Prolactin ELISA Kit

Catalog No. ABIN2815082

Product Details Prolactin ELISA Kit

Target: Prolactin (PRL)

Reactivity: Human, Elephant (Elephantulus), Seal

Detection Method: Colorimetric

Method Type: Sandwich ELISA

Application: ELISA

Purpose: The DetectX® Prolactin kit is designed to quantitatively measure Prolactin present in biological samples and tissue culture media.

Brand: DetectX®

Sample Type: Serum, Plasma, Tissue Culture Medium

Analytical Method: Quantitative

Cross-Reactivity (Details): This kit was developed to measure prolactin in elephant serum and has been tested in both African and Asian elephant sera.

Predicted Reactivity: Cat (Feline)

Components: Clear Coated 96 Well Plate One Plate Clear plastic microplate with break-apart strips coated with mouse anti-Prolactin.
Prolactin Standard 2 Vials Two vials of recombinant human Prolactin at 500 pg. Store at -20°C.
DetectX® Prolactin Conjugate 5 mL A polyclonal antibody to Prolactin labeled with peroxidase. Store at -20°C.
Assay Buffer Concentrate 28 mL A 5X concentrate that should be diluted with deionized or distilled water.
Wash Buffer Concentrate 30 mL A 20X concentrate that should be diluted with deionized or distilled water.
TMB Substrate 11 mL
Stop Solution 5 mL A 1N hydrochloric acid solution. Caustic.
Plate Sealer 2 each

Material not included: Distilled or deionized water.
Polypropylene or glass test tubes.
Repeater pipet and disposable tips capable of dispensing 100 and 50 μL.
A microplate washer.
Colorimetric 96 well microplate reader capable of reading optical density at 450 nm.
Software for converting raw relative optical density readings from the plate reader and carrying out four parameter logistic curve (4PLC) fitting.
Contact your plate reader manufacturer for de- tails.

Application Details

Application Notes: This assay has been validated for human serum, plasma, and tissue culture media (TCM) samples only.
Samples containing visible particulate should be centrifuged prior to using.
This assay detects human, seal and elephant (Loxodonta africana and Elephas maximus) Pro- lactins.
Felids are reported NOT to be detected with this assay.
Other species have not been evaluated in this kit.

Plate: Pre-coated

Protocol: The kit has been tested to measure human and elephant serum prolactin.
Other species have not been measured.
Please read the complete kit insert before performing this assay.
A human Prolactin standard is provided to generate a standard curve for the assay and all samples should be read off the standard curve.
Standards or diluted samples are pipetted into a clear microtiter plate coated with a monoclonal antibody to capture the Prolactin present.
After a 60 minute incubation, the plate is washed and a peroxidase conjugated Prolactin polyclonal antibody is added.
The plate is again incubated for 60 minutes and washed.
Substrate is then added to the plate, which reacts with the bound Prolactin Antibody Conjugate.
After a third incubation, the reaction is stopped and the intensity of the generated color is detected in a microtiter plate reader capable of measuring 450 nm wavelength.
The concentration of the Prolactin in the sample is calculated, after making suitable correction for dilution, using software available with most plate readers.

Reagent Preparation:

Allow the kit reagents to come to room temperature for 30 minutes.
We recommend that all standards and samples be run in duplicate to allow the end user to accurately determine Prolactin concentrations.
Ensure that all samples have reached room temperature and have been diluted as appropriate prior to running them in the kit.
Assay Buffer Dilute Assay Buffer Concentrate 1:5 by adding one part of the concentrate to four parts of deion- ized water.
Once diluted this is stable at 4 °C for 3 months.
Wash Buffer Dilute Wash Buffer Concentrate 1:20 by adding one part of the concentrate to nineteen parts of deionized water.
Once diluted this is stable at room temperature for 3 months.
Standard Preparation Add 500 μL of Assay Buffer to one vial of Prolactin standard to generate the 1,000 pg/mL Stan- dard 1 and mix well.
Label six test tubes as #2 through #7.
Pipet 150 μL of Assay Buffer into tubes #2 to #7.
Add 150 μL of the 1,000 pg/mL Prolactin standard to tube #2 and vortex completely.
Take 150 μL of the Prolactin solution in tube #2 and add it to tube #3 and vortex completely.
Re- peat the serial dilutions for tubes #4 through #7.
The concentration of Prolactin in the vial and tubes #2 through #7 will be 1,000, 500, 250, 125, 62.5, 31.25 and 15.625 pg/mL.
Use all Standards within 2 hours of preparation.

