Microalbumin ELISA Kit

Catalog No. ABIN956069

Product Details

Target: Microalbumin

Reactivity: Dog

Detection Method: Colorimetric

Method Type: Sandwich ELISA

Application: ELISA

Purpose: The Dog Microalbumin ELISA is a highly sensitive two-site enzyme-linked immunoassay (ELISA) for the quantitative determination of microalbumin in dog biological fluids.

Analytical Method: Quantitative

Characteristics: In this assay the albumin present in the sample reacts with the anti-Alb antibody, which has been adsorbed to the surface of polystyrene microtiter wells. After the removal of unbound proteins by washing, anti-Alb antibody conjugated with horseradish peroxidase (HRP) is added. This HRP-conjugated antibody forms a complex with the previously bound albumin. Following another washing step, the enzyme bound to the immunosorbent is assayed by the addition of a chromogenic substrate, 3,3',5,5'- tetramethylbenzidine (TMB). The quantity of bound enzyme is proportional to the concentration of albumin in the sample tested, thus, the absorbance, at 450 nm, is a measure of the concentration of albumin in the test sample. The quantity of albumin in the test sample can be interpolated from the calibration curve constructed from the calibrators and corrected for sample dilution.

Components: 1. Diluent Concentrate: One bottle containing 50 mL of a 5X concentrated diluent running buffer.
2. Wash Solution Concentrate: One bottle containing 50 mL of a 20X concentrated wash solution.
3. Enzyme-Antibody Conjugate Concentrate: One vial containing 150 µL of a 100X concentrated affinity-purified anti-dog albumin antibody conjugated with HRP in a stabilizing buffer.
4. TMB Substrate Solution: One vial containing 12 mL of TMB and hydrogen peroxide in citric acid buffer at pH 3.3.
5. Stop Solution: One vial containing 12 mL of 0.3 M sulfuric acid. WARNING: Avoid contact with skin.
6. Microtiter Plate: Twelve removable eight-well strips in well holder frame. Wells are coated with affinity-purified anti-dog albumin.
7. Dog Microalbumin Calibrator: One vial containing a lyophilized Dog Microalbumin Calibrator.
8. Positive Control: One vial containing 50 µL of serum with 0.1% sodium azide. See the control certificate for the concentration.

Material not included: Precision pipettes (2 µL to 200 µL) for making and dispensing dilutions
Test tubes
Microplate washer/aspirator
Distilled or de-ionized H2O Microplate reader
Assorted glassware for the preparation of reagents and buffer solutions
Timer
Vortex mixer

Application Details

Plate: Pre-coated

Reagent Preparation:

1. Diluent Concentrate: The Diluent solution supplied is a 5X concentrate and must be diluted 1:5 with distilled or de-ionized water.
   2. Wash Solution Concentrate: The Wash Solution supplied is a 20X concentrate and must be diluted 1:20 with distilled or de-ionized water. Crystal formation in the concentrate is not uncommon when storage temperatures are low. Warming of the concentrate to 30-35°C before dilution can dissolve crystals.
   3. Enzyme-Antibody Conjugate Concentrate: Calculate the required amount of working conjugate solution for each microtiter plate test strip by adding 10 µL Enzyme-Antibody Conjugate to 990 µL of 1X Diluent for each test strip to be used for testing. Mix uniformly, but gently. Avoid foaming.
   4. TMB Substrate Solution: Ready to use as supplied.
   5. Stop Solution: Ready to use as supplied.
   6. Microtiter Plate: Ready to use as supplied. Unseal Microtiter Pouch and remove plate from pouch. Remove all strips and wells that will not be used in the assay and place back in pouch and re-seal along with desiccant.
   7. Dog Microalbumin Calibrator: Add 2.0 mL of distilled or de-ionized water to the lyophilized Dog Microalbumin Calibrator and mix gently until dissolved. The calibrator is now at a concentration of 96.8 µg/mL (the reconstituted calibrator should be aliquoted and frozen if future use is intended). Dog Microalbumin Calibrators need to be prepared immediately prior to use. Mix well between each step. Avoid foaming.
   8. Positive Control: The concentration and recommended dilution are provided on the control certificate. Before use, briefly centrifuge the Positive Control to allow all of the liquid to collect in the bottom of the vial.

Calculation of Results:

1. Subtract the average background value from the test values for each sample.
   2. Using the results observed for the calibrators construct a calibration curve. The appropriate curve fit is that of a four- parameter logistics curve, although a second order polynomial (quadratic) or other curve fits may also be used.
   3. Interpolate test sample values from calibration curve. Correct for sample dilution factor to arrive at microalbumin concentration in original sample.
   
   1. Reliable and reproducible results will be obtained when the assay procedure is carried out with a complete understanding of the information contained in the package insert instructions and with adherence to good laboratory practice.
   2. Factors that might affect the performance of the assay include proper instrument function, cleanliness of glassware, quality of distilled or de-ionized water, and accuracy of reagent and sample pipettings, washing technique, incubation time or temperature.

Restrictions: For Research Use only

Handling

Storage: 4 °C

Storage Comment: 1. Complete Kit: The expiration date for the kit is stated on the outer label. The recommended storage temperature is 4°C. Note: See long term storage recommendations below for the Dog Microalbumin Calibrator and Positive Control.
2. Diluent: The 5X Diluent Concentrate is stable until the expiration date. The 1X working solution is stable for at least one week from the date of preparation. Both solutions should be stored at 4°C.
3. Wash Solution: The 20X Wash Solution Concentrate is stable until the expiration date. The 1X working solution is stable for at least one week from the date of preparation. Both solutions can be stored at room temperature (RT, 16-25°C) or at 4°C.
4. Enzyme-Antibody Conjugate: Undiluted anti-Alb-HRP conjugate should be stored at 4°C and diluted immediately prior to use. The working conjugate solution is stable for up to 8 hours.
5. TMB Substrate Solution: The TMB Substrate Solution should be stored at 4°C and is stable until the expiration date.
6. Stop Solution: The Stop Solution should be stored at 4°C and is stable until the expiration date.
7. Microtiter Plate: Anti-dog albumin coated wells are stable until the expiration date, and should be stored at 4°C in the sealed foil pouch with desiccant pack.
8. Dog Microalbumin Calibrator: The lyophilized Dog Microalbumin Calibrator should be stored at 4°C or frozen until reconstituted. The reconstituted calibrator should be aliquoted and stored frozen. Avoid multiple freeze/thaw cycles. The working calibrator solutions should be prepared immediately prior to use and are stable for up to 8 hours.
9. Positive Control: For storage longer than 7 days keep frozen until the expiration date. Storage less than 7 days can be at 4°C. Avoid multiple freeze/thaw cycles.

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

Target Details

Target: Microalbumin

Background: Albumin (Alb) is an amazing polyfunctional protein contributing to homeostasis through mechanisms of hemodynamics, transport and nutrition. Albumin is found both intra- and extra-vascularly in all mammals and many lower vertebrates. It is a molecule of about 67,000 daltons, synthesized by the liver. Normally only very trace amounts of albumin escape reabsorption by kidney glomeruli and are excreted into the urine. Many occult diseases can cause kidney damage that may result in excessive amounts of serum proteins, including albumin, to be excreted by the kidney and into the urine. This ELISA can be used to measure albumin in serum, urine, tissue extracts and other biological fluids.