[Preparation of recombinant serpins B3 and B4 and investigation of their specific interactions with antibodies using hydrogel-based microarrays].
The objective of this work was to obtain preparations of recombinant squamous-cell carcinoma antigens (serpins B3 and B4) and to investigate their interactions with different monoclonal antibodies using hydrogel-based microarrays (biochips). Two genetic constructs encoding full-length serpin B3 and serpin B4 molecules were created to produce recombinant SPB3 and SPB4 proteins carrying a N-terminal His6-tag. Monoclonal antibodies against serpin B3 (H3, C5, H5, H81, and G9) were also obtained. An experimental gel-based biological microchip was designed to contain gel elements that carry immobilized antibodies against SPB3, immobilized commercial monoclonal SCC107 and SCC140 antibodies against squamous-cell carcinoma antigen (SCCA), and gel elements with immobilized SPB3 or SPB4. Judging by the specificity of recombinant SPB3 and SPB4, which bind to monoclonal antibodies against SCCA and, according to the manufacturer's data, can recognize conformational epitopes of both SPB3 and SPB4, it was concluded that the obtained recombinant serpins had the correct tertiary structure. A biochip-based direct immunoassay showed that SPB4 could bind effectively only to SCC107 and SCC140 antibodies, while SPB3 interacted specifically not only with these antibodies, but also with H3 and C5 monoclonal antibodies. Using biochip-based sandwich immunoassay, a pair of monoclonal antibodies SCC107/C5 that interacted specifically with serpin B3 but did not interact with serpin B4 was identified. Thus, it has been demonstrated that serpin B3 can be selectively determined in the presence of highly homologous serpin B4 using a biochip-based assay.
Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Moscow, 119991, Russia.
V I
VI
Butvilovskaya
Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Moscow, 119991, Russia.
M V
MV
Tsybulskaya
Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Moscow, 119991, Russia.|Higher Chemical College of the Russian Academy of Sciences, Mendeleyev University of Chemical Technology, Moscow, 125047, Russia.
A A
AA
Tikhonov
Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Moscow, 119991, Russia.
V O
VO
Talibov
Department of Biology, Lomonosov Moscow State University, Moscow, 119234, Russia.
P V
PV
Belousov
Department of Biology, Lomonosov Moscow State University, Moscow, 119234, Russia.
A Yu
AY
Sazykin
Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Moscow, 119991, Russia.
A M
AM
Schwartz
Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Moscow, 119991, Russia.
L V
LV
Putlyaeva
Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Moscow, 119991, Russia.
S A
SA
Surzhikov
Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Moscow, 119991, Russia.
A A
AA
Stomakhin
Russian Research Center for Molecular Diagnostics and Therapy, Moscow, 117638, Russia.
O N
ON
Solopova
Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Moscow, 119991, Russia.|arubina@biochip.ru.
A Yu
AY
Rubina
D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis / Q000737:chemistry* / Q000276:immunology; D000951:Antigens, Neoplasm / Q000737:chemistry* / Q000235:genetics / Q000276:immunology; D002294:Carcinoma, Squamous Cell / Q000737:chemistry; D003001:Cloning, Molecular; D000939:Epitopes / Q000737:chemistry* / Q000235:genetics / Q000276:immunology; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D006801:Humans; D020100:Hydrogels / Q000737:chemistry*; D056656:Lab-On-A-Chip Devices; D051379:Mice; D008807:Mice, Inbred BALB C; D046228:Microarray Analysis; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D015843:Serpins / Q000737:chemistry* / Q000235:genetics / Q000276:immunology
D000911:Antibodies, Monoclonal; D000951:Antigens, Neoplasm; D000939:Epitopes; D020100:Hydrogels; D011993:Recombinant Fusion Proteins; D015843:Serpins; C051072:squamous cell carcinoma-related antigen
biological microchip; immunoassay; serpin B3; serpin B4; squamous-cell carcinoma antigen (SCCA)