Mouse Prolactin ELISA

Transcription

1 T! C U O D PR ED W IT H Mouse Prolactin ELISA AS E R EF ER TO IN SE R T IN C LU D Catalog Number M R M AT IO N AL U SE O N LY!P LE For the quantitative determination of prolactin in mouse. IN FO For research use only. This product insert must be read in its entirety before using this product.

2 PRINCIPLE OF THE ASSAY This Enzyme Immunoassay (EIA) is based on the competition between unlabelled mouse prolactin and acetylcholinesterase (AChE) linked to mouse prolactin (tracer) for limited specific rabbit anti-mouse prolactin antiserum sites. The complex rabbit antiserum-mouse prolactin (free prolactin or tracer) binds to the mouse monoclonal antirabbit antibody that is attached to the well. The plate is washed to eliminate the unbound tracer and Ellman s reagent (enzymatic substrate for AChE and chromogen) is added to the wells. The AChE tracer acts on the Ellman s reagent to form a yellow compound. The intensity of the color is determined by spectrophotometry and is inversely proportional to the amount of free mouse prolactin present in the well during the immunological incubation. IN30341.R01 products@mdbiosciences.com page 2

3 KIT COMPONENTS Microtiter Plate - The plate contains 12 x 8-well strips coated with mouse anti-rabbit IgG. Ready for use. Standard - 2 vials (250 ng/ml ) mouse prolactin standard. Tracer - 1 vial of mouse prolactin tracer AChE. Antiserum - 1 vial of mouse prolactin antiserum. EIA Buffer - 1 vial of EIA buffer, lyophilized. Wash Buffer - 1 vial of a concentrated wash buffer. Tween 20-1 vial of Tween 20. Quality Control - 2 vials of mouse prolactin quality control. Ellman s Reagent - 2 vials of Ellman s Reagent, lyophilized. Plate Sealer - One self-adhesive plate cover. STORAGE Unopened Kit Opened/Reconstituted Reagents Store at -20 ºC. Do not use past the kit expiration date. Standard Quality Control Store at 2-8 ºC for 1 week. Antiserum Ellman s Reagent Store at 2-8 ºC for 4 days in the dark. Tracer EIA Buffer Store at 2-8 ºC for 1 month. Wash Buffer Return unused wells to the foil pouch containing the desiccant and seal. Store at Microtiter wells 2-8 ºC. SUPPLIES REQUIRED BUT NOT PROVIDED Spectrophotometer plate reader (405 or 414 nm filter) Microplate Washer Orbital Microplate Shaker Pipettes or pipetting equipment with disposable polypropylene tips (10 to 1000 μl) Multi-channel pipette Disposable polypropylene test tubes Glass measuring cylinders Distilled or deionized water IN30341.R01 page 3

4 PRECAUTIONS Each time a new pipette tip is used, aspirate a sample or reagent and dispense it back into the same vessel. Repeat this operation two or three times before distribution. For research laboratory use only. Not for human diagnostic use. Do not pipette liquids by mouth. Do not use kit components beyond the expiration date. Do not eat, drink or smoke in area in which kit reagents are handled. Avoid splashing. Wearing gloves and laboratory coats are recommended when handling immunodiagnostic materials and samples of human origin. COMMENTS Prolactin is a polypeptide made of 197 (22560 Da) amino acid residues. The three dimensional structure is tetra helical. The pituitary gland is the main source of prolactin but other organs such as immune cells, brain and reproductive organs, help to maintain the physiological prolactin level. This polypeptide is defined as a hormone-cytokine with endocrine, paracrin and autocrine functions. More than 300 distinct biological functions of prolactin have been recorded, due to the fact that the receptor is ubiquitous. It could be possible to divide the prolactin effect into 5 different areas: reproduction, immune function, osmoregulation, metabolism and tumorogenesis CRITICAL PARAMETERS Allow samples and all reagents to equilibrate to room temperature (22 25 C) prior to performing the assay. Adhere to recommended incubation temperatures in the protocol as variations may cause inconsistent or poor assay results. It is essential that all wells are washed thoroughly and uniformly. When washing is done by hand, use a squeeze bottle and ensure that all wells are completely filled and emptied at each step. Use only reagents from the same lot for each assay. This is especially important when running more than one plate per sample group. A separate standard curve must be run on each plate. Reagents can easily be trapped in the caps of small microfuge tubes. It is recommended to briefly centrifuge these reagents before use. Mix all reagents thoroughly prior to use, but avoid foaming! Keep the wells sealed with the plate sealer except when adding reagents and during reading. Any variation in the protocol can cause variation in binding! The kit should not be used beyond the expiration date on the kit label. The values obtained by the samples should be within the standard range. If this is not the case, dilute the sample and repeat the assay. We take great care to ensure that this product is suitable for all validated sample types as designated in this protocol. Other sample types may be tested and validated by the user. IN30341.R01 products@mdbiosciences.com page 4

