Chemiluminescence Immunoassay (CLIA) Label Alkaline Phosphatase (ALP)

Release time:2022/11/4 16:43:33

The common labels of chemiluminescent immunoassays is alkaline phosphatase (ALP).

In recent years, chemiluminescence immunoassay (CLIA) has received increasing attention in different fields such as life science, clinical diagnosis, environmental monitoring, food safety and drug analysis because of its high sensitivity, good specificity, wide range of applications, simple equipment and wide linear range.

Conventional immunoassays always require a long incubation time, which in turn results in the entire analysis taking several hours to complete, so the yield and application range is largely limited. Researchers have devised different methods to shorten the analysis time by improving mass transport and reaction kinetics. Immunoassays have expanded the scope of application of CLIA.

CLIA.jpgFigure 1. Chemiluminescence Immunoassay

What Is CLIA?

Chemiluminescence (CL) is defined as the emission of electromagnetic radiation caused by a chemical reaction to produce light. Chemiluminescent immunoassay (CLIA) is an assay that combines  highly sensitive chemiluminescent technology with highly specific immunochemical reactions for the detection and analysis of various antigens, haptens, antibodies, hormones, enzymes, fatty acids, vitamins and drugs. Similar to other labeled immunoassays (RIA, FIA, ELISA), CLIA utilizes a chemical probe to label antibodies by producing light emission through a chemical reaction.

What Is The Principle Of CLIA?

Chemiluminescent immunoassay contains two systems: immunoassay and chemiluminescent assay.

The immunoassay system uses chemiluminescent substances or enzymes as labels to directly label antigens or antibodies, and after the antigen-antibody reaction to form an antigen-antibody immune complex.

Chemiluminescence assay system refers to the process of addition of oxidant or enzyme luminescent substrate at the end of the immune reaction. After the chemiluminescent substance is oxidized by the oxidant, an intermediate in an excited state is formed, which will emit photons to release energy in order to return to the stable ground state, and the luminescence intensity can be detected using a luminescence signal measurement instrument. According to the relationship between chemiluminescent labels and luminescence intensity, the content of the measured substance can be calculated using the standard curve.

Chemiluminescent immunoassays can be divided into three main categories according to the different labels, namely chemiluminescent immunoassays, chemiluminescent enzyme immunoassays and electrochemiluminescent immunoassays. Chemiluminescence immunoassay is a class of immunoassay that uses chemiluminescent agents to directly label antibodies or antigens. At present, the common labels are mainly acridines and alkaline phosphatase (ALP).

ALP - Chemiluminescent Substrate

Alkaline phosphatase (ALP) is an that is present in many parts of the body, but it is primarily found in the liver, bones, intestine, and kidneys. Hepatitis, liver cancer and other diseases can cause sudden changes in ALP levels, which is one of the most commonly used label enzymes in human immunodiagnostic reagent products today.

Alkalinephospharase (AP) is an enzyme that dephosphorylates the corresponding substrate under alkaline conditions, i.e., it removes the phosphate group from the substrate molecule by hydrolyzing the phosphate monoester and generates a phosphate ion and a free hydroxyl group.

The process of removing the phosphate group is called dephosphorylation. The role of phosphatases is the opposite of that of kinases, which are phosphorylating enzymes that can use energy molecules, such as ATP, to add phosphate groups to corresponding substrate molecules. Alkaline phosphatases are commonly found in many organisms.



Application: AMPPD is a chemiluminescent substrate of alkaline phosphatase. Under alkaline conditions, AMPPD is hydrolyzed by AP to generate a fairly stable AMP-D anion, which has a decomposition half-life of 2-30 min and emits continuous light at a wavelength of 470 nm, with its intensity peaking at 15 min and remaining relatively stable from 15 to 60 min.



