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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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

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0.05

0.1

0.15

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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.