S1P (Sphingosine 1 Phosphate) ELISA Kit

Product Name:	S1P (Sphingosine 1 Phosphate) ELISA Kit
Product Code:	AEFI00851
Size:	96 Assays
Detection method:	Competitive, Double Antibody
Sensitivity:	1.875ng/ml
Range:	3.13-200ng/ml
Storage:	2-8°C for 6 months
Note:	For Research Use Only

Recovery:

Matrices listed below were spiked with a certain level of S1P and the recovery rates were calculated by comparing the measured value to the expected amount of S1P in the samples.

Matrix	Recovery range(%)	Average(%)
serum(n=10)	85-104	96
EDTA Plasma(n=10)	87-104	93
Heparin Plasma(n=10)	96-105	93

Linearity:

The linearity of the kit was assayed by testing the samples spiked with appropriate concentration of S1P and their serial dilutions.

Sample	1:2	1:4	1:8
Serum(n=10)	85-105%	92-100%	90-100%
EDTA Plasma(n=10)	85-101%	87-97%	85-101%
Heparin Plasma(n=10)	87-100%	83-100%	81-98%

CV(%):

Intra-Assay: CV<8%
Inter-Assay: CV<10%

Component	Quantity	Storage
ELISA Microplate (Dismountable)	8×12 strips	4°C for 6 months
Lyophilized Standard	2	4°C/-20°C
Sample/Standard Dilution Buffer	20ml	4°C
Biotin-labeled Antibody(Concentrated)	120ul	4°C (Protect 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 (Protect from light)
Stop Solution	10ml	4°C
Wash Buffer(25X)	30ml	4°C
Plate Sealer	5	-

Other materials and equipment 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

Component	Quantity	Storage
ELISA Microplate(Dismountable)	8×12 strips	4°C for 6 months
Lyophilized Standard	2	4°C/-20°C
Sample/Standard Dilution Buffer	20ml	4°C
Biotin-labeled Antibody(Concentrated)	60ul	4°C (Protect 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 (Protect from light)
Stop Solution	10ml	4°C
Wash Buffer(25X)	30ml	4°C
Plate Sealer	5	-

Other materials and equipment 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

*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 before use. When diluting samples and reagents, they must be mixed completely and evenly. It is recommended to plot a standard curve for each test.

Step	Protocol
1.	Set standard, test sample and control (zero) wells on the pre-coated plate respectively, and then, record their positions. It is recommended to measure each standard and sample in duplicate. Wash plate 2 times before adding standard, sample and control (zero) wells!
2.	Add Sample and Biotin-detection antibody: Add 50ÂµL of Standard, Blank or Sample per well. The blank well is added with Sample Dilution Buffer. Immediately add 50 ÂµL of biotin-labelled antibody working solution to each well. Cover with the plate sealer provided. Gently tap the plate to ensure thorough mixing. Incubate for 45 minutes at 37°C. (Solutions are added to the bottom of micro-ELISA plate well, avoid touching plate walls and foaming).
3.	Wash: Aspirate each well and wash, repeating the process three times Wash by filling each well with Wash Buffer (approximately 350µL) using a squirt bottle, multi-channel pipette, manifold dispenser or automated washer. Complete removal of liquid at each step is essential to good performance. After the last wash, remove any remaining Wash Buffer by aspirating or decanting. Invert the plate and pat it against thick clean absorbent paper.
4.	HRP-Streptavidin Conjugate(SABC): Add 100µL of SABC working solution to each well. Cover with a new Plate sealer. Incubate for 30minutes at 37°C.
5.	Wash: Repeat the aspiration/wash process for five times.
6.	TMB Substrate: Add 90µL of TMB Substrate to each well. Cover with a new Plate sealer. Incubate for about 10-20 minutes at 37°C. Protect from light. The reaction time can be shortened or extended according to the actual color change, but not more than 30minutes. When apparent gradient appeared in standard wells, you can terminate the reaction.
7.	Stop: Add 50µL of Stop Solution to each well. Color turn to yellow immediately. The adding order of stop solution should be as the same as the substrate solution.
8.	OD Measurement: Determine the optical density (OD Value) of each well at once, using a microplate reader set to 450 nm. You should open the microplate reader ahead, preheat the instrument, and set the testing parameters.

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. Avoid multiple freeze-thaw cycles. If serum separator tubes are not being used, allow samples to clot overnight at 2-8°C. Centrifuge for 10 minutes at 1,000x g. Remove serum and assay promptly or aliquot and store the samples at -80°C. Avoid multiple freeze-thaw cycles.
Plasma	Collect plasma using EDTA or heparin as an anticoagulant. Centrifuge samples at 4°C for 15 mins at 1000 × g within 30 mins of collection. Collect the plasma fraction and assay promptly or aliquot and store the samples at -80°C. Avoid multiple freeze-thaw cycles. Note: Over haemolysed samples are not suitable for use with this kit.
Urine & Cerebrospinal Fluid	Collect the urine (mid-stream) in a sterile container, centrifuge for 20 mins at 2000-3000 rpm. Remove supernatant and assay immediately. If any precipitation is detected, repeat the centrifugation step. A similar protocol can be used for cerebrospinal fluid.
Cell culture supernatant	Collect the cell culture media by pipette, followed by centrifugation at 4°C for 20 mins at 1500 rpm. Collect the clear supernatant and assay immediately.
Cell lysates	Solubilize cells in lysis buffer and allow to sit on ice for 30 minutes. Centrifuge tubes at 14,000 x g for 5 minutes to remove insoluble material. Aliquot the supernatant into a new tube and discard the remaining whole cell extract. Quantify total protein concentration using a total protein assay. Assay immediately or aliquot and store at ≤ -20 °C.
Tissue homogenates	The preparation of tissue homogenates will vary depending upon tissue type. Rinse tissue with 1X PBS to remove excess blood & homogenize in 20ml of 1X PBS (including protease inhibitors) and store overnight at ≤ -20°C. Two freeze-thaw cycles are required to break the cell membranes. To further disrupt the cell membranes you can sonicate the samples. Centrifuge homogenates for 5 mins at 5000xg. Remove the supernatant and assay immediately or aliquot and store at -20°C or -80°C.
Tissue lysates	Rinse tissue with PBS, cut into 1-2 mm pieces, and homogenize with a tissue homogenizer in PBS. Add an equal volume of RIPA buffer containing protease inhibitors and lyse tissues at room temperature for 30 minutes with gentle agitation. Centrifuge to remove debris. Quantify total protein concentration using a total protein assay. Assay immediately or aliquot and store at ≤ -20 °C.
Breast Milk	Collect milk samples and centrifuge at 10,000 x g for 60 min at 4°C. Aliquot the supernatant and assay. For long term use, store samples at -80°C. Minimize freeze/thaw cycles.