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Human Alkaline Phosphatase / ALPL ELISA Kit

SKU:
HUFI02199
Product Type:
ELISA Kit
Size:
96 Assays
Uniprot:
P05186
Sensitivity:
1.875ng/ml
Range:
3.125-200ng/ml
ELISA Type:
Sandwich
Synonyms:
ALP, Alkaline Phosphatase
Reactivity:
Human
Research Area:
Cell Biology
€599
Frequently bought together:

Description

Human Alkaline Phosphatase / ALPL ELISA Kit

Alkaline Phosphatase / ALPL is an enzyme which helps break down proteins and lipids, and is present in high levels in the liver and bone. Mutations in Alkaline Phosphatase / ALPL can cause a variety of diseases, including liver cancer, skeletal dysplasia, and dwarfism. The Assay Genie Human Alkaline Phosphatase / ALPL ELISA Kit is a highly sensitive assay for the quantitative measurement of Alkaline Phosphatase / ALPL in serum, blood, plasma, cell culture supernatant and tissue samples.

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:

Human Alkaline Phosphatase / ALPL ELISA Kit

Product Code:

HUFI02199

Size:

96 Assays

Alias:

ALP, Alkaline Phosphatase

Detection Method:

Sandwich ELISA, Double Antibody

Application:

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

Sensitivity:

1.875ng/ml

Range:

3.125-200ng/ml

Storage:

4°C for 6 months

Note:

For Research Use Only

Additional Information

Recovery:

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

Matrix

Recovery Range (%)

Average (%)

serum (n=5)

85-97

92

EDTA plasma (n=5)

85-104

95

UFH plasma (n=5)

87-105

98

Linearity:

The linearity of the kit was assayed by testing samples spiked with appropriate concentration of Human ALP 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)

91-104%

92-102%

92-100%

EDTA plasma (n=5)

89-94%

82-100%

86-100%

UFH Plasma (n=5)

85-98%

83-100%

80-97%

CV(%):

Intra-Assay: CV<8%
Inter-Assay: CV<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

Protein Information

Uniprot:

P05186

UniProt Protein Function:

ALPL: This isozyme may play a role in skeletal mineralization. Defects in ALPL are a cause of hypophosphatasia (HOPS). HOPS is an inherited metabolic bone disease characterized by defective skeletal mineralization.

UniProt Code:

P05186

NCBI GenInfo Identifier:

68067533

NCBI Gene ID:

249

NCBI Accession:

P05186.4

UniProt Related Accession:

P41324

Molecular Weight:

51,045 Da

NCBI Full Name:

Alkaline phosphatase, tissue-nonspecific isozyme

NCBI Official Symbol:

ALPL

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 Type

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.

ALPL Background

ALPL, also known as Alkaline Phosphatase, is an essential enzyme found in various tissues throughout the body. It plays a significant role in the regulation of phosphate metabolism. ALPL is involved in numerous physiological processes, including bone mineralization, liver function, and intestinal health. It is present in high levels during periods of rapid bone growth and can serve as a marker for bone disorders and liver diseases.

Role of ALPL in Bone Metabolism

ALPL is particularly crucial for bone metabolism. It functions by hydrolyzing phosphate esters and is involved in the mineralization process of bone. ALPL is present on the surface of osteoblasts, cells responsible for bone formation. It promotes the release of inorganic phosphate, a necessary component for the formation of hydroxyapatite crystals, which give strength and structure to bones. Dysregulation of ALPL activity or levels can lead to skeletal abnormalities and metabolic bone disorders.

ALPL in Liver Diseases

The liver is another major site of ALPL expression. Elevated ALPL levels can indicate liver dysfunction, such as cholestasis or hepatobiliary diseases. ALPL is involved in the transport and metabolism of bile acids, a critical function of the liver. Abnormalities in ALPL expression or activity can disrupt the normal bile flow, leading to the accumulation of bile acids and subsequent liver damage. The measurement of ALPL levels in liver diseases can aid in the diagnosis, monitoring, and management of these conditions.

Genetic Disorders Associated with ALPL

Mutations in the ALPL gene can lead to genetic disorders such as hypophosphatasia, an inherited metabolic bone disease. Hypophosphatasia is characterized by impaired bone mineralization, resulting in skeletal abnormalities and increased susceptibility to fractures. ALPL deficiency due to gene mutations leads to reduced ALPL activity, resulting in the accumulation of substrates that are normally hydrolyzed by ALPL.

ALPL FAQs

What is the ALPL ELISA Kit?

The ALPL ELISA Kit is a specialized tool used for the detection and quantification of Human Alkaline Phosphatase (ALPL) in biological samples.

What are the advantages of using the ALPL ELISA Kit?

The ALPL ELISA Kit offers several advantages, including high specificity and sensitivity, allowing for precise measurement of ALPL levels in biological samples. It provides a simple and efficient workflow, delivering rapid and reproducible results.

Where can I find more information about the ALPL ELISA Kit?

For more detailed information about the ALPL ELISA Kit, including technical specifications, performance characteristics, and ordering details, please refer to the product brochure or contact our customer support team. We are here to assist you with any inquiries you may have.

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