Human 25 Hydroxy Vitamin D (25 Hydroxy Vitamin D) ELISA Kit (AEFI01115)

Product Name:	Human 25 Hydroxy Vitamin D (25 Hydroxy Vitamin D) ELISA Kit
SKU:	AEFI01115
Size:	96T
Reactivity:	Human
Range:	3.125-200ng/ml
Sensitivity:	1.875ng/ml
Detection Method:	Competitive ELISA, Coated with Antigen
Detection Wavelength:	OD450
Reaction Duration:	2 hours
Synonyms:	25 Hydroxy Vitamin D ELISA Kit, 25-HVD ELISA Kit

Kit Components:

Components	Quantity		Storage
	48T	96T
ELISA Microplate(Dismountable)	8×6	8×12	Put the rest strips into a sealed foil bag with the desiccant. Stored for 1 month at 2-8°C; Stored for 6 month at -20°C
Lyophilized Standard	1vial	2vial	Put the rest standards into a desiccant bag. Stored for 1 month at 2-8°C; Stored for 6 month at -20°C
Lyophilized Biotin-labeled Antibody(Concentrated)	1vial	1vial
HRP-Streptavidin Conjugate(SABC, 100X)	60ul	120ul	2-8°C (Avoid Direct Light)
TMB Substrate	5ml	10ml	2-8°C (Avoid Direct Light)
Purified Water	200ul	200ul	2-8°C
Sample Dilution Buffer	10ml	20ml
Antibody Dilution Buffer	5ml	10ml
SABC Dilution Buffer	5ml	10ml
Stop Solution	5ml	10ml
Wash Buffer(Concentrated, 25X)	15ml	30ml
Plate Sealer	3 pieces	5 pieces
Manual	1 copy	1 copy

Other Materials Required (Not provided)

Microplate reader (wavelength: 450nm)
37°C incubator (CO2 incubator for cell culture is not recommenced.)
Automated plate washer or multi-channel pipette/5ml pipettor (for manual washing purpose)
Precision single (0.5-10μL, 5-50μL, 20-200μL, 200-1000μL) and multi-channel pipette with disposable tips(Calibration is required before use.)
Sterile tubes and Eppendorf tubes with disposable tips
Absorbent paper and loading slot
Deionized or distilled water

This kit is based on Competitive-ELISA detection method and takes 2h assay time. The microplate provided in this kit has been precoated with 25 Hydroxy Vitamin D. 25 Hydroxy Vitamin D in the sample or standard competes with a fixed amount of biotin-labeled 25 Hydroxy Vitamin D for sites on a pre-coated antibody specific to 25 Hydroxy Vitamin D. Free components are washed off. After adding HRP-Streptavidin Conjugate (SABC), biotin specifically binds with streptavidin to form immune complexes. Wash off unbound conjugate. Add TMB substrate solution. Then, TMB was catalyzed by HRP to produce a blue color product that turned yellow after adding a stop solution. Read the O.D. absorbance at 450nm in a microplate reader. Compare OD450 value of sample with the standard curve through curve fitting software to determine the concentration of 25 Hydroxy Vitamin D in the sample. The concentration of the target substance is inversely proportional to the OD450 value.

Recovery:

Add a certain amount of 25 Hydroxy Vitamin D into the sample. Calculate the recovery by comparing the measured value with the expected amount of 25 Hydroxy Vitamin D in the sample.

Sample Type	Recovery Range(%)	Average(%)
Serum(n=5)	85-105	95
EDTA Plasma(n=5)	89-103	95
Heparin Plasma(n=5)	88-103	93

Linearity:

Dilute the sample with a certain amount of 25 Hydroxy Vitamin D at 1:2, 1:4 and 1:8 to get the recovery range.

Sample Type	1:2	1:4	1:8
Serum(n=5)	86-103%	85-99%	80-98%
EDTA Plasma(n=5)	90-104%	84-101%	82-99%
Heparin Plasma(n=5)	89-101%	82-97%	83-100%

Precision:

Intra-assay Precision: samples with low, medium and high concentration are tested 20 times on the same plate.

Inter-assay Precision: samples with low, medium and high concentration are tested 20 times on three different plates.

Item	Intra-assay Precision			Inter-assay Precision
Sample	1	2	3	1	2	3
n	20	20	20	20	20	20
Mean (ng/ml)	5.87	25.42	99.7	6.14	24.97	98.26
Standard deviation	0.31	1.19	4.98	0.24	1.28	4.89
CV(%)	5.28	4.68	4.99	3.89	5.11	4.98

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

Step	Protocol
1.	Wash the plate twice before adding the standard and sample.
2.	Add 50ul standard or sample into each well. Immediately add 50ul biotin-labeled antibody working solution into each well(The tip can't be reused after touching the liquid.), gently shake the plate for 1min, seal closely and statically incubate for 45 minutes at 37°C.
3.	Washing: Wash the plate three times and immerse for 1min each time.
4.	Add 100ul HRP-Streptavidin Conjugate (SABC) working solution into each well, seal the plate and statically incubate for 30 minutes at 37°C.
5.	Washing: Wash the plate five times and immerse for 1min each time.
6.	Add 90ul TMB substrate solution, seal the plate and statically incubate for 10-20 minutes at 37°C. (Accurate TMB visualization control is required.)
7.	Add 50ul stop solution. Read at 450nm immediately and calculate.

