Hemoglobin Measurements - Animal Models of Acute Neurological Injuries: Injury and Mechanistic Assessments - page 629

1.6. Advantages
and Limitations

1. Detects Hb in multiple species, including rat, mouse, pig, and
human.
2. Measures Hb levels in brain, serum, plasma, and blood.
3. The assay is simple and high throughput ( 1 ).
4. Assays can be executed in a 96-well plate or a cuvette.
5. Reagents are nontoxic.

1.6.1. Advantages

The major limitation of this assay is detection limit. The assay only
measures from 0.9 to 200 mg/dL Hb.

1.6.2. Limitations

2. Rat Hemoglobin
ELISA

2.1. Materials

The following instruments and materials for this ELISA assay are
recommended:

1. Electronic balance
2. Homogenizer
3. Microplate aspirator
4. Microplate reader
5. Rat Hemoglobin ELISA kit (Kamiya Biomedical Company)
6. Pipetting devices and Pipet Tips
7. 0.1 mol/L PBS
8. Test tubes
9. Centrifuge
10. Distilled water
11. Timer
12. Vortex mixer

2.2. Rat Hb ELISA Kit
Components

1. Diluent concentrate.
2. Wash solution concentrate.
3. Enzyme-antibody conjugate concentrate.
4. TMB substrate solution.
5. Stop solution.
6. Microtiter plate. Wells are coated with affi nity-purifi ed anti-rat Hb.
7. Rat Hb calibrator.

2.3. Reagent
Preparation

1. The diluent solution supplied is a 5× concentrate and must be
diluted 1:5 with distilled or deionized water.
2. The wash solution supplied is a 20× concentrate and must be
diluted 1:20 with distilled or deionized water.