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QB124

Introduction

◆ Lateral-flow immunoassay(LFIA)

・Denaturation and desorption of antibody

・Animal ethics concerns

Lactoferrin 76 kDa

◆ This Study

Expression

by CHO cells

Expression

by E. coli

VH

VL

scFv

Expression

by E. coli

◆ Previous Studies

objective

Development of high sensitive Lateral-flow immunoassay based on

novel Immobilization method of scFv by using PpL1-fused Lactoferrin

Materials and Methods

Results

・Molecular weight analysis by Gel Filtration

Retention Volume [mL]

MYO

BSA

CAB

IgG

monomer

(50 kDa)

① Denatured Protein(reduced)

② Refolded Protein(Non-reduced)

③ Refolded Protein(reduced)　

MW ① ② ③

PpL1-LF

PpL1-LFC can be efficiently recovered from intracellular insoluble fraction

・Refolding of PpL1-fused Lactoferrins

Denatured Protein

Folded Protein

Conclusions

Subjects in conventional LFIA

LFN

333 a.a.

pI 9.22

LFC

357 a.a.

pI 6.91

PpL1 can bind to V_k domain of antibody from

specific species such as human, mouse and so on.

Column：His trap FF Crude

(C.V.：5 mL)

Overnight Express medium 50 mL

(Amp 50 μg/mL, Cam 34 μg/mL)

30 ℃, 200 rpm, 24 hours

pET22b(+)

Vector

Transformation

Culture

Centrifugation

Cell lysis

Solubilization buffer

(8 M Urea-PBS)

Purification

Urea

・Binding Buffer : 8M Urea, 20 mM Imidazole,

2×PBS, pH 8.0

​

・Elution Buffer : 8M Urea, 250 mM Imidazole,

2×PBS, pH 8.0

After purification

Denatured Protein

Dialysis membrane

Refolding

Urea

Dialysis Buffer

1×PBS, pH 8.0

Folded Protein

・High production cost by CHO cell culture

・Require large amount of antibody

Disadvantage

Advantage

・Increase remaining activity after immobilization

・Improvement of orientation of antibodies

・PpL1-fused LFs as scaffold to capture

the rabbit scFvs

・Low-cost production for all components

by E. coli

・Retention of activity after immobilization

Advantage of this study

Column: Superdex G-200 Increase

(C.V.：23 mL)

​

Running Buffer：1×PBS, pH 8.0

​

Flow Rate：0.5 mL/min

Refolded PpL1-LFC was detected at the retention volume which corresponded

10 times higher molecular weight expected

10-mer

(Approx. 510 kDa)

・IgG-binding activity analysis PpL1-LFC by competitive ELISA

Maxisorp

Bt-Human IgG

PpL1-LFC

Refolded PpL1-LFC showed greater IgG-binding activity

・Retention of PpL1-LFC on Nitrocellulose membrane

CBB

staining

washing with

TBS+0.1 % Tween20

Quantification by Image J

R =

P_TBST

P_TBS

P_TBST：Protein amount after washing with TBST

×100

P_TBS ：Protein amount after washing with TBS

R ：Adsorption yield(%)

PpL1-LFC on NC membrane retained at the highest level among proteins tested

・PpL1-LFC was successfully overexpressed and recovered from intracellular insoluble fraction.

・PpL1-LFC efficiently adsorbed and retained on the surface of NC membrane even after

washing with TBST.

・PpL1-LFC formed oligomer after refolding operation, and showed high antibody-binding activity.

・Sensitive antigen detection in LFIA was attained by use of PpL1-LFC-immobilized membrane

and small amount of ^rscFv.

・Antigen detection using rabbit anti-CRP scFv and PpL1-LFC-immobilized

NC membrane

mAb

^rscFv

+

PpL1-LFC

Antigen can be detected with comparable signal intensities even using small amounts of ^rscFv

mAb

^rscFv

+

PpL1-LFC

Development of a New Lateral-flow Immunoassay using

PpL1-fused Lactoferrin and Rabbit scFv

(Kyoto Inst. Tech.) ○Yodai Yamamoto・Jun-ichi Horiuchi・Yoichi Kumada *

KSBB-AFOB Conference 2025

PSP24