FABP3 ELISA Kit

Catalog No. ABIN956149

Pig FABP3 ELISA Kit, Colorimetric assay for quantification of Pig FABP3.

Quick Overview for FABP3 ELISA Kit (ABIN956149)

Target: FABP3 (Fatty Acid Binding Protein 3, Muscle and Heart (FABP3))

Reactivity: Pig

Detection Method: Colorimetric

Method Type: Sandwich ELISA

Application: ELISA

Sample Type: Plasma, Serum

Product Details FABP3 ELISA Kit

Analytical Method: Quantitative

Characteristics: The Pig H-FABP ELISA is based on a solid phase enzyme-linked immunosorbent assay (ELISA). The assay uses affinity purified anti-pig H-FABP antibodies for solid phase (microtiter wells) immobilization and horseradish peroxidase (HRP) conjugated anti-pig H-FABP antibodies for detection. The test sample is diluted and incubated in the microtiter wells for 45 minutes. The microtiter wells are subsequently washed and HRP conjugate is added and incubated for 30 minutes. This results in H-FABP molecules 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. This results 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 H-FABP is proportional to the optical density of the test sample.

Components: Anti-pig H-FABP antibody coated microtiter plate with 96 wells (provided as 12 detachable strips of 8)
Enzyme Conjugate Reagent, 11 mL
Calibrator (lyophilized)
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:

1. Pig serum or plasma samples may need to be diluted prior to assay in order to obtain values within the range of the calibration curve. The dilution factor must be determined empirically. We suggest that the researcher chooses a sample likely to have the highest H-FABP level and run an initial test with that sample to determine an optimum dilution factor. Other samples should subsequently be tested at that dilution.
   2. Dilution should be performed with the yellow diluent provided with the kit.
   3. If samples are to be tested in duplicate a final volume of 250 µL of diluted sample is sufficient.

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. Remove the incubation mixture either with a plate washer or by flicking plate contents into a waste container.
   5. 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.
   6. Strike the wells sharply onto adsorbent paper or paper towels to remove all residual droplets.
   7. Add 100 µL of enzyme conjugate reagent into each well.
   8. Incubate on an orbital micro-plate shaker at 100-150 rpm at room temperature (18-25°C) for 30 minutes.
   9. Wash as detailed in 4 to 5 above.
   10. Strike the wells sharply onto absorbent paper or paper towels to remove residual droplets.
   11. Dispense 100 µL of TMB Reagent into each well.
   12. Gently mix on an orbital micro-plate shaker at 100-150 rpm at room temperature (18-25°C) for 20 minutes.
   13. Stop the reaction by adding 100 µL of Stop Solution to each well.
   14. Gently mix. It is important to make sure that all the blue color changes to yellow.
   15. 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 H-FABP in ng/mL from the calibration curve.
   4. Multiply the derived concentration by the dilution factor to determine the actual concentration of H-FABP 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, samples should be diluted appropriately and re-tested.

Restrictions: For Research Use only

Handling

Storage: 4 °C

Storage Comment: The unused 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 FABP3

Target: FABP3 (Fatty Acid Binding Protein 3, Muscle and Heart (FABP3))

Alternative Name: H-FABP

Background: Fatty acid-binding proteins (FABP) are cytosolic proteins of about 15 kD. They bind long chain fatty acids and play an important role in fatty acid metabolism. Heart, liver and intestinal FABP isoforms exist. Heart has a high content of FABP (10-20 mol % of cytoplasmic proteins) and heart FABP (H-FABP) has proved to be a sensitive biomarker of myocardial necrosis in humans. H-FABP is rapidly released into the circulation from damaged cardiac muscle. Serum/plasma levels are significantly increased within 1-4 hours of muscle injury and values return to normal within 12 to 24 hours. Because H-FABP is also expressed in skeletal muscle, it is necessary to exclude or control for skeletal muscle injury before ascribing H-FABP elevations to cardiac injury.

Pathways: Monocarboxylic Acid Catabolic Process