Sample Preparation:

Serum and plasma samples must be diluted ≥ 1:4 with the provided Assay Buffer prior to running in the kit. Most normal human serum or plasma samples will require a dilution of ≥ 1:10 to allow detection within the standard curve and samples from pregnant humans may have to be diluted up to 1:100. Human. seal and elephant serum samples have been run successfully with this kit. Serum samples from felids have been reported not to work with this kit (see note below). The kit uses a human Prolactin standard and values expressed for other non-human species will be expressed as human Prolactin pg/mL. TCM samples should be diluted in TCM and read off a standard curve generated in the same TCM or diluted ≥ 1:20 in Assay Buffer and read off a standard curve generated in Assay Buffer. Any samples with Prolactin concentrations outside the standard curve range should be diluted further with Assay Buffer or TCM, as appropriate, to obtain readings within the standard curve range. It is up to the end user to determine the appropriate dilution for their samples. Use all samples within 2 hours of dilution.

Assay Procedure:

1. Use the plate layout sheet on the back page of the insert to aid in proper sample and standard identification. Determine the number of wells to be used and return unused wells to foil pouch with desiccant. Seal the ziploc plate bag and store at 4ºC.
   2. Pipet 50 μL of samples or standards into wells in the plate. Pipet 50 μL of Assay Buffer into the zero standard wells.
   3. Cover the plate with the plate sealer and shake at room temperature for 60 minutes.
   4. Aspirate the plate and wash each well 4 times with 300 μL wash buffer. Tap the plate dry on clean absorbent towels.
   5. Add 50 μL of the DetectX® Prolactin Conjugate to each well, using a repeater pipet.
   6. Cover the plate with the plate sealer and shake at room temperature for 60 minutes.
   7. Aspirate the plate and wash each well 4 times with 300 μL wash buffer. Tap the plate dry on clean absorbent towels.
   8. Add 100 μL of the TMB Substrate to each well, using a repeater pipet.
   9. Incubate the plate at room temperature for 30 minutes without shaking. 10. Add 50 μL of the Stop Solution to each well, using a repeater pipet. 11. Read the optical density generated from each well at 450 nm. 12. Use the plate reader's built-in 4PLC software capabilities to calculate Prolactin concentration for each sample.

Calculation of Results:

Average the duplicate OD readings for each standard and sample.
Create a standard curve by reducing the data using computer software capable of generating a four-parameter logistic curve (4PLC) fit.
The sample concentrations should be multiplied by the dilution factor to obtain neat sample values.
Or use the online tool from http://www.myassays.com/arbor-assays-prolactin-detection-kit.assay to calculate the data. *The MyAssays logo is a registered trademark of MyAssays Ltd. tyPical data Sample Mean OD Prolactin Conc. (pg/mL) Standard 1 3.096 1,000 Standard 2 1.696 500 Standard 3 0.953 250 Standard 4 0.591 125 Standard 5 0.409 62.5 Standard 6 0.29 31.25 Standard 6 0.254 15.625 Zero 0.215 0 Sample 1 2.703 856.8 Sample 2 1.04 277.0 Always run your own standard curve for calculation of results.
Do not use this data. 1 ng of human Prolactin is equivalent to 42.94 μunits of the WHO 3rd International Standard (coded 84/500)

Assay Precision: Three human samples were diluted with Assay Buffer and run in replicates of 20 in an assay.
Inter Assay Precision:
Three human samples were diluted with Assay Buffer and run in duplicates in fourteen assays run over multiple days by three operators.

