In this assay the hemoglobin (HM) present in samples reacts with the anti-HM antibodies, which have been adsorbed to the surface of polystyrene microtitre wells. After the removal of unbound proteins by washing, anti-HM antibodies conjugated with horseradish peroxidase (HRP) are added. These enzyme-labeled antibodies form complexes with the previously bound HM. 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 HM in the sample tested; thus, the absorbance, at 450 nm, is a measure of the concentration of HM in the test sample. The quantity of HM in the test sample can be interpolated from the standard curve constructed from the standards, and corrected for sample dilution.
Summary
Sample
Plasma, Serum
Species Reactivity
Dog
Detection Method
ELISA
Intended Use
The HM test kit is a highly sensitive two-site enzyme linked immunoassay (ELISA) for measuring HM in dog biological samples. If the ELISA is to be used outside the intended use, the user may need to optimize for said use.
Contents of Kit
1. ELISA Micro Plate (Antibody Coated) Description: 12 removable 8-well strips, pre-coated. Preparation: Ready to use. Storage: 4–8°C in sealed foil bag with desiccant. Stability: Stable until expiration.
2. Enzyme Conjugated Detection Antibody (100X) Description: 150μL HRP-conjugated antibody in stabilizing buffer. Preparation: Dilute 1:100 before use. Storage: 4–8°C (dark). Stability: Stable until expiration; working solution unstable.
3. Calibrator Description: Lyophilized (see CoA for details). Preparation: Follow CoA; aliquot reconstituted solution. Storage: 4–8°C (lyophilized); ≤–20°C (reconstituted). Stability: Avoid freeze-thaw cycles.
4. Diluent Concentrate (5X) Description: 50 mL buffer. Preparation: Dilute 1:5 for 1X solution. Storage: 4–8°C. Stability: 1X stable ≤1 week; 5X stable until expiration.
5. Wash Solution Concentrate (20X) Description: 50 mL solution. Preparation: Dilute 1:20 for 1X solution. Storage: 4–8°C. Stability: 1X stable ≤1 week; 20X stable until expiration.
6. Chromogen Substrate Solution Description: 12 mL TMB/H₂O₂ in citric acid (pH 3.3). Preparation: Ready to use. Storage: 4–8°C (dark). Stability: Stable until expiration.
7. STOP Solution Description: 12 mL 0.3 M sulfuric acid. Preparation: Ready to use. Storage: 4–8°C. Stability: Stable until expiration.
Storage
The expiration date for the kit and its components is stated on the box label. All components should be stable up to the expiration date if stored and used per this kit protocol insert.
Citations
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