I. INTENDED USE

This Rat/Mouse GIP (Total) ELISA kit is used for the non-radioactive quantification of Rat/Mouse GIP (Total) in rat or mouse serum, plasma, tissue extracts or cell culture media samples.  This kit has 100% crossreactivity to rat GIP and mouse GIP and measures both GIP(1-42) and GIP(3-42). One kit is sufficient to measure 39 unknown samples in duplicate. 
This kit is for research purpose only.

 II.   PRINCIPLES OF PROCEDURE

 This assay is a Sandwich ELISA based, sequentially, on: 1) capture of rat or mouse GIP molecules from samples to the wells of a microtiter plate coated by a pre-titered amount of anti-GIP monoclonal antibodies, 2) wash away of unbound materials from samples, 3) binding of a second biotinylated anti-GIP polyclonal antibody to the captured molecules, 4) wash away of unbound materials from samples, 5) incubation of streptavidin-Horseradish peroxidase conjugate to bind to the immobilized biotinylated antibodies, 6) wash away of free enzyme conjugates, and 7) quantification of immobilized antibody-enzyme conjugates by monitoring horseradish peroxidase activities in the presence of the substrate 3,3’,5,5’-tetramethylbenzidine.  The enzyme activity is measured spectrophotometrically by the increased absorbency at 450 nm, corrected from the absorbency at 590 nm, after acidification of formed products.  Since the increase in absorbency is directly proportional to the amount of captured rat or mouse GIP in the unknown sample, the latter can be derived by interpolation from a reference curve generated in the same assay with reference standards of known concentrations of Rat GIP.

 III.  REAGENTS SUPPLIED

Each kit is sufficient to run one 96-well plate and contains the following reagents: 

 A.  GIP ELISA Plate

Coated with Monoclonal anti GIP Antibodies

Quantity:  1 plate

Preparation:  Ready to Use

 B.  Adhesive Plate Sealer

Quantity:  2 sheets

Preparation:  Ready to Use

 C.  10X HRP Wash Buffer Concentrate

10X concentrate of 50 mM Tris Buffered Saline containing Tween-20.

Quantity:  2 bottles containing 50 ml each

Preparation:  Dilute 1:10 with distilled or deionized water.

       Rat GIP(1-42), 2000 pg/ml, 0.5 ml/vial, lyophilized.

Quantity:  0.5 ml upon hydration

Preparation:  Contents Lyophilized.  Reconstitute with 0.5 ml distilled or deionized water.

 E.   Rat/Mouse GIP Quality Controls 1 and 2

Rat GIP(1-42), 0.5 ml/vial, lyophilized.

      Quantity: 0.5 ml/vial upon hydration

Preparation:  Contents Lyophilized.  Reconstitute with 0.5 ml distilled or deionized water.

Buffer containing BSA and 0.08% Sodium Azide

      Quantity:  12 ml

       Preparation:  Ready to Use

       Pre-titered Biotinylated Rabbit anti GIP Polyclonal Antibody

Quantity:  11 ml

Preparation:  Ready to Use

 H.  Enzyme Solution

Pre-titered Streptavidin-Horseradish Peroxidase Conjugate in Buffer

Quantity:  12 ml

Preparation:  Ready to Use

 I.    Substrate  (Light sensitive, avoid unnecessary exposure to light)

3, 3’, 5, 5’-tetramethylbenzidine in buffer

Quantity:  12 ml

Preparation:  Ready to Use. 

 J.   Stop Solution  (Caution:  Corrosive Solution)

0.3 M HCl

Quantity:  12 ml

Preparation:  Ready to Use

Quantity:  1 ml/vial

Preparation:  Ready to Use

 Prior to use, all components in the kit can be stored up to 2 weeks at 2-8^oC.   For longer storage (> 2 weeks), freeze Wash Buffer, Assay Buffer, Matrix Solution, and reconstituted Standards and Controls at £ –20^oC.  Minimize repeated freeze and thaw of the GIP Standards and Quality Controls.  Refer to expiration dates on all reagents prior to use.  Do not mix reagents from different kits unless they have the same lot numbers. 

