction cascade in the amebocyte lysate reagent. The protease activity of the RCR containing recombinant factor C was much greater than that of recombinant factor C alone, indicating the efficiency of signal amplification in the cascade. Compared with the RCR containing the insect cell-derived factor C, those containing mammalian cell-derived factor C, which features different glycosylation patterns, were less susceptible to interference by the injectable drug components. The standard curve of the RCR containing mammalian cell-derived recombinant factor C had a steeper slope than the curves for those containing natural lysate reagents, suggesting a greater sensitivity to endotoxin. The present study supports the future production of recombinant reagents that do not require the use of natural resources.
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U2 - 10.1177/1753425916681074
DO - 10.1177/1753425916681074
M3 - Article
C2 - 27913792
AN - SCOPUS:85011396142
VL - 23
SP - 136
EP - 146
JO - Innate Immunity
JF - Innate Immunity
SN - 1753-4259
IS - 2
ER -