The Jackson Laboratory

Prepared by:

HS

Date Prepared:

10/22/03

Code:

MP-010

Reviewed by:

RVS

Revision #:

 V3

Date Revised:

 Jan. 31, 2005

0.0

Abstract: This test measures levels of Leptin in mouse plasma in units of ug/L. Leptin levels are determined by using plasma from blood. Blood is collected using 7.5ul Sodium Heparin 1000 units with Heparin coated capillary tubes.

1.0

Experiment layout: This test uses a Spectra Max 190 ELISA plate reader using Softmax software driven by a PC.

2.0

2.2

Supplied materials and materials needed:

The Mouse Leptin ELISA Kit is purchased from Crystal
Chem INC. 1536 Brook Drive, Suite A, Downers Grove, IL 60515.

Kit contains:

(1) Antibody-coated microplate module, 2 packs, (one pack contains 6x8 well strips, i.e., 48 wells per pack),
(2) Mouse leptin standard, Lyophilized, 2.56ng/vial which needs to be reconstituted with 100ul of sample diluent before use.
(3) Washing buffer stock solution (20X), which needs to be diluted with Distilled water by filling to 1000ul,
(4) Frame for fixing microplate module, plastic microplate cover.

All of the following supplied materials are reconstituted and ready for use:
(5) Sample diluent (one bottle)
(6) Guinea pig anti-mouse leptin serum (one 6ml vial)
(7) Anti-guinea pig IgG enzyme conjugate stock solution (one 8.4ml vial)
(8) Enzyme conjugate diluent (one 3.6ml vial)
(9) Enzyme substrate (TMB) solution (one 13ml bottle)
(10) Enzyme reaction stopping solution ((1 N Sulfuric Acid) (one 13ml bottle)

Materials needed:
(1) 5, 50, 100, 500ul micropipette or repeating micropipettes
(2) Containers for reagent preparation, volumetric cylinder: 1L
(3) Distilled water
(4) Polypropylene microtubes: 500ul
(5) Tube racks
(6) Vortex mixer
(7) Aspirator for washing procedure.

3.0

Setup: Make a template as to where your Blank, Standards, Controls and Samples are going to be placed within the microplate. Depending whether single, duplicate, or triplicate samples are run, the plate should be set up this way:

Dilute plasma samples 1:1 using 25ul of Sample diluent.
The plasma is diluted 1:1, to ensure that when levels are read, they will fall within the curve that we produce with our standards. Otherwise the levels would generally be above the generated curve. 
Reconstitute Mouse Leptin Standard with 100ul of distilled water and vortex, this stock is 25,600pg/ml after reconstitution. Prepare working mouse standards in concentrations of 12800, 6400, 3200, 1600, 800, 400, and 200pg/ml by placing 50ul of Sample diluent into 8 microcentrifuge tubes. Place 50ul of Original Mouse Leptin Stock solution into the first tube labeled 12800, vortex to mix, take 50ul of the now 12800 concentration and place into the tube labeled 6400, vortex to mix. Repeat dilution procedure on remaining tubes, but stopping at 0, which should only contain 50ul of sample diluent. See below:

Mouse Leptin Concentration (pg/ml)

*MLSS: Mouse Leptin Stock Solution

**   SD: Sample Diluent

*** Place 50ul of previous dilution into new dilution to create the desired dilution.

4.0

Loading and running: Wash each well of the plate three times using 300ul of washing solution.  Pipette 45ul of Sample diluent and 50ul of Guinea Pig Anti-Mouse leptin serum into each well. Referring to the template that is generated prior to testing, pipette 5ul of each Standard and 5ul of each Sample into appropriate well. Cover plate and let stand overnight (16-20 hours) at 4 °C. Discard the contents of the plate and wash five times with 300ul washing solution per wash. Combine the one bottle of Anti-guinea pig IgG enzyme conjugate stock solution and the one bottle of Enzyme conjugate diluent to form the working Anti-guinea pig IgG enzyme conjugate. Pipette 100ul of the working Anti-guinea pig IgG enzyme conjugate into each well. Cover plate and incubate 3 hours at 4 °C. Discard the contents of the plate and wash each well seven times using 300ul of washing solution. IMMEDIATELY after washing, pipette 100ul of Enzyme Substrate solution into each well, cover and let stand for 30 minutes, protected from light. Pipette 100ul of Enzyme reaction stopping solution into each well. It is advised that caution be practiced at this step. Stop Solution is made of Hydrochloric Acid and you do not want this on your hands. Read optical density at 492nm on a plate reader within 30 minutes of adding the stopping solution. Discard the plate in hazardous waste bin.

5.0

Clean up: After the plate has been read the plate is discarded into a hazardous waste bin. Place all leftover reagents into waste bin. Carefully place all equipment used, back to a location that will be easy to locate during the next test.