 V.    REAGENT PRECAUTIONS

 A.  Sodium Azide

 Sodium azide has been added to certain reagents as a preservative.  Although the concentrations are low, sodium azide may react with lead and copper plumbing to form highly explosive metal azides.  On disposal, flush with a large volume of water to prevent azide build up.

 B.  Hydrochloric Acid

 Hydrochloric Acid is corrosive and can cause eye and skin burns.  It is harmful if swallowed and can cause respiratory and digestive tract burns.  Avoid contact with skin and eyes.  Do not swallow or ingest.

 VI.  MATERIALS REQUIRED BUT NOT PROVIDED

 1.  Pipettes and Pipette Tips:  10 ml - 20 ml or 20 ml - 100 ml

2.      Multi-Channel Pipettes and Pipette Tips:  5 ~ 50 µl and 50 ~ 300 ml

3.      Buffer and Reagent Reservoirs

4.      Vortex Mixer

5.      Deionized Water

6.      Microtiter Plate Reader capable of reading absorbency at 450 nm

7.      Orbital Microtiter Plate Shaker

8.      Absorbent Paper or Cloth

 VII. SAMPLE COLLECTION AND STORAGE

 NOTE:  Although DPPIV inhibitor is not required to be added to serum/plasma samples for measurement of total GIP, we recommend that DPPIV inhibitor be added to the serum/plasma samples during the sample collection so that the same samples could be used in the future for the measurement of intact (1-42) GIP with an assay that is capable of selectively measuring only the intact GIP.

 1.  To prepare serum samples, whole blood is directly drawn into a centrifuge tube that contains no anti-coagulant.  Let blood clot at room temperature for 30 min.

 Promptly centrifuge the clotted blood at 2,000 to 3,000 x g for 15 minutes at 4 ± 2^oC.

 Transfer and store serum samples in separate tubes. Date and identify each sample.

 Use freshly prepared serum or aliquot and store samples at £ –20^oC for later use.  For long-term storage, keep at -70 ^oC.  Avoid freeze/thaw cycles.

2. To prepare plasma samples, whole blood should be collected into centrifuge tubes containing enough K₃EDTA to achieve a final concentration of 1.735 mg/ml and centrifuged immediately after collection.  Observe the same precautions in the preparation of serum samples.

3.  If heparin is to be used as an anticoagulant, the effect on the assay outcome at the dose of heparin used should be pre-determined.

4. Avoid using samples with gross hemolysis or lipemia.

 VIII. SAMPLE PREPARATION

1. No dilution or preparation is needed for normal serum or plasma samples. In the event that any sample is above 2000 pg/ml range, dilutions should be performed using the Matrix Solution provided.

2. Tissue extracts or cell culture samples may require dilution. Dilutions should be performed using the assay buffer provided.

 IX. STANDARD AND QUALITY CONTROLS PREPARATION

A. Rat/Mouse GIP Standard Preparation

1. Use care in opening the lyophilized Standard vial.  Using an Eppendorf pipette, reconstitute the Rat/Mouse GIP Standard with 0.5 ml distilled or deionized water into the glass vial to give a 2000 pg/ml concentration of Standard.  Invert and mix gently, let sit for 5 minutes then mix well.

2. Label five tubes 666.7, 222.2, 74.1, 24.7, and 8.2 pg/ml.  Add 100 ul Assay Buffer to each of the five tubes.  Prepare 3 times serial dilutions by adding 50 µl of the 2000 pg/ml reconstituted standard to the 666.7 pg/ml tube, mix well and transfer 50 µl of the 666.7 pg/ml standard to the 222.2 pg/ml tube, mix well and transfer 50 µl of the 222.2 pg/ml standard to the 74.1 pg/ml tube, mix well and transfer 50 µl of the 74.1 pg/ml standard to the 24.7 pg/ml tube, mix well and transfer 50 µl of the 24.7 pg/ml standard to the 8.2 pg/ml tube and mix well.

Note: Do not use a Repeater pipette.  Change tip for every dilution.  Wet tip with Standard before dispensing.  Unused portions of standard should be stored at £ -20°C.  Avoid multiple freeze/thaw cycles.

