25-Hydroxy Vitamin D3 ELISA Kit is a competitive Enzyme-Linked Immunosorbent Assay (cELISA) designed for the in vitro quantitative determination of 25-Hydroxy Vitamin D3 in serum, plasma, tissue homogenates, and other biological fluids.

Tipo di kit

Competitive (quantitative)

Principio di kit

25-Hydroxy Vitamin D3 ELISA Kit (A303874) employs the competitive enzyme immunoassay technique for the quantitative measurement of 25-Hydroxy Vitamin D3 in serum, plasma, tissue homogenates, and other biological fluids. The 96-well microtiter plate has been pre-coated with 25-Hydroxy Vitamin D3 antigen. During the incubation, 25-Hydroxy Vitamin D3 present in the samples or standards competes with the fixed amount of immobilized 25-Hydroxy Vitamin D3 for binding sites on the Biotinylated Anti-25-Hydroxy Vitamin D3 Antibody. The more 25-Hydroxy Vitamin D3 present in a sample or standard, the less Biotinylated Anti-25-Hydroxy Vitamin D3 Antibody that binds to the plate. Following incubation, unbound Biotinylated Anti-25-Hydroxy Vitamin D3 Antibody is removed by washing, and an HRP-Streptavidin conjugate is added to the wells and the microtiter plate is incubated. Following incubation and washing, TMB substrate solution is then used to visualize the HRP enzymatic reaction by catalysis to produce a blue-coloured product that changes to yellow after addition of acidic stop solution. The density of yellow is inversely proportional to the amount of 25-Hydroxy Vitamin D3 present in each sample or standard. The concentration of 25-Hydroxy Vitamin D3 can then be calculated by reading the O.D. absorbance at 450nm in a microplate reader and referring to the standard curve.

Piattaforma

Pre-coated Microplate (12 x 8 well strips)

Tipo di rilevamento

Colorimetric

Instrument

Colorimetric Microplate Reader

Tipo di campione

Serum, plasma, tissue homogenates, and other biological fluids.

Sensibilità

1.875 ng/ml

Campo di rilevamento

3.125-200 ng/ml

Tempo del kit

2 hours

Reattività

Universal

Recovery

Sample Type	n	Range	Average
Serum	10	95% - 102%	97%
EDTA Plasma	10	95% - 104%	100%
Heparin Plasma	10	88% - 104%	96%

Linearity

Sample Type	n	1:2	1:4	1:8
Serum	10	85-104%	82-97%	81-97%
EDTA Plasma	10	86-104%	84-94%	81-100%
Heparin Plasma	10	90-104%	86-101%	81-98%

Precision

Intra-assay CV: < 8%, Inter-assay CV: < 10%

Note generali

Information online is representative. Always refer to the Product Manual inside the kit.

Conservazione

Store at +4°C. Do not use past expiration date!

Dichiarazione di non responsabilità

Questo prodotto è solo per uso di ricerca. Non è inteso per uso diagnostico o terapeutico.

Kit Components

Item	Quantity	Storage
Pre-Coated 96 Well Microplate	12 x 8 Well Strips	+4°C
Lyopholized Standard	2 Vials	+4°C
Sample Dilution Buffer	20ml	+4°C
Biotinylated Detection Antibody	60µl	+4°C
Antibody Dilution Buffer	10ml	+4°C
HRP-Streptavidin Conjugate	120µl	+4°C
SABC Dilution Buffer	10ml	+4°C
TMB Substrate	10ml	+4°C
Stop Solution	10ml	+4°C
Wash Buffer (25X)	30ml	+4°C
Plate Sealers	5 Adhesive Strips	-
Foil Pouch	1 Zip-Sealed Pouch	-

Required Materials

The following materials are not included in the kit, but are required to run this assay:

Microplate reader capable of measuring absorbance at 450nm ± 10nm.
Squirt bottle, multichannel pipette reservoir, or automated microplate washer.
Graph paper or computer software capable of generating logarithmic functions.
Test tubes and Eppendorf tubes to prepare standards and samples.
Multi- and single-channel pipettes and sterile pipette tips.
Deionized or distilled water.
Absorbent paper towels.
37°C incubator.

Important Notes

Sample concentrations should be estimated before being used in the assay and a proper dilution factor should be selected to make the diluted target protein concentration fall in the optimal detection range of this kit.
Sample matrix components will interfere with the test results. All samples must be diluted at least 1:2 with Sample Dilution Buffer.
Hemolysis can greatly impact the validity of test results. Take care to minimize hemolysis.

Bring all reagents and samples to room temperature before use.

We recommend that all standards and samples be assayed in duplicate.

Step 1

Prepare all reagents, samples, and working standards as directed in the previous sections.

Step 2

Determine the number of microplate strips required for the assay and insert them into the plate frame. Unused strips should be stored in the foil pouch at +4°C.

Step 3

Wash the plate two times with Wash Buffer. Soak wells with 350μl of Wash Buffer for 1-2 minutes each time. Note: 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. Blot the plate onto paper towels or other absorbent material. Do not let the wells dry completely at any time!

Step 4

Add 50μl of standard solutions to the standard wells. Note: A plate layout is provided to record standards and samples assayed.

Step 5

Add 50μl of samples to the sample wells. Note: All samples must be properly diluted in order to produce sample values within the dynamic range of the assay.

Step 6

Immediately add 50μl of Biotinylated Detection Antibody working solution to each well. Gently tap the plate to ensure thorough mixing for 1 minute.

Step 7

Cover with an adhesive plate sealer provided and incubate at 37°C for 45 minutes.

Step 8

Remove the plate sealer, aspirate the liquid from the plate, and wash the plate three times with Wash Buffer. Soak wells with 350μl of Wash Buffer for 1-2 minutes each time.

Step 9

Add 100μl of HRP-Streptavidin Conjugate (SABC) working solution to each well.

Step 10

Cover with a new adhesive plate sealer and incubate at 37°C for 30 minutes.

Step 11

Remove the plate sealer, aspirate the liquid from the plate, and wash the plate five times with Wash Buffer. Soak wells with 350μl of Wash Buffer for 1-2 minutes each time.

Step 12

Add 90μl of TMB Substrate to each well. Cover with a new adhesive plate sealer and incubate at 37°C in the dark for 10-20 minutes. Note: This incubation time is for reference only, the optimal reaction time should be determined by the end-user and can be shortened or extended according to the actual color change. The reaction may be terminated when a gradient is apparent in the standard wells. Do not exceed 30 minutes!

Step 13

Add 50μl of Stop Solution to each well. The color in the wells should change from blue to yellow immediately. Note: If the color in the wells is green or the color change does not appear uniform, gently tap the plate to ensure thorough mixing.

Step 14

Determine the optical density (O.D. value) of each well immediately using a microplate reader set to 450 nm.