Several enzymes have been utilized as detection probes in immunochemistry because of their high sensitivity, simplicity and wide usage applications. The two very common enzyme probes used in immunoassays and chemiluminescence are horseradish peroxidase (HRP) and alkaline phosphatase (AP). These two enzymes have been used to detect biological compounds through chemiluminescence, chromogenic or fluorescent outputs.


Introduction to enzyme probes


Enzymes have been utilized as detection probes for many decades. For many uses, a variety of enzymes have been used to detect foreign protein, antigen or other biological agents after being conjugated with another protein usually an antibody that binds to the target which is usually an antigen. In general, an antibody is bound to an avidin-biotin complex. This is followed by addition of the specific enzyme. The complex is exposed to the foreign protein or antigen and a visible reaction product appears or there may be emission of light or fluorescence. This signal can be detected by either a scanning method or spectrophotometry.


What are the benefits of using enzyme probes?


There are several advantages of using enzyme probes such as HRP and AP to detect a targeted protein and they include the following:


  1. Long shelf life: the enzyme horseradish peroxidase and alkaline phosphatase are relatively stable when stored appropriately. Because these enzymes are light insensitive, they are not affected by the light generated during chemiluminescence.

  2. Good sensitivity: When the chemical reaction occurs, the resulting signal output can be relatively easily detected which allows for identification of even very low levels of target protein or antigen. In addition, there are also other methods available to amplify the signals so that the test is more sensitive. Finally because of the high turnover of the enzymes, this also results in more enzyme binding to the protein and the signal emission can be deleted for a long time.

  3. Versatile system. Today there are many substrates that have properties of chemiluminescence, chromogenesis or fluorescence; thus making the enzyme system very versatile and easy to use.

  4. Probes available. The other feature about alkaline phosphatase and HRP is that both probes are readily available and not prohibitively expensive.


Enhanced chemiluminescence (ECL)


HRP is known to catalyze the oxidation of luminol to 3-aminophthalate via several intermediates. During this reaction, there is generation of low intensity light at 428 nm. However, when certain chemicals are added, the emission of light can be enhanced by a 1000 fold, making the light much easier to detect by spectrophotometers. In addition, the chemicals can also increase the sensitivity of the reaction. This increased generation of light is known as enhanced chemiluminescence. Today there are several types of enhancers that can be utilized in the HRP reaction. The majority of these enhancers are modified phenols (chiefly iodophenol). Enhanced chemiluminescence is now used in many laboratories to detect picograms of nucleic acids in northern and southern blots.The key reason enhanced chemiluminescence is preferred over chromogenic substrates is because the methodology is at least 10-100 times more sensitive because of greater light emission over a wide range of wavelength. On the other hand, when using chromogenic substrates, the colored precipitates have limited light emission only in the visible range.


What are some negatives of using AP and HRP?


1. Need for a substrate. When using AP and HRP, one does require a substrate to detect the foreign protein or antigen and the substrate used may induce a reaction that may be light sensitive.


2. Size interference: In general alkaline phosphatase and HRP are large enzymes and thus size may interfere with the biochemical function of the protein to which they are attached.


3. Background signals: Because the enzymes used to detect target proteins are also found in the cells of the body, this may also result in non specific background signal, unless on takes precautions to inhibit the endogenous enzymes.


Common enzyme probe reporters and chromogenic substrates


Both AP and HRP are used as assay reporters because by reacting with the substrate they produce chemiluminescent or colored fluorescent signals that can be measured and quantified.

The substrate may be either a solid (precipitating) or soluble.


Enzyme probes have many laboratory applications chiefly because they are versatile and can be conjugated to a variety of substrates. Both AP and HRP can be used to detect protein or antigens via an indirect or direct antibody detection test, where the enzyme is conjugated to the primary antibody which then binds to the target antigen or a secondary antibody that attaches to the primary antibody. Both AP and HRP can be conjugated with avidin or biotin for use in avidin biotin signal amplification systems.


