CRP ELISA Kit

Catalog No. ABIN956104

Monkey CRP ELISA Kit, Colorimetric assay for quantification of Monkey CRP.

Quick Overview for CRP ELISA Kit (ABIN956104)

Target: CRP (C-Reactive Protein (CRP))

Reactivity: Monkey

Detection Method: Colorimetric

Method Type: Sandwich ELISA

Application: ELISA

Sample Type: Serum

Product Details CRP ELISA Kit

Purpose: The Monkey High-Sensitive CRP ELISA is based on a solid phase enzyme-linked immunosorbent assay (ELISA).

Analytical Method: Quantitative

Characteristics: The Monkey High-Sensitive CRP ELISA is based on a solid phase enzyme-linked immunosorbent assay (ELISA). The assay uses affinity purified anti-monkey CRP antibodies for solid phase (microtiter wells) immobilization and horseradish peroxidase (HRP) conjugated anti-monkey CRP 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 45 minutes. This results in CRP 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 CRP is proportional to the optical density of the test sample.

Components: Anti-monkey CRP antibody coated microtiter plate with 96 wells (provided as 12 detachable strips of 8)
Enzyme Conjugate Reagent, 11 mL
Monkey CRP Calibrator, lyophilized
Diluent (10X), 25 mL
Wash Solution (20X), 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 tubes
Vortex mixer
Absorbent paper or paper towels
Micro-Plate incubator/shaker mixing speed of ~150 rpm
A microtiter plate reader with an capable of measuring absorbance at 450 nm
Graph paper (PC graphing software is optional)

Application Details

Plate: Pre-coated

Protocol: The assay uses affinity purified anti-monkey CRP antibodies for solid phase (microtiter wells) immobilization and horseradish peroxidase (HRP) conjugated anti-monkey CRP 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 45 minutes. This results in CRP 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 CRP is proportional to the optical density of the test sample.

Sample Preparation:

General Note: We find that CRP is present in normal pooled monkey serum at a concentration of ~5 µg/mL and in-house studies indicate that acute phase concentrations can exceed 150 µg/mL. We suggest that samples initially be diluted 1,000 fold using the following procedure for each sample to be tested:
1. Dispense 999 µL of 1X diluent into one tube for each sample to be tested.
2. Pipette 1.0 µL of the serum sample into the tube containing 999 µL of 1X diluent using a precision micro pipettor and mix. This provides a 1,000 fold diluted sample.
3. 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 diluted samples into the wells (we recommend that samples be tested in duplicate).
   3. Incubate on an orbital micro-plate shaker at 150 rpm at room temperature (18-25°C) for 45 minutes.
   4. Remove the incubation mixture by flicking plate contents into an appropriate Bio-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 wash solution.
   7. Add 100 µL of enzyme conjugate reagent into each well.
   8. Incubate on an orbital micro-plate shaker at 150 rpm at room temperature (18-25°C) for 45 minutes.
   9. Wash as detailed in 4 to 5 above.
   10. Strike the wells sharply onto absorbent paper or paper towels to remove residual wash solution.
   11. Dispense 100 µL of TMB Reagent into each well.
   12. Gently mix on an orbital micro-plate shaker at 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 15 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 CRP in ng/mL from the calibration curve.
   4. Multiply the derived concentration by the dilution factor to determine the actual concentration of CRP in the serum sample.
   5. If available, PC graphing software may be used for the above steps. We find that data usually fit well to a two site binding (hyperbola) equation.
   6. If the A450 values of samples fall outside, or at the extremes, of the calibration curve when tested at a dilution of 1,000, samples should be diluted appropriately and re-tested.

Restrictions: For Research Use only

Handling

Storage: 4 °C/-20 °C

Storage Comment: The lyophilized reference Calibrator should be stored at or below -20°C for optimum stability. 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 CRP

Target: CRP (C-Reactive Protein (CRP))

Alternative Name: CRP

Background: CRP is an acute phase protein in monkeys that is elevated in serum as a result of injury, infection or disease. Normal levels of CRP range from 0-8.3 µg/mL and levels may increase one hundred fold or more during the acute phase response.

Pathways: Carbohydrate Homeostasis