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	Place whole blood sample at room temperature for 2 hours or at 2-8°C overnight. Centrifuge for 20min at 1000xg and collect the supernatant to detect immediately. Or you can aliquot the supernatant and store it at -20°C or -80°C for future’s assay..
Plasma	EDTA-Na2/K2 is recommended as the anticoagulant. Centrifuge samples for 15 minutes at 1000×g 2-8°C within 30 minutes after collection. Collect the supernatant to detect immediately. Or you can aliquot the supernatant and store it at -20°C or -80°C for future’s assay. For other anticoagulant types and uses, please refer to the sample preparation guideline..
Tissue Sample	Generally tissue samples are required to be made into homogenization. Protocol is as below: 3.1. Place the target tissue on the ice. Remove residual blood by washing tissue with pre-cooling PBS buffer (0.01M, pH=7.4). Then weigh for usage. 3.2. Use lysate to grind tissue homogenates on the ice. The adding volume of lysate depends on the weight of the tissue. Usually, 9mL PBS would be appropriate to 1 gram tissue pieces. Some protease inhibitors are recommended to add into the PBS (e.g. 1mM PMSF). 3.3. Do further process using ultrasonic disruption or freeze-thaw cycles (Ice bath for cooling is required during ultrasonic disruption; Freeze-thaw cycles can be repeated twice.) to get the homogenates. 3.4. Homogenates are then centrifuged for 5 minutes at 5000×g. Collect the supernatant to detect immediately. Or you can aliquot the supernatant and store it at -20°C or -80°C for future’s assay. 3.5. Determine total protein concentration by BCA kit for further data analysis. Usually, total protein concentration for Elisa assay should be within 1-3mg/ml. Some tissue samples such as liver, kidney, pancreas which containing a higher endogenous peroxidase concentration may react with TMB substrate causing false positivity. In that case, try to use 1% H2O2 for 15min inactivation and perform the assay again. Notes:PBS buffer or the mild RIPA lysis can be used as lysates While using RIPA lysis, make the PH=7.3. Avoid using any reagents containing NP-40 lysis buffer, Triton X-100 surfactant, or DTT due to their severe inhibition for kits’ working. We recommend using 50mM Tris+0.9%NaCL+0.1%SDS, PH7.3. You can prepare by yourself or contact us for purchasing.
Cell Culture Supernatant	Collect the supernatant: Centrifuge at 2500 rpm at 2-8℃ for 5 minutes, then collect clarified cell culture supernatant to detect immediately. Or you can aliquot the supernatant and store it at -80°C for future’s assay..
Cell Lysate	5.1. Suspension Cell Lysate: Centrifuge at 2500 rpm at 2-8℃ for 5 minutes and collect cells. Then add pre- cooling PBS into collected cell and mix gently. Recollect cell by repeating centrifugation. Add 0.5-1ml cell lysate and appropriate protease inhibitor (e.g. PMSF, working concentration: 1mmol/L). Lyse the cell on ice for 30min-1h or disrupt the cell by ultrasonic disruption. 5.2. Adherent Cell Lysate: Absorb supernatant and add pre-cooling PBS to wash three times. Add 0.5-1ml cell lysate and appropriate protease inhibitor (e.g. PMSF, working concentration: 1mmol/L). Scrape the adherent cell with cell scraper. Lyse the cell suspension added in the centrifuge tube on ice for 30min-1h or disrupt the cell by ultrasonic disruption. 5.3. During lysate process, use the tip for pipetting or intermittently shake the centrifugal tube to completely lyse the protein. Mucilaginous product is DNA which can be disrupted by ultrasonic cell disruptor on ice. (3~5mm probe, 150-300W, 3~5 s/time, 30s intervals for 1~2s working). 5.4. At the end of lysate or ultrasonic disruption, centrifuge at 10000rpm at 2-8℃ for 10 minutes. Then, the supernatant is added into EP tube to detect immediately. Or you can aliquot the supernatant and store it at - 80°C for future’s assay. Notes:Read notes in tissue sample.
Other Biological Sample	Centrifuge samples for 15 minutes at 1000×g at 2-8℃. Collect the supernatant to detect immediately. Or you can aliquot the supernatant and store it at -80°C for future’s assay..

Sample Type:	Serum, Plasma, Cell culture supernatant, Cell or tissue lysate, Other liquid samples
Storage:	2-8°C(Sealed), Don't cryopreserve.
Specificity:	Specifically binds with 25 Hydroxy Vitamin D , no obvious cross reaction with other analogues.