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Mouse ALT ELISA Kit

SKU:
MOFI00634
Product Type:
ELISA Kit
Size:
96 Assays
Sensitivity:
0.094ng/ml
Range:
0.156-10ng/ml
ELISA Type:
Sandwich
Synonyms:
ALT, Alanine Transaminase, ALAT, SGPT, alanine aminotransferase, serum glutamic pyruvic transaminase
Reactivity:
Mouse
$779
Frequently bought together:

Description

Mouse ALT ELISA Kit

Intro paragraph

system_update_alt Datasheet system_update_alt MSDS

Key Features

Save Time Pre-coated 96 well plate
Quick Start Kit includes all necessary reagents
Publication Ready Reproducible and reliable results

Overview

Product Name:

Mouse ALT ELISA Kit

Product Code:

MOFI00634

Size:

96 Assays

Alias:

ALT, Alanine Transaminase, ALAT, SGPT, alanine aminotransferase, serum glutamic pyruvic transaminase

Detection Method:

Sandwich ELISA

Application:

This immunoassay kit allows for the in vitro quantitative determination of Mouse ALT concentrations in serum plasma and other biological fluids.

Sensitivity:

0.094ng/ml

Range:

0.156-10ng/ml

Storage:

4°C for 6 months

Note:

For Research Use Only

Additional Information

Recovery

Matrices listed below were spiked with certain level of Mouse ALT and the recovery rates were calculated by comparing the measured value to the expected amount of Mouse ALT in samples.

Matrix

Recovery Range (%)

Average (%)

serum (n=5)

93-105

99

EDTA plasma (n=5)

91-101

95

UFH plasma (n=5)

87-102

95

Linearity:

The linearity of the kit was assayed by testing samples spiked with appropriate concentration of Mouse ALT and their serial dilutions. The results were demonstrated by the percentage of calculated concentration to the expected.

Sample

1:2

1:4

1:8

Serum (n=5)

87-100%

85-101%

89-99%

EDTA plasma (n=5)

83-100%

85-100%

82-91%

UFH Plasma (n=5)

92-96%

83-89%

83-100%

CV(%)

Intra Assay <8

Inter Assay <10

Kit Components

Component Quantity Storage

ELISA Microplate (Dismountable)

8x12 strips

4°C for 6 months

Lyophilized Standard

2

4°C/ -20°C

Sample/Standard Dlution Buffer

20ml

4°C

Biotin-labeled Antibody (Concentrated)

120ul

4°C (Protection from light)

Antibody Dilution Buffer

10ml

4°C

HRP-Streptavidin Conjugate (SABC)

120ul

4°C (Protect from light)

SABC Dilution Buffer

10ml

4°C

TMB Substrate

10ml

4°C (Protection from light)

Stop Solution

10ml

4°C

Wash Buffer (25X)

30ml

4°C

Plate Sealer

5

-

Other materials required:

  • Microplate reader with 450 nm wavelength filter
  • Multichannel Pipette, Pipette, microcentrifuge tubes and disposable pipette tips
  • Incubator
  • Deionized or distilled water
  • Absorbent paper
  • Buffer resevoir

Protocol

*Note: Protocols are specific to each batch/lot. For the exact instructions please follow the protocol included in your kit.

Before adding to wells, equilibrate the SABC working solution and TMB substrate for at least 30 min at 37°C. When diluting samples and reagents, they must be mixed completely and evenly. It is recommended to plot a standard curve for each test.

Step Procedure

1.

Set standard, test sample and control (zero) wells on the pre-coated plate respectively, and then, record their positions. It is recommended to measure each standard and sample in duplicate. Wash plate 2 times before adding standard, sample and control (zero) wells!

2.

Aliquot 0.1ml standard solutions into the standard wells.

3.

Add 0.1 ml of Sample / Standard dilution buffer into the control (zero) well.

4.

Add 0.1 ml of properly diluted sample ( Human serum, plasma, tissue homogenates and other biological fluids.) into test sample wells.

5.

Seal the plate with a cover and incubate at 37 °C for 90 min.

6.

Remove the cover and discard the plate content, clap the plate on the absorbent filter papers or other absorbent material. Do NOT let the wells completely dry at any time. Wash plate X2.

