Production of a polyclonal antibody against recombinant goldfish prolactin and demonstration of its usefulness in a non-competitive antigen-capture ELISA.
The cDNA encoding the goldfish (Carassius auratus) prolactin was expressed in Escherichia coli using the pRSETA expression vector. The recombinant goldfish prolactin (gfPRL) produced was a fusion protein containing a hexahistidyl sequence, which facilitated its purification on a Ni(2+) column. The fusion protein was overexpressed in the bacteria as inclusion bodies and was successfully purified under denaturing conditions by one-step affinity chromatography. Repeated immunization of rabbits against the purified recombinant gfPRL allowed the production of a high-titer polyclonal antiserum. The IgG fraction of the antiserum was isolated on an immobilized Protein A-agarose column. The antibody recognized recombinant gfPRL, but not recombinant goldfish growth hormone (gfGH) or goldfish somatolactin (gfSL) on Western analyses. The purified antibody was able to recognize gfPRL, but not gfGH or gfSL, in a non-competitive antigen-capture ELISA. The assay was applied in monitoring the purification of native PRL from goldfish pituitaries.
Cheung
Hilda Y S
HY
Department of Biochemistry, The Chinese University of Hong Kong, Shatin, N.T., Hong Kong, PR China.
Chan
K M
KM
Ng
T B
TB
Cheng
Christopher H K
CH
D000818:Animals; D015153:Blotting, Western; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D003429:Cross Reactions; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D004926:Escherichia coli; D029941:Fish Proteins / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification; D006054:Goldfish / Q000276:immunology*; D007106:Immune Sera / Q000096:biosynthesis* / Q000276:immunology*; D008970:Molecular Weight; D010902:Pituitary Gland / Q000378:metabolism; D011388:Prolactin / Q000235:genetics / Q000276:immunology* / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000276:immunology / Q000302:isolation & purification
D029941:Fish Proteins; D007106:Immune Sera; D011994:Recombinant Proteins; D011388:Prolactin