Human CYP27A1 ELISA Kit is a sandwich Enzyme-Linked Immunosorbent Assay (sELISA) designed for the in vitro quantitative determination of human CYP27A1 in serum, plasma, cell culture supernatant, cell or tissue lysate, and other liquid samples.

Assay-Typ

Sandwich (quantitative)

Assay-Prinzip

Human CYP27A1 ELISA Kit (A326516) employs the sandwich enzyme immunoassay technique for the quantitative measurement of human CYP27A1 in serum, plasma, cell culture supernatant, cell or tissue lysate, and other liquid samples. An antibody specific for CYP27A1 has been pre-coated onto a 96-well microtiter plate. The standards and test samples are added into the wells and the CYP27A1 present in each sample is bound to the wells by the immobilized antibody. Following incubation, the wells are washed and then incubated with Biotinylated Anti-CYP27A1 Antibody, which binds the captured CYP27A1 present in each well. Following incubation, unbound biotinylated detection 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 proportional to the amount of CYP27A1 captured in each well. The concentration of CYP27A1 can then be calculated by reading the O.D. absorbance at 450nm in a microplate reader and referring to the standard curve.

Plattform

Pre-coated Microplate (12 x 8 well strips)

Nachweismethode

Colorimetric

Instrument

Colorimetric Microplate Reader

Probentyp

Serum, plasma, cell culture supernatant, cell or tissue lysate, and other liquid samples.

Sensitivität

0.094 ng/ml

Detektionsbereich

0.156-10 ng/ml

Testdauer

4 hours

Reaktivität

Human

Recovery

Sample Type	n	Range	Average
Serum	10	86% - 102%	98%
EDTA Plasma	10	85% - 98%	91%
Heparin Plasma	10	90% - 104%	97%

Linearity

Sample Type	n	1:2	1:4	1:8
Serum	10	87-101%	83-100%	81-92%
EDTA Plasma	10	85-105%	82-95%	96-100%
Heparin Plasma	10	87-105%	83-99%	82-100%

Precision

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

Allgemeine Hinweise

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

Lagerung

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

Synonyms

12-alpha-triol 27-hydroxylase, 5-beta-cholestane-3-alpha, 5-beta-cholestane-3-alpha, 7-alpha, 12-alpha-triol 26-hydroxylase, 5-beta-cholestane-3-alpha, 7-alpha, 12-alpha-triol 27-hydroxylase, 5-beta-cholestane-3-alpha,7-alpha,12-alpha-triol 27-hydroxylase, 7-alpha, Cholestanetriol 26 monooxygenase, CP27, CP27A_HUMAN, CTX, CYP, CYP27, Cytochrome P 450C27/25, Cytochrome P-450C27/25, Cytochrome P450 27, Cytochrome P450 27 mitochondrial, Cytochrome P450 family 27 subfamily A member 1, Cytochrome P450 family 27 subfamily A polypeptide 1, Cytochrome P450 subfamily XXVIIA (steroid 27-hydroxylase cerebrotendinous xanthomatosis) polypeptide 1, mitochondrial, Sterol 26 hydroxylase, Sterol 26 hydroxylase mitochondrial, Sterol 26-hydroxylase, Sterol 27 hydroxylase, Sterol 27-hydroxylase, Vitamin D(3) 25 hydroxylase, Vitamin D(3) 25-hydroxylase

Haftungsausschluss

Dieses Produkt ist nur für Forschungszwecke bestimmt. Es ist nicht für diagnostische oder therapeutische Zwecke bestimmt.

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	120µl	+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

Add 100μl of standard solutions to the standard wells. Note: A plate layout is provided to record standards and samples assayed.

Step 4

Add 100μl 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. All samples must be diluted at least 1:2 with Sample Dilution Buffer.

Step 5

Cover with an adhesive plate sealer provided and incubate at 37°C for 90 minutes.

Step 6

Remove the plate sealer, aspirate the liquid from the plate, and 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 7

Add 100μl of Biotinylated Detection Antibody working solution to each well.

Step 8

Cover with a new adhesive plate sealer and incubate at 37°C for 60 minutes.

Step 9

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 10

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

Step 11

Cover with a new adhesive plate sealer and incubate at 37°C for 60 minutes.

Step 12

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 13

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 14

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 15

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