Vitamin D-Binding Protein ELISA Kit

Catalog No. ABIN956157

Product Details Vitamin D-Binding Protein ELISA Kit

Target: Vitamin D-Binding Protein (GC)

Reactivity: Rat

Detection Method: Colorimetric

Method Type: Sandwich ELISA

Application: ELISA

Sample Type: Plasma, Serum

Analytical Method: Quantitative

Characteristics: The Rat Gc-Globulin ELISA is based on a solid phase enzyme-linked immunosorbent assay (ELISA). The assay uses an affinity purified anti-rat Gc-Globulin antibody for solid phase (microtiter wells) immobilization and a horseradish peroxidase (HRP) conjugated anti-rat Gc-Globulin antibody for detection. The test sample is diluted into actin containing diluent (converting free Gc-globulin to the actin complexed form) and incubated in the microtiter wells for 45 minutes. The microtiter wells are subsequently washed and HRP conjugate is added and incubated for 45 minutes. This results in Gc-Globulin/actin complexes being sandwiched between the immobilization and detection antibodies. The wells are then washed to remove unbound HRP-labeled antibodies and TMB Reagent is added and incubated for 20 minutes at room temperature, resulting in the development of a blue color. Color development is stopped by the addition of Stop Solution, changing the color to yellow, and optical density is measured spectrophotometrically at 450 nm. The concentration of Gc-globulin is proportional to the optical density of the test sample.

Components: Anti-rat Gc-globulin antibody coated microtiter plate with 96 wells (provided as 12 detachable strips of 8)
Enzyme Conjugate Reagent, 11 mL
Calibrator (lyophilized)
Actin (lyophilized), 3 vials
10X Diluent (25 mL)
20X Wash Solution (50 mL)
TMB Reagent (One-Step), 11 mL
Stop Solution (1N HCl), 11 mL.

Material not included: Precision pipettes and tips
Distilled or de-ionized water
Polypropylene or glass tubes
Vortex mixer
Absorbent paper or paper towels
Micro-Plate incubator/shaker mixing speed of ~150 rpm
A microtiter plate reader capable of measuring absorbance at 450 nm, with a bandwidth of 10 nm or less and an OD range of 0-4 OD
Graph paper (PC graphing software is optional)

Application Details

Plate: Pre-coated

Sample Preparation:

General Note: Our studies indicate that Gc-globulin is present in normal rat serum at a concentration of ~1 mg/mL. In order to obtain values within the range of the calibration curve we suggest that samples initially be diluted 10,000 fold in actin containing 1X diluent using the following procedure for each sample to be tested:
1. Dispense 198 µL and 297 µL of actin containing 1X diluent into separate tubes.
2. Pipette and mix 2 µL of the serum/plasma sample into the tube containing 198 µL of diluent. This provides a 100 fold diluted sample.
3. Mix 3 µL of the 100 fold diluted sample with the 297 µL of diluent in the second tube. This provides a 10,000 fold dilution of the sample.
4. Repeat this procedure for each sample to be tested.

Assay Procedure:

1. Secure the desired number of coated wells in the holder.
   2. Dispense 100 µL of calibrators and samples into the wells (we recommend that samples be tested in duplicate).
   3. Incubate on an orbital micro-plate shaker at 100-150 rpm at room temperature (18-25°C) for 45 minutes.
   4. Wash and empty the microtiter wells 5 times with 1X wash solution. This may be performed using either a plate washer (400 µL/well) or with a squirt bottle. The entire wash procedure should be performed as quickly as possible.
   5. Strike the wells sharply onto adsorbent paper or paper towels to remove all residual droplets.
   6. Add 100 µL of enzyme conjugate reagent into each well.
   7. Incubate on an orbital micro-plate shaker at 100-150 rpm at room temperature (18-25°C) for 45 minutes.
   8. Wash as detailed in 4 above.
   9. Strike the wells sharply onto adsorbent paper or paper towels to remove all residual droplets.
   10. Dispense 100 µL of TMB Reagent into each well.
   11. Gently mix on an orbital micro-plate shaker at 100-150 rpm at room temperature (18-25°C) for 20 minutes.
   12. Stop the reaction by adding 100 µL of Stop Solution to each well.
   13. Gently mix. It is important to make sure that all the blue color changes to yellow.
   14. Read the optical density at 450 nm with a microtiter plate reader within 5 minutes.

Calculation of Results:

1. Calculate the average absorbance values (A450) for each set of reference calibrators, and samples.
   2. Construct a calibration curve by plotting the mean absorbance obtained from each reference calibrator against its concentration in ng/mL on linear graph paper, with absorbance values on the vertical or Y-axis and concentration on the horizontal or X-axis.
   3. Using the mean absorbance value for each sample, determine the corresponding concentration of Gc-globulin in ng/mL from the calibration curve.
   4. Multiply the derived concentration by the dilution factor to determine the actual concentration of Gc-globulin in the serum/plasma sample.
   5. If available, PC graphing software may be used for the above steps.
   6. If the OD450 values of samples fall outside of the calibration curve when tested at a dilution of 10,000, samples should be diluted appropriately and re-tested.

Restrictions: For Research Use only

Handling

Storage: -20 °C

Storage Comment: Lyophilized actin should be stored at or below -20°C. The remainder of the kit should be stored at 4°C and the microtiter plate should be kept in a sealed bag with desiccant to minimize exposure to damp air. Test kits will remain stable until the expiration date provided that the components are stored as described above.

Expiry Date: The expiry date is stated on the label.

Target Details for Vitamin D-Binding Protein

Target: Vitamin D-Binding Protein (GC)

Alternative Name: Gc-Globulin (GC Products)

Synonyms: DBP ELISA Kit, DBP/GC ELISA Kit, GRD3 ELISA Kit, VDBG ELISA Kit, VDBP ELISA Kit, zgc:110389 ELISA Kit, zgc:92753 ELISA Kit, GC ELISA Kit, Gc ELISA Kit, DBP02 ELISA Kit, VDB ELISA Kit, Vdbp ELISA Kit, VTDB ELISA Kit, gc ELISA Kit, GC, vitamin D binding protein ELISA Kit, D-box binding PAR bZIP transcription factor ELISA Kit, D site albumin promoter binding protein ELISA Kit, group-specific component (vitamin D binding protein) ELISA Kit, group specific component ELISA Kit, group-specific component (vitamin D binding protein) S homeolog ELISA Kit, GC ELISA Kit, Dbp ELISA Kit, gc ELISA Kit, Gc ELISA Kit, gc.S ELISA Kit

Background: Gc-globulin, also known as vitamin D-binding protein, is an acute phase protein that is synthesized in the liver and serves as an actin scavenger in blood. It has recently been identified as a potentially useful early marker of liver toxicity and is reportedly useful as an indicator of skeletal muscle injury.

Pathways: Metabolism of Steroid Hormones and Vitamin D, Monocarboxylic Acid Catabolic Process