Hemoglobin (Hb) is made of four globin chains each carrying a heme group. It is carried by red blood cells and transports oxygen from the lungs to the peripheral tissues to maintain the viability of cells. Quantitation of blood hemoglobin has been a key diagnostic parameter for various diseases such as anemia, polycythemia and dehydration.
Simple, direct and automation-ready procedures for measuring hemoglobin concentration are becoming popular in Research and Drug Discovery. Hemoglobin assay kit is based on an improved Triton/NaOH method, in which the hemoglobin is converted into a uniform colored end product. The intensity of color, measured at 400 nm, is directly proportional to hemoglobin concentration in the sample. The optimized formulation exhibits high sensitivity and substantially reduces interference by substances in the raw samples.
Applications:Direct Assays: total hemoglobin in blood, serum, plasma, urine, etc
Pharmacology: effects of drugs on hemoglobin metabolism
Drug Discovery: HTS for drugs that modulate hemoglobin levels.
Key Features:Sensitive and accurate. Linear detection range 0.9–200 mg /dL hemoglobin in 96-well plate assay
Simple and high-throughput. The “mix-and-read” procedure involves addition of a single working reagent and reading the optical density. Can be readily automated as a high-throughput assay in 96-well plates for thousands of samples per day
Safety. Reagents are non-toxic
Versatility. Assays can be executed in 96-well plate or cuvet.
Kit Contents: (250 tests in 96-well plates)Reagent: 50mlCalibrator: 10mlStorage conditions. The kit is shipped at room temperature. Store reagent and calibrator at 4°C. Shelf life: 12 months after receipt. Precautions: reagents are for research use only. Normal precautions for laboratory reagents should be exercised while using the reagents. Please refer to Material Safety Data Sheet for detailed information.
Procedures:Procedure using 96-well plate:1. Blank and Calibrator. Pipette 50ul water (Blank) and 50ul Calibrator into wells of a clear bottom 96-well plate. Transfer 200ul water into the Blank and Calibrator wells. The diluted calibrator is equivalent to 100 mg/dL hemoglobin
2. Samples. Serum and plasma samples can be assayed directly (n=1). Blood samples should be diluted 100-fold in distilled water (n=100). Transfer 50ul samples into wells (Important: avoid bubble formation during the pipetting steps). Add 200ul Reagent to sample wells and tap plate lightly to mix
3. Incubate 5 min at room temperature. Read OD at 390-405nm (peak 400nm)
Procedure using cuvette:1. Transfer 100ul sample and 1000ul Reagent into a cuvet and tap lightly to mix. Read OD 400 nm against water
2. Transfer 100ul Calibrator and 1000ul water to cuvet. Read OD at 400nm against water.
Calculation:Subtract blank OD (water) from the Calibrator and Sample OD values. The hemoglobin concentration of Sample is calculated as=ODSAMPLE–ODBLANKODCALIBRATOR–ODBLANKX 100 x n (mg/dL)ODSAMPLE, ODCALIBRATOR and ODBLANK are OD values of the sample,the Calibrator and water. 100 mg/dL is the equivalent hemoglobinconcentration of the diluted calibrator. n is the dilution factor (100 forblood samples)
Conversions: 1mg/dL Hb equals 0.156uM, 0.001% or 10 ppm.
Materials Required, But Not Provided:Pipetting devices and accessories
Procedure using 96-well plate: Clear-bottom 96-well plates (e.g. Corning Costar) and plate reader
Procedure using cuvette: Cuvets and spectrophotometer.
Examples:Hb was determined using the 96-well plate protocol. The values were 43.4± 0.4 mg/dL for rat serum, 11.2 ± 1.1 mg/dL for human plasma and 15.4 ± 0.7 g/dL for a mouse whole blood sample.