5 SAMPLE COLLECTION AND STORAGE This assay may be used to measure mouse prolactin in culture media. All samples must be free of organic solvents prior to the assay. Samples should be assayed immediately after collection or stored at -20 C. REAGENT PREPARATION Note: All reagents should be stored at the recommended temperatures. Bring all reagents to room temperature (22-25 C) before use. Reagents can easily be trapped in the caps of small microfuge tubes. It is recommended to briefly centrifuge these reagents before use. EIA buffer - Reconstitute one vial with 50 ml of distilled or deionized water. Allow it to stand 5 minutes until completely dissolved and then mix thoroughly by gentle inversion. Reconstituted buffer is stable at 4 C for 1 month. Standard - In tube 1, reconstitute the vial with 1 ml of distilled water. Allow it to stand 5 minutes until completely dissolved and then mix thoroughly by gentle inversion. The concentration of this standard is 250 ng/ ml. Label seven propylene tubes (for the seven remaining standards) and add 500 μl of culture media to each tube. Add 500μL of the first tube (containing the first standard) to the second tube. Continue to make a dilution series as shown in the diagram below. Stability at 4 C: 1 day Serial Dilutions using 500μL ng/ml ng/ml ng /ml ng/ml ng/ml ng/ml ng/ml Quality Control - Reconstitute the vial with 1 ml of culture media used in the experiment. Allow to stand 5 minutes until completely dissolved and then mix thoroughly by gentle inversion. Stable for 24 hours at 4 C. Mouse prolactin-ache Tracer -Reconstitute the vial with 5 ml of mouse prolactin EIA buffer. Allow to stand 5 minutes until completely dissolved and then mix thoroughly by gentle inversion. Stable for 30 days at 4 C. Mouse prolactin Antiserum - Reconstitute the vial with 5 ml of mouse prolactin EIA buffer. Allow to stand 5 minutes until completely dissolved and then mix thoroughly by gentle inversion. Stable for 1 week at 4 C. Wash Buffer - Dilute 1 ml of the concentrated wash buffer to 400 ml with distilled or deionized water. Add 200 μl of Tween 20 (Use a magnetic stirrer to mix the contents). Wash Buffer is stable for 1 week at 4 C. Ellman s Reagent - Five minutes before use, reconstitute with 50 ml of distilled water per vial. Mix the tube contents thoroughly. Ellman s Reagent is stable for 4 days at 4 C in the dark. IN30341.R01 page 5

6 ASSAY PROTOCOL Read the entire protocol before beginning the assay. It is recommended that all standards and samples be assayed in duplicate. Reagents can easily be trapped in the caps of small microfuge tubes. It is recommended to briefly centrifuge these reagents before use. Note: All samples and reagents must reach room temperature prior to performing the assay. See the Reagent and Sample Preparation sections before proceeding. 1. Prepare all reagents and samples as described in the previous sections. 2. Remove any excess microtiter strips from the plate frame and return them to the foil pouch containing the desiccant pack and store at -20 C. 3. Before beginning the assay, wash each well 5 times with Wash Buffer (300 μl/well). Empty the plate by turning over and shaking. Blot the plate on paper towels to remove residual fluid from the plate. Standard/Sample Incubation 4. Leave 2 wells empty for blanks. 5. Pipette 100 μl of EIA Buffer into duplicate wells for Non-Specific Binding (NSB). 6. Pipette 50 μl of EIA Buffer into duplicate wells for Maximum Binding (B 0 ). 7. Pipette 50 μl of Mouse prolactin standard from each of the eight standards (S1 to S8) into duplicate wells. 8. Pipette 50 μl into duplicate wells for each sample or qulity control. 9. Pipette 50 μl of Tracer into each well, except the Blank (B) wells. 10. Pipette 50 μl of Antiserum into each well except the Blank (B) wells and the NSB wells. 11. Incubate for hours at room temperature (22-25 C). Wash 12. Reconstitute the wash buffer and Ellman s reagent as indicated in the reagent preparation section. 13. Empty the plate by turning it over and shaking it. Blot the plate on paper towels to remove residual fluid. 14. Wash each well five times with 300 μl Wash Buffer. Substrate Incubation 14. Dispense 200 μl of Ellman s Reagent to each well including the blank wells and the NSB wells. 15. Incubate in the dark (plate covered with an aluminum sheet) at room temperature. Optimal development is obtained using an orbital shaker at 300 rpm. 16. The plate should be read between 405 and 414 nm (yellow color) when the Maximum Binding (B 0 ) wells reach an absorbance of 200 mau after blank subtraction. IN30341.R01 products@mdbiosciences.com page 6

