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QuantiChromTM Hemoglobin Assay Kit
(DIHB-250)
Colorimetric Determination of Total
Hemoglobin at 400 nm
DESCRIPTION
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. BioAssay Systems' QuantiChromTM 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: 50 mL
Calibrator: 10 mL
Storage conditions .
The kit is shipped at room temperature. Store reagent
and calibrator at 4°C. Shelf life: at least 6 months
(see expiry dates on labels).
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 50 μL water (Blank) and 50 μL Calibrator into
wells of a clear bottom 96-well plate. Transfer 200 μL
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 50 μL samples into wells (Important:
avoid bubble formation during the pipetting steps). Add
200 μL 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 100 μL sample and 1000 μL
Reagent into a cuvet and tap lightly to mix. Read OD 400
nm against water.
2. Transfer 100 μL Calibrator and
1000μL 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 ,
ODCALIBRATOR
and ODBLANK
are OD
values of the sample, the Calibrator and water. 100
mg/dL is the equivalent hemoglobin concentration of the
diluted calibrator.
n is
the dilution factor (100 for
blood
samples).
Conversions: 1mg/dL Hb equals
0.156 μM ,
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.
PUBLICATIONS
1. Qin, Z. et al (2007). Hyperbaric
oxygen-induced attenuation of hemorrhagic transformation
after experimental focal transient cerebral ischemia.
Stroke 38:1362-1367.
2. Thaker, P.H. et al (2006). Chronic
stress promotes tumor growth and angiogenesis in a mouse
model of ovarian carcinoma. Nature Med. 12 (8): 939-944.
3. Burne-Taney, M.J. et al (2006).
Decreased capacity of immune cells to cause tissue
injury mediates kidney ischemic preconditioning. J.
Immunology 176: 7015–7020. |
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