6.0

Data reduction: Once the results have been calculated by the plate reader you can view the results for the Unknowns, Standards or the Standard Curve individually or as a whole depending on which set of data points you have open at any one time.  To open any set of data simply click on the name of the sample that you wish to view and the software opens the data requested.  All data can be printed out by selecting print and then selecting the corresponding data set you have open. All data is reviewed, outliers are excluded and is then sent to Mutajax by exporting the file in a text format to the Leptin folder under “Mutaprd Peggy”. 

7.0

Safety: This test should be conducted wearing safety gloves. Although it is unlikely to be infected by mouse plasma caution should be taken as though it were possible. This test also uses hydrochloric acid in the stop procedure and therefore it is hazardous.  Caution is practiced, and all material is dispensed into hazardous waste bins.

8.0

Time and capacity:

26 samples: Bench Time 1 –1? hours
26 samples: Calendar Time 21-26 hours depending on initial incubation

Diluting of all samples and standards, washing the plate three times, loading of all standards and samples (in duplicate or triplicate) and the reagents for the first reaction into a 96 well plate usually takes approximately an hour and a half, which is then followed by a 16-20 hour incubation at 4 °C. The wash following the incubation takes about 5 minutes using a plate washer. Preparing and loading the working Anti-guinea pig IgG enzyme conjugate takes approximately 3 minutes for one plate, which is followed by a 3-hour incubation at 4 °C. Following the second incubation the plate is washed seven times which takes approximately 5 minutes using a plate washer.  Enzyme Substrate solution is then added (approximately 3 minutes per plate) and incubated in a dark place for 30 minutes. Stop solution is added (3 minutes per plate) and plate is read within 30 minutes. Reading the plate with the plate reader takes approximately 20 minutes, loading the sample names as well as standards takes the longest for they are entered by hand. Actual time for plate reader to calculate results is about 30 seconds. Total bench time is between one and a half hours to 2 hours; Total test time is between 21 and 26 hours depending on how long the first incubation is.

A summary of procedures follow:

-    Make Template Sheet
-    Dilute Plasma Samples 1:1 with Sample Diluent.
-    Prepare Mouse Leptin Standard
-    Prepare working mouse standards
-    Wash each well 3 times with 300ul of Washing buffer
-    Pipette 45ul of Sample diluent into each well
-    Pipette 50ul of Guinea Pig anti-mouse leptin to each well.
-    Pipette 5ul of each standard into each corresponding well from the template.
-    Pipette 5ul of each sample into the wells that correspond to the template.
-    Cover plate with plastic microplate cover and let stand overnight (16-20 hours) at 4°C.
-    Wash 5 times with 300ul of Washing buffer.
-    Pipette 100ul of Anti-guinea pig IgG enzyme conjugate to each well.
-    Cover plate and incubate for 3 hours at 4°C.
-    Wash 7 times with 300ul of washing buffer each time.
-    Pipette 100ul of Enzyme Substrate Solution
-    Incubate for 30 minutes, protect from light.
-    Add 100ul of Enzyme reaction stopping solution to each well.
-    Measure absorbance within 30 minutes, at 450nm with subtracting wavelength 630nm.

9.0

9.1


















9.2

Protocols and quality control:

 Protocols: Bring test strips and all reagents to room temperature before start. All reagents, once opened, are only good for one week; planning to use the entire kit at once is advised. Make template sheet of samples and standards that will be run. Prepare washing solution by mixing distilled water and the washing buffer stock solution to 1000ml. Dilute plasma samples 1:1 using sample diluent and prepare mouse leptin stock by adding 100ul of sample diluent. Prepare working mouse standards 12800, 6400, 3200, 1600, 800, 400, and 200pg/ml respectively using reconstituted mouse leptin stock. Add 45ul of sample diluent and 50ul of Guinea Pig anti-mouse leptin to each well. Add 5ul of each standard into representing wells. Add 5ul of each diluted sample into representing wells. Cover plate and incubate 16-20 hours at 4 °C. Discard contents of plate, wash five times with 300ul of Washing buffer with plate washer. Add 100ul of Anti-guinea pig IgG enzyme conjugate to each well. Cover plate and incubate 3 hours at 4 °C. Discard contents of wells and wash 7 times with 300ul of washing buffer using plate washer. IMMEDIATELY after washing add 100ul of Enzyme Substrate Solution and incubate in dark drawer ***PROTECT FROM LIGHT*** for 30 minutes. Add 100ul of Enzyme reaction stopping solution to each well ***Wear gloves, stop solution is Hydrochloric Acid*** Read optical density at 492nm within 30 minutes.

 Quality Control: Standards are checked for quality assurance by checking if the controls and standards fall in range of the test. All data is reviewed, outliers are excluded and is then sent to Mutajax by exporting the file in a text format to the Leptin folder under “Mutaprd Peggy”. 
Rejected samples are repeated when possible, and are stored at -20°C until they can be repeated. Sample data is rejected when two or more data points do not fall within the standard curve. If only one out of three data points, for any sample, is out of range, that data point is rejected and the two other data points for that sample remain and are sent to Muta Jax.