Luteinizing Hormone ELISA 试剂盒

• Pre-coated, ready to use 96-well strip plate, flat buttom
• Plate sealer for 96 wells
• Reference Standard
• Reference Standard & Sample Diluent
• Biotinylated Detection Antibody (100 x concentrate)
• HRP Conjugate (100 x concentrate)
• Biotinylated Detection Antibody Diluent
• HRP Conjugate Diluent
• Substrate Reagent
• Stop Solution
• Wash Buffer (25 x concentrate)
• Instruction manual

Information on standard material:
The formulation of the standard is 0.01 M PBS. The standard contains additives (1 % BSA).

Information on reagents:
Reagents include 1 M SO₂. Azide, thimerosal, 2-mercaptoethanol (2-ME) or any other poisonous materials are not used.

Information on antibodies:
The provided antibodies and their host vary in different kits. All antibodies are affinity purified

The sensitivity of this assay, or Lower Limit of Detection (LLD) was defined as the lowest protein concentration that could be differentiated from zero. It was determined by adding two standard deviations to the mean optical density value of twenty zero standard replicates and calculating the corresponding concentration.

1. Add 100 µL standard or sample to each well. Incubate for 90 min at 37 °C.
2. Remove the liquid. Add 100 µL Biotinylated Detection Antibody. Incubate for 1 hour at 37 °C.
3. Aspirate and wash 3 times.
4. Add 100 µL HRP Conjugate. Incubate for 30 min at 37 °C.
5. Aspirate and wash 5 times.
6. Add 90 µL Substrate Reagent. Incubate for 15 min at 37 °C.
7. Add 50 µL Stop Solution. Read at 450 nm immediately.
8. Calculation of results.

1. Bring all reagents to room temperature (18-25 °C) before use. If the kit will not be used up in one assay, please only take out the necessary strips and reagents for present experiment, and store the remaining strips and reagents at required condition.
2. Wash Buffer: Dilute 30 mL of Concentrated Wash Buffer with 720 mL of deionized or distilled water to prepare 750 mL of Wash Buffer. Note: if crystals have formed in the concentrate, warm it in a 40 °C water bath and mix it gently until the crystals have completely dissolved.
3. Standard working solution: Centrifuge the standard at 10,000xg for 1 min. Add 1.0 mL of Reference Standard &Sample Diluent, let it stand for 10 min and invert it gently several times. After it dissolves fully, mix it thoroughly with a pipette. This reconstitution produces a working solution of 10mIU/mL(or add 1.0 mL of Reference Standard &Sample Diluent, let it stand for 1-2 min and then mix it thoroughly with a vortex meter of low speed. Bubbles generated during vortex could be removed by centrifuging at a relatively low speed). Then make serial dilutions as needed. The recommended dilution gradient is as follows: 10, 5, 2.5, 1.25, 0.63, 0.32, 0.16, 0mIU/mL. Dilution method: Take 7 EP tubes, add 500μL of Reference Standard & Sample Diluent to each tube. Pipette 500μL of the 10mIU/mL working solution to the first tube and mix up to produce a 5mIU/mL working solution. Pipette 500μL of the solution from the former tube into the latter one according to this step. The illustration below is for reference. Note: the last tube is regarded as a blank. Don't pipette solution into it from the former tube.
4. Biotinylated Detection Antibody working solution: Calculate the required amount before the experiment (100 μL/well). In preparation, slightly more than calculated should be prepared. Centrifuge the Concentrated Biotinylated Detection Antibody at 800xg for 1 min, then dilute the 100x Concentrated Biotinylated Detection Antibody to 1x working solution with Biotinylated Detection Antibody Diluent(Concentrated Biotinylated Detection Antibody: Biotinylated Detection Antibody Diluent= 1: 99).
5. HRP Conjugate working solution: Calculate the required amount before the experiment (100 μL/well). In preparation, slightly more than calculated should be prepared. Centrifuge the Concentrated HRP Conjugate at 800xg for 1 min, then dilute -6- the 100x Concentrated HRP Conjugate to 1x working solution with HRP Conjugate Diluent(Concentrated HRP Conjugate: HRP Conjugate Diluent= 1: 99). 10 5 2.5 1.25 0.63 0.32 0.16 0