ELISA kit with HRP or ALP – what to choose?

Published: September 20, 2023

Updated: March 19, 2024

Our ELISA Flex kits are almost always available in two versions: they rely on either ALP or HRP for the colorimetric reaction. If you are wondering which one to choose, this is for you. 


Horseradish peroxidase (abbreviated HRP) is an enzyme found in the roots of horseradish. Alkaline phosphatase (abbreviated ALP or AP) is an enzyme that comes in different variants: Mabtech kits use ALP from calf intestine. HRP and ALP catalyze different reactions and require different substrates. Nonetheless, both HRP and ALP enzymes can catalyze a colorimetric reaction, which is the typical readout of an ELISA. In our ELISA kits, both enzymes are used either as enzyme conjugates or directly conjugated to the detection antibody (Figure 2).


We prefer HRP for ELISA because of its rapid kinetics, resulting in a shorter assay time for a kit with HRP than the corresponding kit with ALP. The ALP reaction is slower than that of HRP. However, ALP has the advantage of having a linear activity. When using ALP, you can perform continuous readings to catch the optimal time point for your readout, which might be favorable in certain contexts. So, even if we recommend choosing HRP, it might be beneficial for your setup to choose ALP. At the end of the day, the differences are rather subtle and often simply a matter of taste. Good luck with your next ELISA experiment. 


Enzyme Horseradish peroxidase (HRP)Alkaline phosphatase (ALP) 
ELISA substrateELISA substrate: TMB for HRPELISA substrate: pNPP for ALP
KineticsVery fast, typically within 5 minutesMore linear, signal plateau typically at 60 minutes
AdvantagesRapid kinetics,
TMB substrate is less chemically hazardous than pNPP
More linear activity – allows for readings at different time points

Endpoint reading only

pNPP substrate contains diethanolamine which is more chemically hazardous than TMB

Add stop solutionYes No
Recommended reading timeStop reaction after 15 minutes (shorten to 5 minutes if very fast), then read.Read at 60 minutes. (Try 30, 60, and 90 minutes when setting up a new experiment.) 
Wavelength450 nm405 nm
InterferenceHRP can be degraded by microorganisms and antibacterial agents. The enzyme can be inhibited by cyanides, sulfides, and azides.ALP can be inhibited by cysteine, cyanides, arsenate, inorganic phosphate, and divalent cation chelators, such as EDTA.




Differences in the ELISA Flex protocol between kits using ALP or HRP

Figure 1. Differences in Mabtech's ELISA Flex protocol between kits utilizing either Streptadivin-HRP (SA-HRP) or Streptadivin-ALP (SA-ALP).


As our ELISA Flex kits don’t include buffers or substrate, make sure to obtain the complementary products required (if you don’t have them already available in your lab). 



ELISA Flex kits, sandwich, two- or one-step detection, ALP or HRP

Figure 2. Mabtech’s ELISA Flex kits enable you to set up a sandwich ELISA. Most of our kits are based on a two-step detection process (Fig. 2A-B) utilizing an enzyme conjugate, either A) Streptadivin-HRP (SA-HRP) or B) Streptadivin-ALP (SA-ALP). Selected ELISA Flex kits are based on a one-step detection process (Fig. 2C-D), containing a detection antibody directly conjugated to either C) HRP or D) ALP. 



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