[Production of a monoclonal antibody against Cry j 2].
The peptide possessing N-terminal 10 amino acid sequence of Cry j 2 was chemically synthesized and conjugated to KLH-carrier protein. Rabbits were immunized with this haptenized protein and the antibody (N-10 antibody) was obtained. Cross-blot analysis revealed that N-10 antibody recognized the hapten moiety. Purification of Cry j 2 was carried out with monitoring its reactivity with N-10 antibody. The substance reactive to N-10 antibody existed in the DEAE-Sephadex unadsorbed fraction. The substance reactive to N-10 antibody was subjected to CM-Sephadex equilibrated with 10 mM acetate buffer (pH 5.0). In contrast to the previous report on Cry j 2, the substance reactive to N-10 antibody existed in the CM-Sephadex unadsorbed fraction. The CM-Sephadex unadsorbed fraction was concentrated with 80% saturated ammonium sulfate, and applied to a Superdex pg 200 column. The major peak of protein was regarded as the final preparation and was subjected to SDS-PAGE. The substance reactive to N-10 antibody had a MW of 40 kDa under reducing and 37 kDa under non-reducing conditions, respectively. Furthermore, the substance reactive to N-10 antibody showed potent allergenic activity. These data completely agreed with the previous data on Cry j 2 and strongly suggested that the final preparation contained a large amount of Cry j 2. BALB/c mice were immunized intraperitoneally with the final preparation to produce a monoclonal antibody against Cry j 2. Eleven clones reacting to the final preparation were obtained, and the antibodies produced from these clones did not react with purified Cry j 1.(ABSTRACT TRUNCATED AT 250 WORDS)
Homma
R
R
Section of Allergology, Torii Pharmaceutical Co., Ltd.
Uesato
N
N
Miyahara
A
A
Kimura
H
H
Hantani
Y
Y
Ito
G
G
Hayashibe
M
M
Ando
T
T
Ino
Y
Y
Yamakawa
H
H
D000485:Allergens / Q000276:immunology*; D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis*; D051379:Mice; D008807:Mice, Inbred BALB C; D010446:Peptide Fragments / Q000276:immunology; D010940:Plant Proteins / Q000276:immunology*; D011058:Pollen / Q000276:immunology*; D011817:Rabbits; D014197:Trees
D000485:Allergens; D000911:Antibodies, Monoclonal; C065291:Cry j II protein, Cryptomeria japonica; D010446:Peptide Fragments; D010940:Plant Proteins