Horseradish Peroxidase


The enzyme horseradish peroxidase (HRP) is commonly found in the roots of the horseradish plant. This metalloenzyme with several isoforms is widely used in a variety of biochemical applications. Essentially the enzymes catalyses the oxidation of hydrogen peroxide to water and oxygen gas.


Substrates for HRP


When HRP is used in biochemical reactions its presence is usually made visible by a substrate that when oxidized by HRP using the oxidizing agent hydrogen peroxide, it produces a classic color change that is detected by spectrophotometer methods


Several substrates for HRP have been developed and fall into two basic groups:


  1. HRP catalyze the conversion of chromogenic substrate TMB, DAB, ABTS into colored byproducts

  2. Produce light when acted upon by luminol or chemiluminescent substrate.


Enzyme label


peroxidase (HRP)



3,3'-diaminobenzidine (DAB)

3,3',5,5'-tetramethylbenzidine (TMB)

2,2'-Azinobis [3-ethylbenzothiazoline-6-sulfonic acid] (ABTS)

o-phenylenediamine dihydrochloride (OPD)


HRP as an enzyme reporter for probes


Horseradish peroxidase (HRP) has the ability to catalyze the transfer of two electrons from a  substrate to hydrogen peroxide to generate water and an oxidized substrate. HRP is frequently used in conjugates to detect the presence of a protein target. For example, when HRP is conjugated to an antibody it can be used to detect trace amounts of a specific protein in a western blot. The antibody provides the specificity to locate the antigen of interest and the HRP in the presence of a substrate, generates a detectable signal. HRP is used in immunohistochemistry and ELISA because it generates colored compounds. For detection of an antigen or protein molecule, HRP substrates have been designed so that they will  generate a chemiluminescent, chromogenic, or fluorescent signal upon oxidation.


Advantages of using HRP


Small Size: The molecular weight of HRP is 40,000, which is relatively small compared to many other enzyme conjugates. The small size also is an advantage as it allows for more effective penetration into the sample cells and decreases the probability of interference with the conjugated protein function. In addition, HRP has four lysine molecules, each of which can be conjugated, which further enhances the efficiency of cross linking to the antigen or protein being assayed.


High Turnover: The other key feature about HRP is that it has a high rate of turnover and this results in an efficient reaction, producing an abundance of reactant products in a short span of time at normal pH. Studies show that HRP bound to IgG is much more efficient than alkaline phosphatase because of constant conjugate formation due to its higher specific enzymatic activity (ie there is more HRP /mole of antibody).


Less steric hindrance: Further HRP conjugates have enhanced immunologic reactivity because of their small size, which results in lesser steric hindrance and more effective signals.


Stable: The most important feature of HRP conjugates is that they are stable at neutral pH for a long time.


Cost: Overall, HRP is less expensive than AP.


Drawbacks of HRP as an enzyme in chemiluminescence


1.The first minor drawback of using HRP is that it may result in non-specific staining that may occur from the endogenous peroxidase activity found in certain tissues or cells. When thin frozen section of tissues are made, they often reveal a mild to moderate amount of endogenous peroxidase activity. However, to get by this problem there are commercial peroxidase inhibitors available that can decrease or completely eliminate the endogenous peroxidase activity in the tissues.


2.Another minor negative of HRP is related to its sensitivity to breakdown. Some microorganisms as well as some antibiotics may enhance the breakdown of HRP. One potent inhibitor of HRP is sodium azide but to get around this problem, one can use 0.01% thimerosal. However it is important to be aware that the activity of HRP can also be inhibited by sulfides, cyanides and azides.


3. A final negative of HRP is that there are reports that some of the reaction products of HRP substrates may have the potential to be mutagenic or carcinogenic. So far there are only a few reports in small animals. However, if this is a concern than one should use other substrates like AP.


Use of alkaline phosphatase in chemiluminescence


Alkaline phosphatase (AP)  contains five cysteine residues, one magnesium atom and two zinc atoms that are needed for its enzymatic function. The enzyme is optimally active at an alkaline pH. Alkaline phosphatase essentially plays a role in dephosphorylation of compounds; the enzyme is widely found in the body and even bacteria. Alkaline phosphatase is heat stable and found in several isoforms in the body. Because of its high concentration in the liver and bone it is often used as a biomarker to determine for presence of liver injury or even bone disorders like osteomalacia.


