I.   INTENDED USE

 This Human GIP (Total) ELISA kit is used for the non-radioactive quantification of Human GIP in human serum, plasma, tissue extract and cell culture samples.  This kit has 100% cross reactivity to human 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 human 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 human 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 human 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 anti-GIP Monoclonal Antibodies

Quantity:  1 plate

Preparation:  Ready to Use

Note:  Unused strips should be resealed in the foil pouch with the desiccant provided and stored at 2-8°C.

 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.

D.  Human GIP Standard

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

Quantity:  0.5 ml/vial upon hydration

Preparation:  Reconstitute with 0.5 ml distilled or deionized water.

E.   Human GIP Quality Controls 1 and 2

Human 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-Human 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

K.  Matrix Solution

Quantity:  1 ml/vial

Preparation:  Ready to Use

IV. STORAGE AND STABILITY

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.  Unused microtiter strips should be resealed in the foil pouch with the desiccant provided and stored at 2-8°C.  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 media samples may require dilution. Dilutions should be performed using the assay buffer provided.

IX. STANDARD AND QUALITY CONTROLS PREPARATION

A. Human GIP Standard Preparation

1.  Use care in opening the lyophilized Standard vial.  Using an Eppendorf pipette, reconstitute the Human 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, and 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 µl Assay Buffer to each of the five tubes.  Perform 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.