• BMB Reports
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• 제30권3호
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• pp.177-181
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• 1997

We constructed a single-chain immunoglobulin in which the carboxyl end of the heavy chain variable domain is covalently joined to the amino terminus of the light chain variable domain via peptide linker and the carboxyl end of the light chain variable domain is linked to human ${\gamma}1$ Fc region through the hinge region. The molecule was expressed in Chinese hamster ovary cells, assembled into a dimeric molecule and secreted into the culture medium. The dimeric molecule (2E11) was purified from the culture supernatant by affinity chromatography on Protein G-Sepharose column. The size of the unreduced or reduced protein was the expected molecular weight of approximately 120 or 60 kDa, respectively, as assessed by SDS-polyacrylamide gel electrophoresis. The antigen-binding affinity of 2E11 was almost the same as that of a native antibody counterpart (CS131A), suggesting that the single-chain immunoglobulin may function like a native antibody.

• 미생물학회지
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• 제47권4호
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• pp.328-334
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• 2011

Leucine zipper motif는 여러 개의 주기적인 leucine 잔기로 구성되어 amphipathic alpha helix형태의 구조를 나타내며 소수성 결합에 의해 이량체를 형성한다. 이 leucine zipper motif를 single chain Fv 항체의 C-terminus에 도입하면 leucine zipper motif의 소수성 결합에 의해 amphipathic alpha helix의 이량체가 형성되면서 융합된 single chain Fv 항체의 이량체 (Dimer) 형성 또한 유도할 수 있다. 이량체 형태의 single chain Fv 항체는 2개의 항원 결합부위를 갖게 되므로 단량체 형태의(monomer) single chain Fv 항체에 비해 항원 결합력(Avidity)이 증가 될 것이다. 이 개념에 기초하여 이전 연구에서 제조된 단량체 형태인 닭 single chain Fv 항체인 8C3 ScFv 항체의 C-terminus에 leucine zipper motif를 도입하여 이량체 형태의 8C3 ScFv 항체를 개발하였다. 이량체 8C3 ScFv 항체는 가금류의 대표적인 기생충 질병인 coccidiosis를 유발하는 Eimerian sporozoite에 특이적으로 결합하는 기능을 나타내었다. 또한 이량체 8C3 ScFv 항체는 avidity 증가로 인하여 단량체에 비해 항원 결합력이 약 3배 증가됨을 확인할 수 있었으며 단백질 회수율 또한 2배 증가되는 부수적인 효과를 얻을 수 있었다.

• BMB Reports
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• 제40권6호
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• pp.875-880
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• 2007

Glucose oxidase (GOD) is an oxidoreductase catalyzing the reaction of glucose and oxygen to peroxide and gluconolacton (EC 1.1.3.4.). GOD is a widely used enzyme in biotechnology. Therefore the production of monoclonal antibodies and antibody fragments to GOD are of interest in bioanalytics and even tumor therapy. We describe here the generation of a panel of monoclonal antibodies to native and heat inactivated GOD. One of the hybridomas, E13BC8, was used for cloning of a single chain antibody(scFv). This scFv was expressed in Escherichia coli XL1-blue with the help of the vector system pOPE101. The scFv was isolated from the periplasmic fraction and detected by western blotting. It reacts specifically with soluble active GOD but does not recognize denatured GOD adsorbed to the solid phase. The same binding properties were also found for the monoclonal antibody E13BC8.

• Biotechnology and Bioprocess Engineering:BBE
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• 제4권1호
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• pp.72-77
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• 1999

An antibody containing a genetically engineered lipid group at the N-termunus and a hexahistidinyl tag at the C-terminus (Lpp-scF-His6) was immobilized in an oriented manner on the surface of liposome. Liposomes, consisting of antibody and phosphatidyl-choline, have been prepared and imaged by AFM. For AFM visualization, the resulting liposomes were bound on the surface of mica by two different mechanisms. The histidine tags present in the antibody molecules of the immonuliposome were anchored to the NiCl2 treated mica surface. Alternatively, the immunoliposomes were immunochemically bound on antigen-coated mica surface. Both approaches yielded liposomes which were clearly imaged without damage by AFM in ambient condition.

• Journal of Plant Biotechnology
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• 제8권1호
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• pp.9-14
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• 2006

As an alternative method to produce low cost reagents for immunodiagnosis and protect the plants from viral disease, a gene encoding a single chain variable fragment(scFv) recombinant antibody targeted to the coat protein of cucumber mosaic virus (CMV) was expressed in Nacotiana tabacum. The source of the scFv recombinant antibody gene was from spleen tissue of an immunized mouse. The gene was initially cloned into the pCANTAB5E phagemid and expressed in E. coli. In the following study, the antibody gene was subcloned into the plant expression vector, pCAMBIA-1301 and introduced into tobacco leaf tissue via Agrobacterium tumefacients mediated transformation. After transformation, 56 out of 58 plants were shown to carry the desired anti-CMV scFv gene by PCR analysis. Overall, only 12.5% of the 56 putative transgenic plants were found to express the antibody to a detectable level.

