100% Solvent FreeAlert® K-Blue® Aqueous is specifically designed for use with horseradish peroxidase (HRP) based sandwich immunoassays and also has been proven to work in competitive peroxidase-based immunoassays. The substrate is not intended for use with assays requiring a precipitating substrate. Alert K-Blue Aqueous utilises an indicator system that clearly indicates when substrate and acid stop have been added to an assay’s microwells.

The substrate is based on Neogen’s original K-Blue Aqueous formula which is a revolutionary 100% solvent free, non-volatile TMB substrate. It contains 3,3’,5,5’ tetramethylbenzidine (TMB) and hydrogen peroxide (H2O2) in a one-bottle format with long-term stability and high activity. The addition of acid to the substrate produces a colour change that can be measured on a microplate reader or observed visually. This colour change increases the absorbance value at 540nm and changes the appearance of the substrate colours to pink.

Sensitivity: This is highly sensitive TMB substrate has low background development and offers excellent signal-to-noise ratio.

Stability and Storage: Alert K-Blue Aqueous is stable for 36 months when stored at 4°C.

Appearance: Yellow to golden yellow solution.

Custom Packaging Service: Neogen can package Alert K-Blue Aqueous in custom bottle sizes and volume fills to meet your specific packaging requirements. This service is a time and cost saving feature for any test kit manufacturer. For details on this service, please contact a Neogen representative.

Order Details

Product # Product Description
334175 Alert K-Blue Aqueous TMB Substrate, 200 mL
334176 Alert K-Blue Aqueous TMB Substrate, 500 mL
334177 Alert K-Blue Aqueous TMB Substrate, 1 L
334257 Alert K-Blue Aqueous TMB Substrate, 20 L (1 x 20 L)

For research and manufacturing use only.


Individual well management is improved by substituting a Neogen Alert substrate for the traditional TMB substrate because the chance of a well returning a false negative due to reagent addition errors is reduced. This substrate change allows for an additional wavelength check to ensure that the only valid results are from wells that return a signal (pink) at 540 nm. As the figure below demonstrates, non-verifiable and false negatives with a traditional TMB substrate are recorded as invalid results when using the Alert TMB system.

Traditional TMB Substrate with Acid Stop
OD Measured at 450 nM - 650 nM
Reagent Added: Well contains sample that is:
Substrate Acid NEGATIVE Result POSITIVE Result
YES YES Colourless TN Yellow TP
YES NO Colourless N Blue Invalid or FN
NO YES Colourless N Colourless FN
NO NO Colourless N Colourless FN
Alert TMB Substrate with Acid Stop
OD Measured at 450 nM - 650 nM
Reagent Added: Well contains sample that is:
Substrate Acid NEGATIVE Result POSITIVE Result
YES YES Colourless/No Pink TN Yellow/Pink TP
YES NO Colourless/No Pink Invalid Blue/No Pink Invalid
NO YES Colourless/No Pink Invalid Colourless/No Pink Invalid
NO NO Colourless/No Pink Invalid Colourless/No Pink Invalid

TN = True Negative
N = Negative, but not verifiable
TP = True Positive
FN = False Negative
Invalid = Value returned in outside acceptable parameters

Please check with your local representative for availability of these products within your region.