Hemoglobin (HB) BioAssay™ ELISA Kit (Sheep)

The BioAssay™kit is a competitive inhibition enzyme immunoassay technique for the in vitro quantitative measurement of hemoglobin in ovine serum, plasma, erythrocyte lysates and other biological fluids.

Detection Range:
6.17-500ug/ml

Sensitivity:
2.63ug/ml

Specificity:
This assay has high sensitivity and excellent specificity for detection of Hemoglobin (HB). No significant cross-reactivity or interference between Hemoglobin (HB) and analogues was observed.

Precision:
Intra-Assay %CV: <10%
Inter-Assay %CV: <12%

Test Principle:
This assay employs the competitive inhibition enzyme immunoassay technique. A monoclonal antibody specific to hemoglobin has been pre-coated onto a microplate. A competitive inhibition reaction is launched between biotin labeled hemoglobin and unlabeled hemoglobin (Standards or samples) with the pre-coated antibody specific to hemoglobin. After incubation the unbound conjugate is washed off. Next, avidin conjugated to Horseradish Peroxidase (HRP) is added to each microplate well and incubated. The amount of bound HRP conjugate is reverse proportional to the concentration of hemoglobin in the sample. After addition of the substrate solution, the intensity of color developed is reverse proportional to the concentration of hemoglobin in the sample.

Kit Components:
Pre-coated, ready to use 96-well strip plate
Standard, 2x vials
Detection Reagent A, 1x120ul
Detection Reagent B, 1x120ul
TMB Substrate, 1x9ml
Wash Buffer (30X): 1x20ml
Standard Diluent, 1x20ml
Assay Diluent A, 1x12ml
Assay Diluent B, 1x12ml
Stop Solution, 1x6ml

Storage and Stability:
Store Microtiter Strips, Standard, Detection Reagents and Positive Control at -20°C. After reconstitution of Positive Control, store at 4°C and use within 5 days. Store all the other components at 4°C. Unused kit is stable for 6 months after receipt. Once kit components are opened, it is highly recommended to use remaining reagents within 1 month provided this is within the expiration date of the kit. For maximum recovery of product, centrifuge the original vials after thawing and prior to removing the cap.

Assay Procedure Summary:
1. Prepare all reagents, samples and standards.
2. Add 100ul standard or sample to each well. Incubate 1 hour at 37°C.
3. Aspirate and add 100ul prepared Detection Reagent A. Incubate 1 hour at 37°C.
4. Aspirate and wash 3 times.
5. Add 100ul prepared Detection Reagent B. Incubate 30 minutes at 37°C.
6. Aspirate and wash 5 times.
7. Add 90ul Substrate Solution. Incubate 10-20 minutes at 37°C.
8. Add 50ul Stop Solution. Read at 450nm immediately.