Transgenic (TG) silkworms that produce recombinant monoclonal antibodies
Recombinant antibodies synthesized by middle silk glands are secreted to the sericin layers of the silk threads.
The antibodies can be extracted from cocoons, and purified using a simple method.
Hybridomas producing antibodies
Light chain gene
Isolation of antibody genes
Heavy chain gene
Insertion into vector
Vector
Transduction into silkworms
Transgenic silkworms
Changeover of antibodies for ELISA kit:::: From mice to silkworms
Performance of silkworm-produced anti-amyloidββββ
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60
80
100
120
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60
80
100
120
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15
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25
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35
0 5 10 15 20 25 30 35
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0 5 10 15 20 25 30 35
Mouse-produced antibody Mouse-produced antibody
Silk
wor
m-p
rodu
ced
antib
ody
Silk
wor
m-p
rodu
ced
antib
ody
Serum Cerebral fluid
Correlation of reactivity between silkworm- and mouse-produced antibodies
High correlation was observed.
Additional merits・・・・Low production cost・・・・Production of high quality antibodies with lot-to-lot consistency
HAMA (Human Anti-Mouse Antibody)・Human antibody recognizes mouse antibody as an antigen
・Detection rate in human serum samples: A few percent to a few dozen percent
・Cause of false-positive reaction in two-site ELISA
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Issue of false-positive reaction caused by HAMA
Presence of antigen in serum sample
Presence of HAMA in serum sample
Antigen
Capture antibody
Capture antibody
HAMA
Addition ofdetection antibody
Positive
False positive
Centrifuge
Addition ofdetection antibody
Centrifuge
Inhibition of HAMA-reaction by using mouse-human chimeric antibody
Variable region from mouse antibody
Constant region from mouse antibody
Variable region from mouse antibody
Constant region from human antibody
Mouse antibody Mouse-human chimeric antibody
Decrease in reaction
React
HAMA
HAMAHAMA reacts to the constant region of mouse antibody.
HAMA-reaction is inhibited by using a mouse-human chimeric antibody in which the constant region of the mouse antibody is replaced by the constant region of the human antibody.
TG silkworm producing chimeric antibody
Mouse antibody gene
Human antibody gene
Chimeric antibody gene
Ligation of two genes
Transduction to silkworm
TG silkworm producing chimeric antibody
TG silkworm
Chimeric antibody
HAMA serum Normal serum HAMA serum Normal serum
×2 0.340 -0.005 0.012 -0.003
×4 -0.005 -0.008 0.002 0.000
×8 -0.006 0.000 0.012 0.005
×16 -0.004 -0.006 0.014 0.016
×32 -0.008 -0.005 0.008 0.003
×64 -0.004 -0.004 0.006 0.006
×128 0.495 -0.003 0.005 0.003
blank 0.007 0.003 0.006 0.000
Chimeric antibodyDeletion
Mouse antibody
0
0.05
0.1
0.15
0.2
0.25
0.3
0.35
Abs
orba
nce
at 4
50 n
m
Mouse antibody Chimeric antibody
HAMA serum (X2)
Normal serum (X2)
Evaluation of TG silkworm-produced chimeric antibody
Normal serum HAMA-containing serum
ELISA system was constructed by using two independent antibodies which each react to different antigens.
The chimeric antibody was used as a capture antibody.
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False positive
HAMA-reactivity decreased to 3% of that for mouse antibody.
Hybridoma culture
Mouse ascites
TG silkworm
Cost
High
Low
Low*
Relatively low
Low
High
Comparison to other antibody production system
No
Yes
No
Short
Short
Lot-to-lotconsistency
Ethical problemProduction period
Longat first production
*Production cost in TG silkworm is nearly comparable to the production of mice ascites.
Antibody production using TG silkworms- Solution for the production of antibodies for diagnostic use -
・・・・No ethical problems. Best alternative to mice ascites production.
・・・・High lot-to-lot consistency. High quality.・・・・Low cost when producing orders of more than a gram.
・・・・Low background. Inhibition of HAMA-reaction.