Human alpha 1 Antitrypsin ELISA Kit is a sandwich Enzyme-Linked Immunosorbent Assay (sELISA) designed for the in vitro quantitative determination of human alpha 1 Antitrypsin in serum, plasma, tissue homogenates, and other biological fluids.

Tipo di kit

Sandwich (quantitative)

Principio di kit

Human alpha 1 Antitrypsin ELISA Kit (A75959) employs the sandwich enzyme immunoassay technique for the quantitative measurement of human alpha 1 Antitrypsin in serum, plasma, tissue homogenates, and other biological fluids. An antibody specific for alpha 1 Antitrypsin has been pre-coated onto a 96-well microtiter plate. The standards and test samples are added into the wells and the alpha 1 Antitrypsin 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-alpha 1 Antitrypsin Antibody, which binds the captured alpha 1 Antitrypsin 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 alpha 1 Antitrypsin captured in each well. The concentration of alpha 1 Antitrypsin 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

4.688 ng/ml

Campo di rilevamento

7.813-500 ng/ml

Tempo del kit

4 hours

Reattività

Human

Recovery

Sample Type	n	Range	Average
Serum	5	87% - 100%	96%
EDTA Plasma	5	87% - 105%	95%
Heparin Plasma	5	90% - 99%	94%

Linearity

Sample Type	n	1:2	1:4	1:8
Serum	5	85-98%	87-95%	86-103%
EDTA Plasma	5	89-96%	82-88%	88-101%
Heparin Plasma	5	86-100%	80-95%	87-99%

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!

Sinonimi

A1A, A1AT, A1AT_HUMAN, AAT, Alpha 1 antiproteinase, Alpha 1 antitrypsin null, Alpha 1 protease inhibitor, Alpha-1 protease inhibitor, Alpha-1-antiproteinase, alpha1 proteinase inhibitor, Alpha1AT, Dom1, PI, PI1, PRO2275, Serine (or cysteine) proteinase inhibitor clade A member 1, Serine protease inhibitor 1-1, Serine protease inhibitor A1a, Serpin A1, Serpin A1a, Serpin peptidase inhibitor clade A member 1, Serpina1, Short peptide from AAT, SPAAT, Spi1-1

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	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.

Immagini (1)

Clicca sull'immagine per la visualizzazione estesa

Figure 1: Standard Curve - Human alpha 1 Antitrypsin ELISA Kit (A75959)

Representative standard curve for Human alpha 1 Antitrypsin ELISA Kit (A75959).

Pubblicazioni (1)

Visualizza pubblicazione

Relationship between ß 1-AA and AT1-AA and Cardiac Function in Patients with Hypertension Complicated with Left Ventricular Diastolic Function Limitation

Riferimenti: Human alpha 1 Antitrypsin ELISA Kit (A75959)

Astratto:

Objective: To investigate the association between ß1 adrenergic receptor autoantibodies (ß1-AA) and angiotensin II type-1 receptor autoantibodies (AT1-AA) and cardiac function in patients with hypertension complicated with left ventricular diastolic function limitation.

Methods: A total of 120 patients with essential hypertension who were not taking drug treatment and were hospitalised in the Department of Cardiology at the authors' hospital from April 2018 to December 2018 were enrolled in this study and divided into a diastolic dysfunction group (65 cases) and a normal diastolic group (55 cases) according to their left ventricular diastolic function. The levels of cardiac parameters, ß1-AA, AT1-AA, and other indicators were compared. Logistic regression analysis was used to analyse the related factors affecting left ventricular diastolic dysfunction (LVDD). The diagnostic efficacy of related factors in the diagnosis of diastolic dysfunction was evaluated.

Results: Univariate analysis demonstrated that the left ventricular posterior wall diameter (10.29 ± 1.23 vs. 9.12 ± 1.53), left ventricular systolic dysfunction (10.56 ± 1.37 vs. 9.43 ± 1.44), systolic blood pressure (152.37 ± 10.24 vs. 140.33 ± 5.99), diastolic blood pressure (95.66 ± 6.34 vs. 87.33 ± 7.28), ß1-AA (33 vs. 9 cases), and AT1-AA (35 cases vs. 12 cases) were higher in the dysfunction group than in the control group (all P < 0.05). Multivariate regression analysis showed that ß1-AA (odds ratio (OR) = 1.96, 95% confidence interval (CI): 1.369-4.345) and AT1-AA (OR = 2.02, 95% CI: 1.332-6.720) were independent risk factors for cardiac diastolic dysfunction (P < 0.05). Both autoimmune antibodies had a certain predictive value, and the combined prediction value of the two was the highest, with an area under the curve of 0.942 (95% CI: 0.881~0.985).

Conclusion: The positive rate of ß1-AA and AT1-AA in essential hypertension patients with LVDD was higher than that in the normal group. Both ß1-AA and AT1-AA could be used as early markers of LVDD in essential hypertension patients.

Visualizza pubblicazione