Key Features

	Save Time	Pre-coated 96 well plate
	Quick Start	Kit includes all necessary reagents
	Publication Ready	Reproducible and reliable results

Overview


Product Name:	Mouse Histamine (HIS) ELISA Kit
Product Code:	MOEB2524
Size:	96 Assays
Alias:	Histamine, HIS
Detection Method:	Competitive
Reactivity:	General
Range:	7.8-500 ng/mL
Storage:	4°C for 6 months
Note:	For Research Use Only

Kit Components

Component	Quantity	Storage
ELISA Microplate (Dismountable)	8x12 strips	4°C for 6 months
Lyophilized Standard	2	4°C/ -20°C
Sample/Standard Dlution Buffer	20ml	4°C
Biotin-labeled Antibody (Concentrated)	120ul	4°C (Protection from light)
Antibody Dilution Buffer	10ml	4°C
HRP-Streptavidin Conjugate (SABC)	120ul	4°C (Protect from light)
SABC Dilution Buffer	10ml	4°C
TMB Substrate	10ml	4°C (Protection from light)
Stop Solution	10ml	4°C
Wash Buffer (25X)	30ml	4°C
Plate Sealer	5	-

Other materials required:

Microplate reader with 450 nm wavelength filter
Multichannel Pipette, Pipette, microcentrifuge tubes and disposable pipette tips
Incubator
Deionized or distilled water
Absorbent paper
Buffer resevoir

Protocol

*Note: Protocols are specific to each batch/lot. For the correct instructions please follow the protocol included in your kit.

Equilibrate the TMB substrate for at least 30 min at 37°C beforeuse. When diluting samples and reagents, they must be mixed completely andevenly. It is recommended to plot a standard curve for each test.

Step	Procedure
1.	Add 50µL of Standard, Blank, or Sample per well. The blank well is added with Sample diluent. Solutions are added to the bottom of micro ELISA plate well, avoid inside wall touching and foaming as possible.
2.	Immediately add 50µL of Detection Reagent A working solution to each well. Cover with the Plate sealer. Gently tap the plate to ensure thorough mixing. Incubate for 1 hour at 37°C. Note: if Detection Reagent A appears cloudy warm to room temperature until solution is uniform.
3.	Aspirate each well and wash, repeating the process three times. Wash by filling each well with Wash Buffer (approximately 400µL) (a squirt bottle, multi-channel pipette, manifold dispenser or automated washer are needed) and let it sit in the well for 1-2 minutes. Complete removal of liquid at each step is essential. After the last wash, completely remove remaining Wash Buffer by aspirating or decanting. Invert the plate and pat it against thick clean absorbent paper.
4.	Add 100µL of Detection Reagent B working solution to each well. Cover with the Plate sealer. Incubate for 45 minutes at 37°C.
5.	Repeat the wash process for five times as conducted in step 3.
6.	Add 90µL of Substrate Solution to each well. Cover with a new Plate sealer and incubate for 10-20 minutes at 37°C. Protect the plate from light. The reaction time can be shortened or extended according to the actual color change, but this should not exceed more than 30 minutes. When apparent gradient appears in standard wells, user should terminate the reaction.
7.	Add 50µL of Stop Solution to each well. If color change does not appear uniform, gently tap the plate to ensure thorough mixing.
8.	Determine the optical density (OD value) of each well at once, using amicro-plate reader set to 450 nm. User should open the micro-plate reader in advance, preheat the instrument, and set the testing parameters.
9.	After the experiment, store all reagents according to the specified storage temperature respectively until their expiry.

Sample Preparation

When carrying out an ELISA assay it is important to prepare your samples in order to achieve the best possible results. Below we have a list of procedures for the preparation of samples for different sample types.

Sample Type

Protocol

Serum

If using serum separator tubes, allow samples to clot for 30 minutes at room temperature. Centrifuge for 10 minutes at 1,000x g. Collect the serum fraction and assay promptly or aliquot and store the samples at -80°C. A

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