Alkaline phosphatases are a family of enzymes that can hydrolyze phosphates from proteins and nucleotides. This family class functions best at alkaline pH (around 9) and are activated by divalent cations like calcium and inhibited by cyanide, cysteine, inorganic phosphate, arsenate and divalent chelating agents like EDTA.


In humans the two predominant forms of alkaline phosphatase include one that is distributed in the organs and the other one found only in the gastrointestinal tract. The two subtypes of alkaline phosphatases are influenced differently heat and chemical inhibitors. It is established that levamisole in the substrate buffer will suppress activity of endogenous tissue AP. On the other hand, intestinal AP is inhibited by 20% acetic acid, followed by potassium borohydride. Thus, when using AP, it is important to know the source of the enzyme.


In most laboratories calf intestinal alkaline phosphatase conjugates are ideal in applications where high endogenous peroxidase levels contraindicate the use of HRP conjugates, such as with cryostat sections where peroxidase inhibitors are ineffective.


Enzyme label

Alkaline Phosphatase



Combination of nitro blue tetrazolium chloride

(NBT)and 5-bromo-4-chloro-3-indolyl phosphate (BCIP)

p-Nitrophenyl Phosphate (PNPP)


Advantages of Calf Intestinal AP


Unlike HRP, the molecular weight of calf intestinal APis about 3.5 times more, or 140,000. Some of the advantages of using calf AP include the following:


  1. The reaction rates are linear and easily measured

  2. The sensitivity of the reaction can be enhanced by permitting the reaction to occur for a long period of time

  3. Unlike HRP, the activity of calf intestinal AP is not altered by exposure to antibiotics and other agents like thimerosal or sodium azide. This means that the enzyme can be stored for a definite period of time, even in a non-sterile environment.

  4. Finally since the non-intestinal AP activity can be suppressed by levamisole, antibodies labeled with this enzyme may be used as biological markers in a number of different cell types.


Uses of alkaline phosphatase in the laboratory


Alkaline phosphatase has many applications in the molecular biology laboratory.


  1. Removing phosphate groups. Because DNA normally contains phosphate groups on the 5- terminal end, removing these end phosphates prevents the DNA from forming a coil. Thus, this keeps the DNA molecule linear during the preparation phase. Further removal of the phosphate groups by alkaline phosphatase also permits radiolabeling and this allows one to measure for the presence of labeled DNA in experiments.

  2. Enzyme immunoassays: Alkaline phosphatase is now widely used as a label for enzyme immunoassays.

  3. Immunohistochemical staining is an invaluable technique for detecting specific antigens in cells and tissues. To perform the immunohistochemical staining, first the tissue section has to be deparaffinized and then rehydrated before soaking it with the primary antibody. Enzyme-conjugated secondary antibodies are then applied to the section and the specific staining can be visualized once the enzyme-specific substrate is added. Sometimes, there may be little or no staining observed, and in such cases one may need to unmask the antigen by enzyme digestion.

  4. Since many differentiated cells contain high concentrations of alkaline phosphatase on the cell surface, alkaline phosphatase staining can be used to detect for presence or recurrence of certain cancers

  5. Alkaline phosphatase is also found in bone and the liver and measurement of its concentration in the blood may reveal a disease process.




Overall, both alkaline phosphatase and HRP conjugates can be used to obtain chemiluminescent results in western or other immunoblot tests, immunochemistry or ELISA. HRP is a heme containing enzyme that catalyses the oxidation of luminol resulting in low intensity light emission at 428. This light emission can be enhanced by a 100 fold by adding certain chemicals. Alkaline phosphatase dephosphorylate proteins, nucleotides and alkaloids in an alkaline environment. The alkaline phosphatase is usually derived from the calf intestine and used in immunoassay results in a chromogenic detection. When the AP substrate is added, the conjugate results in a color change that can be visible at 405 and easily quantified.