Development and Characterization of Cholinephosphotransferase Antibody D. Chatterjee, S. Mukherjee, and S. K. Das 1 Department of Biochemistry, Meharry Medical College, 1005 D.B. Todd Boulevard, Nashville, Tennessee 37210 Received June 3, 2001 In the present study, we generated antibodies in rabbits against two synthetic peptides, one based on peptide sequence from yeast CPT cDNA (position 86 to 98 of the amino acid sequence) and the other from our guinea pig CPT cDNA (it corresponds to amino acid positions 119 to 130 according to yeast CPT gene). The antibody titers were measured by both dot blot anal- ysis and ELISA using Keyhole limpets hemocyanin coupled CPT peptides. The CPT antibody recognized a single band by Western blot analysis of proteins from guinea pig liver mitochondria and microsomes. The molecular weight of the protein recognized by West- ern blot analysis is close to the predicted molecular weight (46 kDa) of yeast CPT. Further analysis re- vealed that the antibody inhibited CPT activity in both subcellular fractions in a dose dependent man- ner, thus confirming the specificity of the antibody against both subcellular CPT. © 2001 Academic Press Key Words: cholinephosphotransferase; peptides; an- tibody; ELISA; Western blots; enzyme assay; immuno inhibition. Cholinephosphotransferase (CPT) is a key enzyme for production of lung surfactant phospholipids and maintenance of normal lung functions (1). The bio- chemical properties of CPT has not been well charac- terized because of the difficulties to purify the enzyme by conventional techniques. The major problem is the loss of enzyme activity during different steps of purifi- cation (2). Recently, the full sequence for two human CPT genes has been reported (3, 4). One of the clones (3) showed only 47% homology with yeast CPT gene, but the recombinant product of this clone had both CPT and EPT activities. We did not try to produce antibody from any sequence of this human CPT gene as the CPT gene is different from the CPT/EPT genes (2, 5). We had also characterized a partial CPT clone from guinea pig liver cDNA library (6). This clone has 96% homology with a partial sequence of yeast CPT (5, 7). In the present study, we have synthesized two pep- tides, one on the basis of the partial sequence of our guinea pig CPT clone (6) and the other based on yeast CPT gene sequence (5, 7). Our main objective is to produce and characterize a highly specific antibody against these two peptides and use these antibodies to identify CPT during its purification at different steps. MATERIALS AND METHODS Conjugation of the synthetic peptide. Synthetic peptides were obtained from PeptidoGenic Research & Co., Inc. (Liverman, CA). One of the CPT peptide (peptide “A”) sequence was obtained from yeast CPT gene sequence (position 86 to 98 of the amino acid se- quence) (5, 7) which is exposed well out side the membrane, i.e., the most hydrophobic surface antigen was chosen. The other peptide (peptide “B”) was synthesized on the basis of our partial guinea pig cDNA clone (6). This peptide had sequence homology to the yeast CPT (position 119 to 130 of the amino acid sequence of yeast gene) and it was close to the transmembrane domain, i.e., less hydrophobic. The synthetic peptide was coupled to BSA using maleimide acti- vated BSA carrier and the conjugated peptide was purified according to the instruction manual of PIERCE imject activated super carrier system kit. Immunization of the rabbits. The conjugated peptide (0.5 mg in 1 ml of Tris-buffered saline, pH 7.4) was mixed with 0.5 ml of alum adjuvant supplied in the PIERCE conjugation kit and emulsified thoroughly by passing through a 21-gauge needle. The rabbits were injected (subcutaneous, 0.7 ml of the antigen emulsion per rabbit) every week. After fourth injection, blood was taken and the serum was tested for the presence of antibody. Dot blot analysis. Keyhole limpets hemocyanin (KLH) conjugated peptides were coated on to nitrocellulose membrane (10 g/slot) and the membrane was blocked with 5% nonfat dry milk. After blocking, the membrane was incubated with immune rabbit serum followed by anti-rabbit IgG-peroxidase conjugate. The membrane was washed and then the color was developed using 4-chloro-1-naphthol as a substrate (Sigma Chemicals, St. Louis, MO). Preimmune serum was used as a control. ELISA assay for antibody titer. To determine the antibody titer, we carried out ELISA test using KLH conjugated peptide as an antigen. The antigen was coated onto 96-well ELISA plate (5 g/ well), blocked with 5% nonfat dry milk. Antisera or preimmune sera 1 To whom correspondence should be addressed at Fax: (615) 327 6442. E-mail: sdas@mmc.edu. Biochemical and Biophysical Research Communications 285, 965–968 (2001) doi:10.1006/bbrc.2001.5283, available online at http://www.idealibrary.com on 965 0006-291X/01 $35.00 Copyright © 2001 by Academic Press All rights of reproduction in any form reserved.