Restrictions: For Research Use only

Handling

Precaution of Use: As with all such products, this kit should only be used by qualified personnel who have had labo- ratory safety instruction.
The complete insert should be read and understood before attempting to use the product.
The antibody coated plate needs to be stored desiccated.
The silica gel pack included in the foil ziploc bag will keep the plate dry.
The silica gel pack will turn from blue to pink if the ziploc has not been closed properly.
This kit utilizes a peroxidase-based readout system.
Buffers, including other manufacturers Wash Buffers, containing sodium azide will inhibit color production from the enzyme.
Make sure all buffers used for samples are azide free.
Ensure that any plate washing system is rinsed well with deionized water prior to using the supplied Wash Buffer as prepared on Page 8.
The Stop Solution is acid.
The solution should not come in contact with skin or eyes.
Take appro- priate precautions when handling this reagent.

Storage: -20 °C,4 °C

Storage Comment: Store kit at -20°C until first thaw. All components of this kit, except the Prolactin standard and Conjugate, should then be stored at 4 °C until the expiration date of the kit. The Prolactin standard and Conjugate MUST be stored at -20°C.

Target Details for Prolactin

Target: Prolactin (PRL)

Alternative Name: Prolactin (PRL Products)

Synonyms: PRL ELISA Kit, prolactin ELISA Kit, PRLB ELISA Kit, PRLSD1 ELISA Kit, Prl1a1 ELISA Kit, Prol ELISA Kit, RATPRLSD1 ELISA Kit, RNPROL ELISA Kit, AV290867 ELISA Kit, prl ELISA Kit, prolactin ELISA Kit, prolactin, gene 2 L homeolog ELISA Kit, PROLACTIN protein ELISA Kit, prolactin-like ELISA Kit, PRL ELISA Kit, prl.2.L ELISA Kit, Prl ELISA Kit, LOC100136792 ELISA Kit, LOC100136580 ELISA Kit, PROLACTIN ELISA Kit, LOC101843376 ELISA Kit

Background: Prolactin (PRL) is a polypeptide hormone that is synthesized and secreted from specialized cells of the anterior pituitary gland. The hormone was given its name based on the fact that an extract of bovine pituitary gland would cause growth of the crop sac and stimulate the production of milk in pigeons or promote lactation in rabbits (1). However it is now appreciated that prolactin has over 300 separate biological activities (2). Prolactin has multiple roles in reproduction other than lactation, and it also plays multiple homeostatic roles in the organism. Furthermore, the synthesis and secretion of prolactin is not restricted to the anterior pituitary gland, but multiple other or- gans and tissues in the body have this capability. The prolactin gene is composed of 5 exons and 4 introns (3, 4). Transcription of the prolactin gene is regulated by two independent promoter regions. The human prolactin cDNA is 914 nucleotides long and generates the prolactin prohormone of 227 amino acids. After cleavage of the 28 amino acid signal peptide, mature human prolactin is composed of 199 amino acids (5). The prolactin molecule is arranged in a single chain of amino acids with three intramolecular disulfide bonds between six cysteine residues (Cys4-Cys11, Cys58-Cys174, and Cys191-Cys199 in humans) (3). The se- quence homology can vary from up to 97 % among primates to as low as 56 % between primates and rodents (5). In rats (6) and mice (7), pituitary prolactin consists of 197 amino acids, whereas in sheep, pigs, cattle, and humans (8) it consists of 199 amino acids with a molecular weight of about 23,000 Da. Prolactin is synthesized by the anterior pituitary, placenta, brain, uterus, dermal fibroblasts, de- cidua, B cell, T cells, NK cells, and breast cancer cells. Originally characterized as a lactogenic hormone, studies have demonstrated broader roles in breast cancer development, regulation of reproductive function, and immuno-regulation. Various cells have been shown to express the pro- lactin receptor. Three forms of the receptor, generated by differential splicing, have been identi- fied. These isoforms differ in the length of their cytoplasmic domains. It is believed that the short cytoplasmic form is non functional. Prolactin signal transduction involves the JAK/STAT families and Src kinase family

Pathways: JAK-STAT Signaling, Peptide Hormone Metabolism, Response to Growth Hormone Stimulus, Protein targeting to Nucleus