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The Quantitative Determination of Prolactin Hormone Concentration in Human Serum by a Microplate Immunoenzymometric assay. Measurements obtained by this device are used in the diagnosis and treatment of disorders of the anterior pituitary gland or the hypothalamus portion of the brain.

Monobind Inc., registration number 2020726, plans to introduce into commercial distribution an enzymelmmunoassay (IEMA) kit for the determination of prolactin (PRL) in human serum. The proprietary name is Prolactin (PRL) ELISA and the usual name is PRL IEMA. This device classification name is - prolactin test system - product code CFT (per 21 CRF section 862.1625). The Monobind ELISA method is based on two-site immunoassay (sandwich) technology utilizing the streptavidin-blotin reaction to effect separation. Upon mixing monoclonal blotinylated anti-PRL antibody, the enzyme-labeled anti-PRL antibody and a serum containing the native antigen (PRL), reaction results between the native antigen (PRL) and the antibodies, without competition or steric hindrance, to form a soluble sandwich complex. Simultaneously, the complex is deposited to the well through the high affinity reaction of streptavidin and biotinylated antibody. After incubation is complete, decantation or aspiration separates the bound fraction. The enzyme activity on the well is directly proportional to the native antigen (PRL) concentration. By utilizing several different serum references of known antigen values, a dose response curve can be generated from which the antigen concentration of an unknown can be ascertained.

The AquaLite® Prolactin Bioluminescent Immunoassay (BIA) Kit (or the AquaLite® Prolactin assay) is intended to be used in clinical laboratories for the quantitative determination of human prolactin levels in sera and plasma. The AquaLite® Prolactin assay is for in vitro diagnostic use.

The AquaLite® Prolactin Bioluminescent Immunoassay Kit uses a mixture of mouse monoclonal and rabbit polyclonal with anti-prolactin activity that is pre-coated onto polystyrene tubes (solid phase). Samples (serum or plasma), or appropriate calibrators or controls, are pipetted (25 uL) into the pre-coated tubes. Anti-prolactin conjugate consisting of mouse monoclonal antibody covalently linked to AquaLite® (150µL) is then added to the tubes. The conjugate uses the photoprotein, AquaLite® (recombinant aequorin: U.S. Patent Nos. 5,422,266 and 5,486,455) which is covalently linked to an anti-prolactin polyclonal antibody. Prolactin in the sample simultaneously combines with polyclonal antibody on the solid phase and conjugate antibody to form an immune complex or "sandwich" bound to the solid phase. Complex formation is complete after a 60-minute incubation period at room temperature (18° to 25°C) on a standard orbital shaker. The tubes are then washed to remove unbound conjugate. The washed tubes are placed in a luminometer that is capable of reading a triggered. flash-type reaction in 12 x 75 mm tubes. An injected calcium trigger solution causes AquaLite® to oxide its self-contained luciferin molecule. This reaction produces a twosecond flash of light at 469 nm, which is measured by the luminometer. The intensity of the light is directly proportional to the concentration of the prolactin in the sample. To calculate results, the luminometer uses a cubic spline curve fit applied to a logit-log transformation of the light intensity (in relative light units, RLU) of the prolactin calibrators versus prolactin concentration (in ng/mL). Note: Samples that generate signals greater than the signal from the highest calibrator are off-scale. These samples must be diluted and re-assayed. Remember to multiply the results from diluted samples by the dilution factor used.

Immunoassay for the in vitro quantitative determination of human prolactin in human serum and plasma.

Sandwich principle. Total duration of assay: 18 minutes (37 °C). • 1st incubation (9 min.): 10µL of sample, a biotinylated monoclonal prolactin- specific antibody (75 µL), and a monoclonal prolactin-specific antibody labeled with a ruthenium complex (75 µL)** react to form a sandwich complex. •2nd incubation (9 min.): after addition of streptavidin-coated microparticles (40 µL), the complex becomes bound to the solid phase via interaction of biotin and streptavidin. **Tris(2,2'-bipyridyl)ruthenium(II) complex (Ru(bpy)2+3) •The reaction mixture is aspirated into the measuring cell where the microparticles are magnetically captured onto the surface of the electrode. Unbound substances are then removed with ProCell. Application of a voltage to the electrode then induces chemiluminescent emission which is measured by a photomultiplier (0.4 second read frame). •Results are determined via a calibration curve which is instrument specifically generated by 2-point calibration and a master curve provided via the reagent bar code.