Enzyme immunoassay using a rat prolactin-alkaline phosphatase recombinant tracer.
This paper describes a competitive enzyme immunoassay of rat prolactin (rPrl) using a recombinant conjugate as a colorimetric tracer. rPrl was inserted into the N-terminal end of Escherichia coli alkaline phosphatase (AP), using an expression vector which allows insertion of foreign DNA sequences between codons +6 and +7 of the phoA gene. The assay was performed in 96-well microtiter plates coated with a mouse monoclonal antibody raised against rabbit immunoglobulin G. Each component (recombinant tracer, rabbit antiserum against rPrl, and rPrl standard) was added in a volume of 50 microL. The sensitivity of the assay was sufficiently high to allow titration of rPrl in plasma. The detection threshold was 15 pg (0.3 ng/mL) and the B/B0 50% value was 150 pg (3 ng/mL). The intraassay coefficient of variation was less than 10% over a wide range of rPrl concentrations (2.9-50 ng/mL). The interassay coefficient of variation was less than 15% for rat plasma samples in the concentration range of 4-40 ng/mL. The good parallelism observed between the standard curve and sample dilution curves showed that the immunoreactivity in rat plasma behaves like standard rPrl. Together with recovery experiments, these results indicated that assay without extraction is possible. A single immunoreactive peak that comigrates with standard rPrl is observed after molecular sieve fractionation of plasma samples. The reliability of the assay was confirmed by good correlation with conventional radioimmunoassay (r = 0.996, slope 0.978).
Gillet
D
D
Département d'Ingénierie et d'Etude des Protéines, (DIEP), Commissariat à l'Energie Atomique, Gif/Yvette, France.
Ezan
E
E
Ducancel
F
F
Gaillard
C
C
Ardouin
T
T
Istin
M
M
Ménez
A
A
Boulain
J C
JC
Grognet
J M
JM
D000469:Alkaline Phosphatase / Q000737:chemistry; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D004247:DNA / Q000737:chemistry; D004926:Escherichia coli / Q000201:enzymology; D005260:Female; D007124:Immunoenzyme Techniques; D008297:Male; D008969:Molecular Sequence Data; D011388:Prolactin / Q000097:blood* / Q000737:chemistry / Q000235:genetics; D051381:Rats; D017207:Rats, Sprague-Dawley; D011994:Recombinant Proteins / Q000737:chemistry; D012680:Sensitivity and Specificity
D011994:Recombinant Proteins; D011388:Prolactin; D004247:DNA; D000469:Alkaline Phosphatase