Human HLA DRB5 ELISA Kit is a competitive Enzyme-Linked Immunosorbent Assay (cELISA) designed for the in vitro quantitative determination of human HLA DRB5 in serum, plasma, tissue homogenates, and other biological fluids.

Assay-Typ

Competitive (quantitative)

Assay-Prinzip

Human HLA DRB5 ELISA Kit (A74409) employs the competitive enzyme immunoassay technique for the quantitative measurement of human HLA DRB5 in serum, plasma, tissue homogenates, and other biological fluids. The 96-well microtiter plate has been pre-coated with HLA DRB5 antigen. During the incubation, HLA DRB5 present in the samples or standards competes with the fixed amount of immobilized HLA DRB5 for binding sites on the Biotinylated Anti-HLA DRB5 Antibody. The more HLA DRB5 present in a sample or standard, the less Biotinylated Anti-HLA DRB5 Antibody that binds to the plate. Following incubation, unbound Biotinylated Anti-HLA DRB5 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 HLA DRB5 present in each sample or standard. The concentration of HLA DRB5 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, tissue homogenates, and other biological fluids.

Sensitivität

0.938 ng/ml

Detektionsbereich

1.563-100 ng/ml

Testdauer

2 hours

Reaktivität

Human

Recovery

Sample Type	n	Range	Average
Serum	5	89% - 103%	94%
EDTA Plasma	5	85% - 102%	91%
Heparin Plasma	5	92% - 105%	98%

Linearity

Sample Type	n	1:2	1:4	1:8
Serum	5	85-105%	85-103%	88-105%
EDTA Plasma	5	83-99%	84-96%	85-96%
Heparin Plasma	5	81-94%	81-99%	88-99%

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

DR 9, DR beta 5, DR beta 5 chain, DR beta-5, DR beta5, DR2 beta 2, DR2-beta-2, DR9, DRB5_HUMAN, Dw2, FLJ76359, HLA class II histocompatibility antigen, HLA class II histocompatibility antigen DR beta 5 chain, HLA class II histocompatibility antigen DRB1-9 beta chain, HLA class II histocompatibility antigen, DR-5 beta chain, HLA DRB, HLA-DRB5, Human leucocyte antigen DRB5, Leukocyte antigen class II, Major histocompatibility complex, class II, DR beta 51, MHC class II antigen DRB1*9, MHC class II antigen DRB5, MHC class II HLA beta chain, MHC HLA-DR-beta cell surface glycoprotein, MHC HLA-DR-beta chain

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	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.

Produktbilder (1)

Bild Vergrößern

Figure 1: Standard Curve - Human HLA DRB5 ELISA Kit (A74409)

Representative standard curve for Human HLA DRB5 ELISA Kit (A74409).

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