Clusterin ELISA Kit

Catalog No. ABIN956107

Mouse Clusterin ELISA Kit, Colorimetric assay for quantification of Mouse Clusterin.

Quick Overview for Clusterin ELISA Kit (ABIN956107)

Target: Clusterin (CLU)

Reactivity: Mouse

Detection Method: Colorimetric

Method Type: Sandwich ELISA

Application: ELISA

Sample Type: Plasma, Serum

Product Details Clusterin ELISA Kit

Analytical Method: Quantitative

Characteristics: The Mouse Clusterin ELISA uses two different affinity purified antibodies. One is used for solid phase immobilization (on the microtiter wells). The second is conjugated to horse radish peroxidase (HRP). The samples (diluted serum, plasma or urine) are mixed in the microtiter wells with HRP conjugated antibody and clusterin is sandwiched between the solid phase and HRP-conjugated antibodies. After incubation on a plate shaker for one hour at room temperature the wells are washed to remove unbound HRP-conjugated antibodies. A solution of tetramethylbenzidine (TMB), an HRP substrate, is then added and incubated for 20 minutes, resulting in the development of blue color. The color development is stopped by the addition of 1N HCl changing the color to yellow. The concentration of clusterin is proportional to the absorbance at 450 nm.

Components: Anti-mouse clusterin coated wells (1 plate, 96 wells)
Mouse Clusterin Calibrator: Lyophilized clusterin (reconstitute with 0.20 mL H2O)
Diluent (10X), 25 mL
Wash Solution (20X), 50 mL
Anti-clusterin HRP Conjugate, 11 mL
TMB Reagent (HRP substrate solution), 11 mL
Stop Solution (1N HCl), 11 mL.

Material not included: Distilled or de-ionized water
Pipettes: P-10, P-200 & P-1000 or equivalent
Disposable pipette tips
Plate reader capable of reading OD at 450 nm
Vortex mixer
Absorbent paper
Graph paper or appropriate PC graphing software
Polypropylene microcentrifuge tubes (1.5 mL)

Application Details

Plate: Pre-coated

Sample Preparation:

Serum or plasma should be prepared as quickly as possible after blood collection. If samples cannot be assayed immediately they should be frozen at -70°C and thawed only once prior to use. Samples must not contain azide because this inactivates the HRP conjugate. Clusterin is generally present in normal mouse serum or plasma at a concentration of ~ 10 µg/mL. In order to obtain values within the range of the calibration curve we suggest that samples be diluted 100 fold by mixing 3 µL of sample with 297 µL of 1X diluent.

Assay Procedure:

1. Secure the desired number of coated wells in the holder.
   2. Dispense 100 µL of samples into the appropriate wells (diluted samples must be added before the HRP conjugate).
   3. Dispense 100 µL of HRP conjugate into each well.
   4. Incubate on an orbital shaker (150 rpm) at room temperature (18-25°C) for 60 minutes.
   5. Remove the incubation mixture using a plate washer or by flicking plate contents into a Bio-waste container.
   6. Wash and empty the microtiter wells 6 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.
   7. Strike the wells sharply onto absorbent paper or paper towels to remove all residual droplets.
   8. Dispense 100 µL of TMB Reagent into each well.
   9. Incubate on an orbital micro-plate shaker (150 rpm) at room temperature for 20 minutes.
   10. Stop the reaction by adding 100 µL of Stop Solution to each well.
   11. Gently mix until all the blue color changes to yellow.
   12. Read absorbance at 450 nm with a plate reader within 15 minutes. Please Note: Due to plate reader differences, the high calibrator absorbance values may be out of range occasionally. If this occurs, absorbance values may be determined at 405 nm instead.
   13. If absorbance values exceed the high calibrator, the samples should be appropriately diluted and re-determined.

Calculation of Results:

1. Calculate the mean absorbance value (A450) for the calibrators and samples.
   2. Construct a calibration curve by plotting A450 values obtained for each reference calibrator against its concentration in ng/mL on graph paper, with absorbance on the vertical (Y) axis and concentration on the horizontal (X) axis.
   3. Using the A450 values for each sample, determine the corresponding concentration of clusterin (ng/mL) from the calibration curve.
   4. Multiply the derived clusterin value by the dilution factor to obtain the concentration in the original sample.
   5. If available, graphing software may be used to analyze the data. Depending on the range of the calibration curve used, we find that good fits of the data may be obtained with linear regression analysis or using a two-site binding model. Alternatively, calibration curves may be generated using a point-to-point fit.

Restrictions: For Research Use only

Handling

Storage: 4 °C

Storage Comment: Store the kit at 4°C. Keep the microtiter plate in a sealed bag with desiccant to minimize exposure to damp air. The kit expiration date is indicated on the box label.

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

Target Details for Clusterin

Target: Clusterin (CLU)

Alternative Name: Clusterin

Background: Clusterin, also referred to as apolipoprotein J, sulfated glycoprotein-2, glycoprotein III and testosterone-repressed prostate message-2, is a glycoprotein of 70 - 80 kDa, composed of one -subunit and one -subunit, derived from proteolytic cleavage of a precursor peptide. Clusterin is expressed in many tissues and is found in serum, seminal fluid and urine. It has been identified as a potential biomarker of various forms of renal injury and prostate disease.

Pathways: Apoptosis, Negative Regulation of intrinsic apoptotic Signaling