7.

Add 0.1 ml of Biotin- detection antibody working solution into the above wells (standard, test sample & zero wells). Add the solution at the bottom of each well without touching the side wall.

8.

Seal the plate with a cover and incubate at 37°C for 60 min.

9.

Remove the cover, and wash plate 3 times with Wash buffer. Let wash buffer rest in wells for 1 min between each wash.

10.

Add 0.1 ml of SABC working solution into each well, cover the plate and incubate at 37°C for 30 min.

11.

Remove the cover and wash plate 5 times with Wash buffer, and each time let the wash buffer stay in the wells for 1-2 min.

12.

Add 90 µl of TMB substrate into each well, cover the plate and incubate at 37°C in dark within 10-20 min. (Note: This incubation time is for reference use only, the optimal time should be determined by end user.) And the shades of blue can be seen in the first 3-4 wells (with most concentrated standard solutions), the other wells show no obvious color.

13.

Add 50 µl of Stop solution into each well and mix thoroughly. The color changes into yellow immediately.

14.

Read the O.D. absorbance at 450 nm in a microplate reader immediately after adding the stop solution.

Sample Preparation

When carrying out an ELISA assay it is important to prepare your samples in order to achieve the best possible results. Below we have a list of procedures for the preparation of samples for different sample types.

Sample Type Protocol

Serum

If using serum separator tubes, allow samples to clot for 30 minutes at room temperature. Centrifuge for 10 minutes at 1,000x g. Collect the serum fraction and assay promptly or aliquot and store the samples at -80°C. Avoid multiple freeze-thaw cycles.

If serum separator tubes are not being used, allow samples to clot overnight at 2-8°C. Centrifuge for 10 minutes at 1,000x g. Remove serum and assay promptly or aliquot and store the samples at -80°C. Avoid multiple freeze-thaw cycles.

Plasma

Collect plasma using EDTA or heparin as an anticoagulant. Centrifuge samples at 4°C for 15 mins at 1000 × g within 30 mins of collection. Collect the plasma fraction and assay promptly or aliquot and store the samples at -80°C. Avoid multiple freeze-thaw cycles. Note: Over haemolysed samples are not suitable for use with this kit.

Urine & Cerebrospinal Fluid

Collect the urine (mid-stream) in a sterile container, centrifuge for 20 mins at 2000-3000 rpm. Remove supernatant and assay immediately. If any precipitation is detected, repeat the centrifugation step. A similar protocol can be used for cerebrospinal fluid.

Cell culture supernatant

Collect the cell culture media by pipette, followed by centrifugation at 4°C for 20 mins at 1500 rpm. Collect the clear supernatant and assay immediately.

Cell lysates

Solubilize cells in lysis buffer and allow to sit on ice for 30 minutes. Centrifuge tubes at 14,000 x g for 5 minutes to remove insoluble material. Aliquot the supernatant into a new tube and discard the remaining whole cell extract. Quantify total protein concentration using a total protein assay. Assay immediately or aliquot and store at ≤ -20 °C.

Tissue homogenates

The preparation of tissue homogenates will vary depending upon tissue type. Rinse tissue with 1X PBS to remove excess blood & homogenize in 20ml of 1X PBS (including protease inhibitors) and store overnight at ≤ -20°C. Two freeze-thaw cycles are required to break the cell membranes. To further disrupt the cell membranes you can sonicate the samples. Centrifuge homogenates for 5 mins at 5000xg. Remove the supernatant and assay immediately or aliquot and store at -20°C or -80°C.

Tissue lysates

Rinse tissue with PBS, cut into 1-2 mm pieces, and homogenize with a tissue homogenizer in PBS. Add an equal volume of RIPA buffer containing protease inhibitors and lyse tissues at room temperature for 30 minutes with gentle agitation. Centrifuge to remove debris. Quantify total protein concentration using a total protein assay. Assay immediately or aliquot and store at ≤ -20 °C

Breast Milk

Collect milk samples and centrifuge at 10,000 x g for 60 min at 4°C. Aliquot the supernatant and assay. For long term use, store samples at -80°C. Minimize freeze/thaw cycles.