7 SUMMARY Prepare reagents and samples as previously described. Prior to assay, wash the plate 5 times with 300 µl of Wash Buffer. ] ] ] ] ] ] Leave 2 wells empty for blanks. Pipette 100 µl EIA Buffer into duplicate NSB wells and 50 µl into duplicate B 0 wells. Pipette 50 µl standards, quality control, or sample into duplicate wells. Add 50 µl of Tracer to each well, except the Blank (B) wells. Add 50 µl Antiserum to each well except the Blank (B) wells and the NSB wells. Incubate for hours at room temperature (22-25 C). Aspirate and wash 5 times with 300 µl of Wash Buffer. Add 200 µl of Ellman s Reagent to each well. Incubate in the dark at RT on a shaker at 300 rpm. Read absorbance (between 405 and 414 nm) when B 0 wells reach an absorbance of 200 mau after blank subtraction. IN30341.R01 page 7

8 CALCULATION OF RESULTS Make sure that your Plate Reader has subtracted the absorbance readings of the blank well (absorbance of Ellman s reagent) from the absorbance readings of the rest of the plate. If not, do it now. Calculate the average absorbance for each NSB, B 0, standards and samples. Calculate the B/B 0 (%) for each standard and sample: (average absorbance of standards or sample - average absorbance of NSB) divide by (average absorbance of B 0 - average absorbance of NSB) & multiplied by 100. Using a semi-log graph paper, plot the B/B 0 (%) for each standard point (y axis) versus the concentration (x axis). Draw a best-fit line through the points. To determine the concentration of your samples, find the B/B 0 (%) value on the y axis. From the point of intersection on the line, read the corresponding value on the x axis which is the concentration of your unknown sample. Samples with a concentration greater than 250 ng/ml should be re-assayed after dilution in culture media. Most plate readers are supplied with curve-fitting software capable of graphing this type of data (autospline, logit/log or 4-parameter). If you have this type of software, we recommend using it. Refer to it for further information. TYPICAL DATA The following data is to be used for demonstration purpose only. Your data may be different and still correct. This data was obtained using all reagents as supplied in this kit under the following conditions: 1 hour developing at 20 C and reading samples at 414 nm. A four-parameter logistic curve fitting was used to determine the concentrations. NSB Bo Standard 250 ng/ml Standard125 ng/ml Standard 62.5 ng/ml Standard 31.2 ng/ml Standard 15.6 ng/ml Standard 7.81 ng/ml Standard 3.9 ng/ml Standard 1.95 ng/ml mau B/B0 (%) N/A N/A Acceptable Range: Ratio NSB absorbance / B 0 absorbance: < 0.1. B 0 absorbance: >200 mau after blank subtraction in the conditions indicated above. 50% B/B 0 (%): 25 ng/ml IN30341.R01 products@mdbiosciences.com page 8

9 ASSAY CHARACTERISTICS The enzyme immunoassay of mouse prolactin has been validated to be used in cell culture media. Cross-reactivity TSH (mouse) < 1 % LH (mouse) < 1 % GH (mouse) < 1 % Prolactin (rat) 1.4 % The limit of detection The limit of detection is a calculated value using the concentration of mouse prolactin obtained from the B 0 minus 3 standard deviations. Precision Intra-assay Precision QC level Concentration (ng/ml) CV (%) QC High 94 12% QC Medium 59 6% QC Low 4 16% Inter-assay Precision QC level Concentration (ng/ml) CV (%) QC High 97 13% QC Medium 62 10% QC Low 5 22% Limit of Quantification Limit of detection: 1.7 ng/ml REFERENCES 1. Kohmoto K, Tsunasawa S, Sakiyama F. Complete amino acid sequence of mouse prolactin. Eur; J. Biochem Hallgeir Rui. Prolactin. Review Department of pathology, Uniformed Services University Bethesda. 3. Michael J Soares. The prolactin and growth hormone families: Pregmancy specific hormones / cytolkines at the maternal-fetal interface. Review Reproductive Biology and endocrinology. 4. I Fernandez, P Touraine and V.Goffin. Prolactin and Human tumourogenesis. Review Journal of Neuroendocrinology IN30341.R01 page 9

10 ASSAY TROUBLESHOOTING Problem B 0 value too low NSB too high High dispersion of duplicates QC value IC 50 out of range Recommendation Always follow proper incubation times and temperature. Increase Incubation time of Ellman s Reagent. Ensure addition of all reagents (Tracer, Antiserum, Ellman s Reagent) as per protocol. Contamination of NSB wells with Mouse prolactin antiserum. To reduce contamination always use new pipette tips. Use separate reservoirs for pipetting different solutions with multichanned pipettes. Ensure efficient washing procedure. Check pipette calibration. Ensure accurate pipetting. Ensure efficient washing procedure. Ensure correct preparation of standards. IN30341.R01 products@mdbiosciences.com page 10

11 NOTES IN30341.R01 page 11

12 T! C U O D PR IT H ED W U SE O N AS E LE!P LY International/Headquarters MD Bioproducts Division of MD Biosciences GmbH Gewerbestrasse Egg b Zurich Switzerland Tel: Fax: R EF ER TO IN SE R T IN C LU D North America MD Bioproducts Divison of MD Biosciences, Inc. 1 Imation Way Oakdale, MN USMDBIO Tel: Fax: IN FO R M AT IO N AL products@mdbiosciences.com