Human brain pyridoxal-5'-phosphate phosphatase: production and characterization of monoclonal antibodies.
We cloned and expressed human pyridoxal-5\'-phosphate (PLP) phosphatase, the coenzymatically active form of vitamin B6, in Escherichia coli using pET15b vector. Monoclonal antibodies (mAb) were generated against purified human brain PLP phosphatase in mice, and four antibodies recognizing different epitopes were obtained, one of which inhibited PLP phosphatase. The binding affinities of these four mAbs to PLP phosphatase, as determined using biosensor technology, showed that they had similar binding affinities. Using the anti-PLP phosphatase antibodies as probes, we investigated their cross-reactivities in various mammalian and human tissues and cell lines. The immunoreactive bands obtained on Western blots had molecular masses of ca. 33 kDa. Similarly fractionated extracts of several mammalian cell lines all produced a single band of molecular mass 33 kDa. We believe that these PLP phosphatase mAbs could be used as valuable immunodiagnostic reagents for the detection, identification, and characterization of various neurological diseases related to vitamin B6 abnormalities.
Kim
Dae Won
DW
Department of Biomedical Science and Research Institute for Bioscience and Biotechnology, Hallym University, Chunchon 200-702, Korea.
Eum
Won Sik
WS
Choi
Hee Soon
HS
Kim
So Young
SY
An
Jae Jin
JJ
Lee
Sun Hwa
SH
Sohn
Eun Joung
EJ
Hwang
Seok-Il
SI
Kwon
Oh-Shin
OS
Kang
Tae-Cheon
TC
Won
Moo Ho
MH
Cho
Sung-Woo
SW
Lee
Kil Soo
KS
Park
Jinseu
J
Choi
Soo Young
SY
D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry*; D001671:Biochemistry / Q000379:methods; D015374:Biosensing Techniques; D001921:Brain / Q000201:enzymology*; D018076:DNA, Complementary / Q000378:metabolism; D018604:Epitope Mapping; D000939:Epitopes / Q000737:chemistry; D004926:Escherichia coli / Q000378:metabolism; D015723:Gene Library; D005822:Genetic Vectors; D006801:Humans; D007150:Immunohistochemistry / Q000379:methods; D010744:Phosphoric Monoester Hydrolases / Q000737:chemistry* / Q000378:metabolism*; D051381:Rats
D000911:Antibodies, Monoclonal; D018076:DNA, Complementary; D000939:Epitopes; C022493:pyridoxal-5-phosphatase; D010744:Phosphoric Monoester Hydrolases