100% Solvent FreeAlert® K-Blue® Aqueous is specifically designed for use with HRP based sandwich immunoassays but is proven to work in competitive peroxidase based immunoassays as well. The substrate is not intended for use with assays requiring a precipitating substrate. Alert K-Blue Aqueous utilises a patent-pending 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 with low background development.

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