Application: APS-5 is a chemiluminescent substrate based on 9,10-dihydroacridine, which is mainly used in ELISA for the detection of compounds with alkaline phosphatase AP conjugation. The 9,10-dihydroacridine structure of APS-5 provides stable and efficient luminescence in the presence of AP. APS-5 is well suited for phosphatase activity solution assays and phosphatase immunoassays.

APS-5 has the following advantages.

1. High sensitivity: it can detect protein markers below 5 pg.

2. High luminescence intensity: peak luminescence can be reached in a short time.

3. Continuous and stable luminescence: its luminescence process is not affected by temperature within 25-35°C and does not require temperature control.

Huateng Pharma can provides APS-5 from milligram to kilogram scale. Contact us at for more details.


  • Name: Bis-MUP | Bis(4-methylumbelliferyl)phosphate

  • CAS No: 51379-07-8

  • Molecular Weight: 414.30

  • Ex(nm): 360nm

  • Em(nm): 448nm


Application: Bis-MUP is an alkaline phosphatase (ALP) chemiluminescent substrate.

4-Methylumbelliferyl phosphate

  • Name: 4-Methylumbelliferyl phosphate

  • CAS No.: 3368-04-5

  • Molecular Weight:256.15

  • Ex(nm): 360nm

  • Em(nm): 448nm


Application: 4-Methylumbelliferyl phosphate is used as a fluorescent substrate for phosphatases. It is widely used for the detection of phosphatases in solution. However, this phosphatase substrate is not suitable for live cell or serial assays because 4-Methylumbelliferyl phosphate is an enzymatic hydrolysate that develops maximum fluorescence only at pH > 10. Therefore, it is also difficult to use 4-Methylumbelliferyl phosphate to detect phosphatases with an acidic optimal pH range, such as acid phosphatase.


  • Name: 4-MUP | 4-Methylumbelliferyl phosphate disodium salte

  • CAS No.: 22919-26-2

  • Molecular Weight: 300.11

  • Ex(nm): 360nm

  • Em(nm): 448nm


Uses: 4-MUP is a fluorescent substrate for phosphatases and it is widely used for the detection of phosphatases in solution. However, this phosphatase substrate is not suitable for live cell or continuum analysis, because 4-MUP is an enzymatic hydrolysate and only emits maximum fluorescence at pH >10. Therefore the detection of phosphatases in the acidic range with MUP remains a challenge, for example the detection of acid phosphatases.

CDP star

  • Name: CDP star

  • CAS No.: 160081-62-9

  • Molecular Weight: 495.2


Uses: CDP star is currently the most advanced alkaline phosphatase substrate, which emits light signals at a sustained rate through baidioxetane activated by alkaline phosphatase. CDP star is highly sensitive and the luminescence time can extend for days starting from a few minutes, so multiple exposures can be made and the exposure time can be optimized. It is the fastest and most sensitive chemiluminescent substrate available, with an exposure time of only 15-60 seconds. When diluted 1:100 in detection buffer on nylon membranes, CDP-Star's light signal reaches its maximum within 15 minutes and decays slowly for more than 3 days. It is particularly suitable for the detection of single copy mammalian genes, detection of very small amounts of target DNA, e.g. for fingerprinting and forensic analysis.


  • Name: CSPD

  • CAS No.: 142456-88-0

  • Molecular Weight: 460.7


Application: CSPD is an upgrade to AMPPD, a state-of-the-art ultra-sensitive alkaline phosphatase substrate that reacts quickly and provides correct and reliable results in a very short time. CSPD chemiluminescent substrate allows you to detect alkaline phosphatase and alkaline phosphatase-labeled molecules with outstanding sensitivity, speed and ease of use.

CSPD is mostly used for protein or nucleic acid blotting on nitrocellulose membranes. This versatile chemiluminescent substrate has shown high sensitivity in membrane based applications such as Southern, Northern and Western blotting. These substrates can also be used in solution-based assays such as immunoassays, DNA probe assays, enzyme assays, and reporter gene assays. The maximum light level is reached in about 10 minutes and the glow emission lasts for several hours.

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