B.     Rat/Mouse GIP Quality Control 1 and 2 Preparation

1. Use care in opening the lyophilized Quality Control vials.  Using an Eppendorf pipette, reconstitute each of the Rat/Mouse GIP Quality Control 1 and Quality Control 2 with 0.5 ml distilled or deionized water into the glass vials.  Invert and mix gently, let sit for 5 minutes then mix well.

 X.   ASSAY PROCEDURE

 Pre-warm all reagents to room temperature prior to setting up the assay. 

1. Dilute the 10X concentrated Wash Buffer 10 fold by mixing the entire contents of both buffer bottles with 900 ml deionized or distilled water.

2. Remove the microtiter assay plate from the foil pouch and fill each well with 300 ml of diluted Wash Buffer. Incubate at room temperature for 5 minutes. Decant Wash Buffer and remove the residual amount from all wells by inverting the plate and tapping it smartly onto absorbent towels several times. Do not let wells dry before proceeding to the next step.  If an automated machine is used for the assay, use a gentle wash program for all washing steps described in this protocol.   

3. Add in duplicate, 90 µl Assay Buffer to the blank wells and sample wells.  (See plate well map for suggested well orientation).

4. Add in duplicate, 80 µl Assay Buffer to Standard wells, QC1 and QC2 wells. 

5. Add 10 µl Matrix Solution to Blank wells, Standard wells, and QC1 and QC2 wells.

6. Add in duplicate, 10 ml Rat/Mouse GIP Standards in the order of ascending concentration to the appropriate wells.  Add in duplicate, 10 ml QC1 and 10 ml QC2 to the appropriate wells.  Add sequentially, 10 µl of the unknown samples in duplicate to the remaining wells. For best result all additions should be completed within 30 minutes.  

7. Cover the plate with plate sealer and incubate at room temperature for 1.5 hours on an orbital microtiter plate shaker set to rotate at moderate speed, approximately 400 to 500 rpm.

8. Remove plate sealer and decant solutions from the plate.  Tap as before to remove residual solutions in the wells.

9. Wash wells 3 times with diluted Wash Buffer, 300 µl per well per wash.  Decant and tap firmly after each wash to remove residual buffer.

10. Add 100 µl Detection Antibody to all wells. Cover the plate with plate sealer and incubate at room temperature for 1 hour on an orbital microtiter plate shaker set to rotate at moderate speed, approximately 400 to 500 rpm.

11. Remove plate sealer and decant solutions from the plate.  Tap as before to remove residual solutions in the wells.

12. Wash wells 3 times with diluted Wash Buffer, 300 µl per well per wash.  Decant and tap firmly after each wash to remove residual buffer.

13. Add 100 µl Enzyme Solution to each well.  Cover plate with sealer and incubate with moderate shaking at room temperature for 30 minutes on the microtiter plate shaker.

14. Remove sealer, decant solutions from the plate, and tap plate to remove the residual fluid. 

15. Wash wells 3 times with diluted Wash Buffer, 300 µl per well per wash. Decant and tap firmly after each wash to remove residual buffer.

16. Add 100 µl of Substrate Solution to each well, cover plate with sealer and shake on the plate shaker for approximately 5 to 18 minutes (A longer development time may be needed if using a plate washer).  Blue color should be formed in wells of GIP standards with intensity proportional to increasing concentrations of GIP. 

NOTE:  Please be aware that the color may develop more quickly or more slowly than the recommended incubation time depending on the localized room temperature.  Please visually monitor the color development to optimize the incubation time.  One can monitor color development using 370 nm filter, if available on the spectrophotometer.  When the absorbance is between 1.2 and 1.8 at 370 nm, the stop solution can be added to terminate the color development.

17. Remove sealer and add 100 µl Stop Solution [CAUTION: CORROSIVE SOLUTION] and shake plate by hand to ensure complete mixing of solution in all wells.  The blue color should turn to yellow after acidification.  Read absorbance at 450 nm and 590nm in a plate reader within 5 minutes and ensure that there are no air bubbles in any well.  Record the difference of absorbance units. The absorbance of the highest GIP standard should be approximately 2.0-3.2, or not to exceed the capability of the plate reader used.