Alanine Transaminase (ALT) Background

Alanine Transaminase (ALT) / glutamate-pyruvate transaminase (GPT)

Alanine Transaminase (ALT), also known as glutamate-pyruvate transaminase (GPT), is an enzyme that plays a crucial role in amino acid metabolism. It is primarily found in the liver and is involved in the reversible transfer of an amino group between alanine and alpha-ketoglutarate. ALT is important for the breakdown of proteins and the production of energy.

ALT Structure:

The three-dimensional structure of ALT in mice has been determined through X-ray crystallography. The protein adopts a dimeric form, where two identical subunits come together to form an active enzyme. Each subunit consists of multiple domains, including the N-terminal domain, the pyridoxal-5'-phosphate (PLP) binding domain, and the C-terminal domain. The PLP molecule is essential for ALT's enzymatic activity, serving as a cofactor that participates in the transfer of amino groups.

Predicted Structure of Mouse ALT1. Source: Uniprot

ALT/ GPT Function:

The primary function of ALT is to facilitate the interconversion of alanine and alpha-ketoglutarate. By transferring the amino group from alanine to alpha-ketoglutarate, ALT generates pyruvate and glutamate. This transamination reaction is a crucial step in amino acid metabolism, allowing for the utilization of alanine as an energy source and the synthesis of other important molecules.

ALT Clinical Significance

ALT is commonly measured in blood tests to assess liver health. Serum GPT refers to the measurement of the enzyme Alanine Transaminase (ALT) in the bloodstream. The terms "serum GPT" and "ALT" are often used interchangeably in clinical practice.

The clinical significance of measuring serum GPT/ALT lies in its role as a biomarker for liver health. ALT is primarily found in the liver, and its levels in the blood can indicate liver injury or damage. When liver cells are damaged or destroyed, ALT is released into the bloodstream, causing an increase in serum GPT/ALT levels.

Elevated serum GPT/ALT levels are commonly seen in various liver conditions, including viral hepatitis (such as hepatitis B or C), alcoholic liver disease, non-alcoholic fatty liver disease (NAFLD), cirrhosis, drug-induced liver injury, and liver cancer. Monitoring serum GPT/ALT levels helps in diagnosing and assessing the severity of these liver diseases.

Elevated Alanine Transaminase

Normal range of ALT is 29–33 U/L for males and 19–25 U/L for females in healthy adults.

Elevated serum GPT/ALT levels are commonly seen in various liver conditions, including viral hepatitis (such as hepatitis B or C), alcoholic liver disease, non-alcoholic fatty liver disease (NAFLD), cirrhosis, drug-induced liver injury, and liver cancer. Monitoring serum GPT/ALT helps in diagnosing and assessing the severity of these liver diseases.

Mouse ALT ELISA Kit FAQs

Q: What is the purpose of the Mouse ALT ELISA kit?

This ELISA kit is primarily used for research purposes to study liver function and diseases in mouse models. It enables researchers to measure and compare ALT levels across different samples, investigate the impact of experimental treatments or conditions on liver health, and assess the efficacy of potential therapeutic interventions.

Q: What type of samples can be used with the Mouse ALT ELISA kit?

The Mouse ALT ELISA Kit is validated for use with mouse serum and plasma samples. It can also be used with other mouse biological samples, such as liver tissue homogenates, as long as appropriate sample preparation steps are followed.

Q: Can the Mouse ALT ELISA kit be used for diagnostic purposes?

No, the Mouse ALT ELISA Kit is intended for research purposes only and should not be used for diagnostic or clinical applications. Diagnostic testing requires validated assays with specific regulatory approvals.

Q: Is the Mouse ALT ELISA kit compatible with other species?

This specific kit is designed for the quantitative measurement of mouse ALT. For other species or sample types, there may be variations in ALT isoforms or concentrations, which could affect assay performance. It is recommended to use a kit specifically validated for the species and sample type of interest.

Q: Where can I find additional technical support or assistance with the Mouse ALT ELISA kit?

For any technical inquiries or assistance regarding the Mouse ALT kit, you can reach out to our team. They will be available to answer your questions and provide the necessary guidance to ensure a successful experiment.

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