Hemoglobin ELISA Kit

Catalog No. ABIN956136

The Rat Hemoglobin ELISA Kit (ABIN956136) is a Colorimetric ELISA Kit designed to quantify Rat Hemoglobin.

Quick Overview for Hemoglobin ELISA Kit (ABIN956136)

Target: Hemoglobin

Reactivity: Rat

Detection Method: Colorimetric

Method Type: Sandwich ELISA

Application: ELISA

Product Details Hemoglobin ELISA Kit

Purpose: The Rat Hemoglobin ELISA is a highly sensitive two-site enzyme-linked immunoassay (ELISA) for the quantitative determination of Hemoglobin in rat biological samples.

Analytical Method: Quantitative

Characteristics: In this assay the Hemoglobin present in sample reacts with the anti-Hemoglobin antibodies which have been adsorbed to the surface of polystyrene microtiter wells. After the removal of unbound proteins by washing, anti-Hemoglobin antibodies conjugated with horseradish peroxidase (HRP), are added. These enzyme-labeled antibodies form complexes with the previously bound Hemoglobin. 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 varies directly with the concentration of Hemoglobin in the sample tested, thus, the absorbance, at 450 nm, is a measure of the concentration of Hemoglobin in the test sample. The quantity of Hemoglobin 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-rat Hemoglobin 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-rat Hemoglobin.
7. Rat Hemoglobin Calibrator: One vial containing a Rat Hemoglobin Calibrator.

Material not included: Test tubes
Precision pipette (2 µL to 200 µL) for making and dispensing dilutions.
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. Rat Hemoglobin Calibrator: The Rat Hemoglobin Calibrator is now at a concentration of 192 µg/mL. The Rat Hemoglobin calibrators need to be prepared immediately prior to use. Mix well between each step. Avoid foaming.

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. A second order polynomial (quadratic) or other curve fits may also be used.
   3. Interpolate test sample values from the calibration curve. Correct for sample dilution factor to arrive at Hemoglobin concentration in original sample.

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 Rat Hemoglobin Calibrator.
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 horseradish peroxidase anti-Hemoglobin 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-rat Hemoglobin coated wells are stable until the expiration date and should be stored at 4°C in the sealed foil pouch with a desiccant pack.
8. Rat Hemoglobin Calibrator: Long Term Storage : Upon receipt, aliquot the calibrator and store frozen. It will be stable until expiration date. Short Term Storage : Upon receipt, aliquot the calibrator and store frozen. It will be stable until expiration date.

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

Target Details for Hemoglobin

Target: Hemoglobin

Background: Hemoglobin (HM) is the iron containing oxygen transport protein in red blood cells. It picks up oxygen in the lungs and delivers it to the tissues to maintain viability of the cells. Each subunit of hemoglobin is a globular protein with an embedded heme group. Each heme group contains an iron atom and is responsible for the binding of oxygen. Decrease of hemoglobin leads to symptoms of anemia.