Mouse Alanine Transaminase ELISA Kit is a sandwich Enzyme-Linked Immunosorbent Assay (sELISA) designed for the in vitro quantitative determination of mouse Alanine Transaminase in serum, plasma, tissue homogenates, cell lysates or other biological fluids.

Type de test

Sandwich (quantitative)

Principe du test

Mouse Alanine Transaminase ELISA Kit (A2389) employs the sandwich enzyme immunoassay technique for the quantitative measurement of mouse Alanine Transaminase in serum, plasma, tissue homogenates, cell lysates or other biological fluids. An antibody specific for Alanine Transaminase has been pre-coated onto a 96-well microtiter plate. The standards and test samples are added into the wells and the Alanine Transaminase 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-Alanine Transaminase Antibody, which binds the captured Alanine Transaminase present in each well. Following incubation, unbound biotinylated detection antibody is removed by washing, and an HRP-Avidin 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 Alanine Transaminase captured in each well. The concentration of Alanine Transaminase can then be calculated by reading the O.D. absorbance at 450nm in a microplate reader and referring to the standard curve.

Plate-forme

Pre-coated Microplate (12 x 8 well strips)

Méthode de détection

Colourmetric

Instrument

Colorimetric Microplate Reader

Type d'échantillon

Serum, plasma, tissue homogenates, cell lysates or other biological fluids.

Sensibilité

0.057 ng/ml

Gamme de detection

0.156-10 ng/ml

Durée du test

4h 30m

Reactivité

Mouse

Recovery

Sample Type	n	Range	Average
Serum	5	80% - 102%	91%
EDTA Plasma	5	81% - 100%	90%
Heparin Plasma	5	80% - 89%	84%

Linearity

Sample Type	1:2	1:4	1:8	1:16
Serum (n=5)	87-91%	87-107%	74-101%	92-97%
EDTA Plasma (n=5)	90-105%	84-101%	90-101%	79-108%
Heparin Plasma (n=5)	84-95%	92-105%	82-105%	89-91%

Precision

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

Notes générales

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

Stockage

Store kit components at +4°C or -20°C as instructed. Do not use past expiration date!

Synonymes

AAT1, Alanine aminotransferase, Alanine aminotransferase 1, ALAT1_HUMAN, ALT1, Glutamate pyruvate transaminase 1, Glutamic alanine transaminase 1, Glutamic pyruvate transaminase (alanine aminotransferase), Glutamic pyruvic transaminase 1, Glutamic--alanine transaminase 1, Glutamic--pyruvic transaminase 1, gpt, GPT 1, GPT1

Avertissement

Ce produit est uniquement destiné à la recherche. Il n'est pas destiné à un usage diagnostique ou thérapeutique.

Kit Components

Item	Quantity	Storage
Pre-Coated 96 Well Microplate	12 x 8 Well Strips	-20°C
Lyopholized Standard	2 Vials	-20°C
Detection Solution A	120μl	-20°C
Detection Solution B	120µl	-20°C
Wash Buffer (30X)	20ml	+4°C
Sample Dilution Buffer	45ml	-20°C
TMB Substrate	9ml	+4°C
Stop Solution	6ml	+4°C
Plate Sealers	5 Adhesive Strips	-

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.
Hemolysis can greatly impact the validity of test results. Take care to minimize hemolysis.
Samples should be brought to room temperature before starting the assay.

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 a foil pouch at -20°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.

Step 5

Cover with an adhesive plate sealer provided and incubate at 37°C for 2 hours.

Step 6

Remove the plate sealer and aspirate the liquid from the plate.

Step 7

Add 100μl of working Detection Solution A 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 300μl 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 10

Add 100μl of working Detection Solution B 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 300μl of Wash Buffer for 1-2 minutes each time.

Step 13

Add 90μl of TMB Substrate to each well. Cover with a new plate sealer and incubate at 37°C in the dark for 15-25 minutes (do not exceed 30 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.

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.

Images (1)

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Figure 1: Standard Curve - Mouse Alanine Transaminase ELISA Kit (A2389)

Representative standard curve for Mouse Alanine Transaminase ELISA Kit (A2389).

Références (1)

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Modulation of Cytosolic Phospholipase A2 as a Potential Therapeutic Strategy for Alzheimer's Disease

Références : Mouse Alanine Transaminase ELISA Kit (A2389)

Résumé :

Background: Alzheimer's disease (AD) is a neurodegenerative disorder lacking any curative treatment up to now. Indeed, actual medication given to the patients alleviates only symptoms. The cytosolic phospholipase A2 (cPLA₂-IVA) appears as a pivotal player situated at the center of pathological pathways leading to AD and its inhibition could be a promising therapeutic approach.

Objective: A cPLA₂-IVA inhibiting peptide was identified in the present work, aiming to develop an original therapeutic strategy.

Methods: We targeted the cPLA₂-IVA using the phage display technology. The hit peptide PLP25 was first validated in vitro (arachidonic acid dosage [AA], cPLA₂-IVA cellular translocation) before being tested in vivo. We evaluated spatial memory using the Barnes maze, amyloid deposits by MRI and immunohistochemistry (IHC), and other important biomarkers such as the cPLA₂-IVA itself, the NMDA receptor, AßPP and tau by IHC after i.v. injection in APP/PS1 mice.

Results: Showing a high affinity for the C2 domain of this enzyme, the peptide PLP25 exhibited an inhibitory effect on cPLA₂-IVA activity by blocking its binding to its substrate, resulting in a decreased release of AA. Coupled to a vector peptide (LRPep2) in order to optimize brain access, we showed an improvement of cognitive abilities of APP/PS1 mice, which also exhibited a decreased number of amyloid plaques, a restored expression of cPLA₂-IVA, and a favorable effect on NMDA receptor expression and tau protein phosphorylation.

Conclusions: cPLA₂-IVA inhibition through PLP25 peptide could be a promising therapeutic strategy for AD.

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