• Journal of Microbiology and Biotechnology
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• 제9권3호
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• pp.376-380
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• 1999

Constructs, encoding a single-chain variable fragment of a catalytic antibody 4f4f (scFv-4f4f) with glycosidase activity, were made by combining the coding sequences for the heavy and light chain variable domains with a sequence encoding a linker (GGGGS). Using three different plasmid systems, single-chain antibodies were expressed separately in Escherichia coli, demonstrating significant differences in the expression level and amounts in soluble form of the recombinant protein. The protein expression from pET3a-scFv-4f4f was up to 20% of the total soluble proteins and, more importantly, the proteins were mostly found in a soluble form. An SDS-PAGE analysis of the purified single-chain proteins, yielding higher than 5mg from a 1-1 culture, showed a single band corresponding to its molecular weight of 29,100. A preliminary study shows that the expressed scFv-4f4f is catalytically active. The catalytic parameters for the hydrolysis of p-nitrophenyl-$\beta$-D-glucopyranoside by scFv-4f4f are being investigated.

• 대한의생명과학회지
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• 제9권3호
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• pp.111-121
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• 2003

Recombinant single chain Fv (scFv) antibodies offer many advantages over mouse monoclonal antibodies such as faster clearance from blood, improved tumor localization, reduced human anti-mouse antibody (HAMA) response, and the availability to manipulate the scFv through genetic approaches. The recombinant phage display was constructed using lym-l hybridoma cells as a source of genetic starting material. mRNA was isolated from the corresponding antibodies hybridoma cells. VH and VL cDNA were amplified with RT-PCR and linked with ScFv by linker DNA to form ScFv DNA, which then were inserted into phagemid pCANTAB5E. The phage of positive clones selected with tube containing raji lymphoma cell and infected by competent E. coli HB2151 to express soluble scFv. The scFv lym-l was secreted into the cytosol and culture supernatant and shown to be of expected size (approximately 32 kDa) by western blot. An active scFv lym-l could be produced in E. coli with soluble form and high yield from hybridoma cell line, using phage display system. Immunoreactivity indicated that scFv lym1 showed a potential biding affinity against the raji lymphoma cell as its parental antibody (intact lym-l Ab).

• 대한방사성의약품학회지
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• 제6권1호
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• pp.39-45
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• 2020

Recently, single-domain antibodies (sdAb) are bioengineered for molecular imaging applications. Single-domain antibody, obtained from naturally occurring antibodies in camelid species and cartilaginous fish is the smallest fully functional antigen-binding antibody fragments of heavy-chain. Since their discovery, they have been investigated extensively in clinical therapeutics, monitoring and diagnostics. Their small size is important advantage for high solubility, high stability, fast blood clearance and rapid targeting. This review article summarizes the recent status of this new antibody to visualize, diagnose or inhibit specific targets of cancer.

• 한국가금학회지
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• 제38권4호
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• pp.323-330
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• 2011

닭의 단일 클론 항체인 13C8 항체는 조류의 콕시듐증을 유발하는 것으로 알려진 Eimeria acervulina의 포자충(sporozoites) 항원에 결합하는 닭 항체이다. 그러나 닭 하이브리도마 유전자의 불안정성 때문에 분비되는 항체의 생산량이 낮은 단점이 있다. 이러한 단점을 보완하기 위해 hybridoma로 부터 항체의 중사슬 가변 부위(VH)유전자와 경사슬 가변 부위(VL) 유전자를 증폭하여 linker peptide로 연결해준 재조합 ScFv 항체 유전자를 구축하고, E. coli를 형질 전환시켜 재조합 단백질로 발현 정제하였다. ELISA 분석 결과 재조합 13C8 ScFv 항체는 세 종류의 Eimeria spp.에 대해 모두 항원 결합력을 나타내었으며, 염기서열 분석을 수행하여 germline sequence와 비교한 결과 닭 항체유전자의 다양성(diversity)은 pseudogene들의 gene conversion 기작에 의해 이루어짐을 제시해 주었다.

• 생명과학회지
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• 제30권11호
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• pp.1012-1020
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• 2020

생쥐의 가변부위가 인간의 정상부위에 연결되어 제조된 바이오시밀러 자연항체치료제인 레미케이드는 암괴사인자-알파(TNF-α)에 특이적인 항체로써 카이메릭 단일클론항체이며 류마티스 관절염치료를 위해 개발되었다. 바이오시밀러 레미케이드 항체의 생물학적 기능을 연구하기 위해 우리는 단백질 데이터 은행을 이용한 생물정보학 분석을 수행하여 레미케이드 자연항체와 암괴사인자-알파 항원간의 결합기작특징을 분석하였다. 자연항체를 생산하는 세포의 유전적 불안정성 때문에 레미케이드 항체생산이 제한되므로 우리는 중 사슬 가변부위를 다중펩타이드 링커에 의해 경 사슬 가변부위에 연결된 레미케이드 ScFv항체(Remicade)를 제조하였다. 더욱이 더 높은 생산과 더 높은 항원결합력을 위해 레미케이드 ScFv를 leucine zipper에 융합시켰다. Remicade와 RemicadeScZip ScFv는 대장균에서 발현되었고 Ni⁺-NTA-아가로스 컬럼으로 정제하였다. 정제된 단백질들은 예상한대로 sodium dodecyl sulfate-polyacrylamide electrophoresis에서 28.80 kDa과 33.96 kDa을 나타내었다. Remicade는 ELISA, western blot에서 TNF-α 항원에 대한 결합력이 관찰되지 않았으나 RemicadeScZip은 항원결합력을 나타내었다. 추가적인 BLI분석으로 RemicadeScZip의 TNF-α 항원에 대한 결합력을 재확인시켜주었으며 이 결과는 Leucine zipper가 레미케이드 ScFv의 접힘을 안정화시키고 TNF-α 항원에 대한 결합력을 개선시켰음을 제시해주고 있다.