s(HS)TMB (#sTMB)

s(HS)TMB, soluble (High Sensitivity) TMB Substrate for ELISA, #sTMB, is a peroxidase
substrate containing
3,3\',5,5\'-tetramethylbenzidine (TMB) and hydrogen peroxide in an aqueous buffer
formulation. This product is supplied as a one component ready-to-use reagent.
Intact/unreacted substrate is clear colorless to slightly yellowish in appearance. Reaction
with peroxidase results in converting substrate into soluble blue (~ 650 nm) product which
after stopping with strong acid further turns to super-oxidized soluble yellow product.
Yellow product can be measured at 450 nm (measuring at 450 nm vs.
620-650 nm reference wavelength will additionally improve readout pattern in ELISA).

s(HS)TMB is optimal for 15-min. substrate development reaction in ELISA. Shaker mode
will improve overall performance.

s(HS)TMB substrate yields exceptionally high signals with HRP conjugates and therefore
is particularly recommended for Ultra-Sensitive ELISA tests.

It does not contain high density components, is not viscous, has no detergent or colloidal
activity. It does not foam and does not stick to hydrophobic surfaces. This allows easy
and precise dispensing: standard PP and HD-PE pipette tips repel s(HS)TMB restlessly,
almost as it were pure water.

After stopping, fixed reaction remains stable during several following hours, also under
direct light. There is no fading, neither increase in color. This concerns both low signals
(blank, background, negative control) and high positive signals (no precipitation in wells
with OD up to 3.500) and makes later postponed reading in ELISA-spectrophotometer

s(HS)TMB is stable two years at +2°C/+8°C or one year at ambient room temperature (15

Both storage regimens are equally applicable depending on desired duration of storage.
Within first year, storage in refrigerator will neither improve, nor worsen product
performance. +4°C is however a preferred storage regimen providing better safety in
cutting off adverse influence of higher temperatures.

Warm up s(HS)TMB substrate, if stored at +4°C, to assay temperature (optimum = 17°C-
18°C) prior to use.

Avoid exposure to light, air and extreme temperatures.

For repackaging bulk product use only clean intact amber Nunc/Nalge(n) or Wheaton HD-
PE bottles. Bottle substrate in light-protected (weak diffused light) and clean area
gravimetrically relying on d20°C = 1 g/ml. Alternatively use peristaltic pump with certified
compatible tubing (e.g. Norprene or Cole-Parmer/Millipore C-Flex tubing). Do not re-filter.

For dispensing smaller volumes, use close-design syringe dispensers (e.g. CombiTips of

s(HS)TMB substrate does not contain volatile/aprotic organic solvents and is non caustic
and non corrosive to plastic, glass and metal materials used in automatic dispensing and
robotic ELISA instumentation. It is a completely water base product added with non-toxic
proprietary stabilizers and enhances that are not health-hazardous. This product is non-
hazardous, non-toxic, non-carcinogenic, environmentally safe unrestricted product.

s(HS)TMB, stable soluble 1-component TMB
Substrate for High Sensitivity ELISA applications,
100% aqueous formula, ready-to-use          

s(HS)TMB Weakener is complementary reagent.

s(HS)TMB Weakener (#sTMB-W)

Some customers who do not really need very high analytical sensitivity in their test (typical
case is determination of specific antibodies in indirect antigen sandwich ELISA with anti-
species HRP conjugate) or formerly used in their systems weaker TMB products and do
wish to retain signal levels they used to have, ask us whether it would be possible to lower
signals with s(HS)TMB through e.g. diluting it with blank substrate buffer.

Although it is generally not recommended to dilute TMB substrates, instead of producing
tailored weaker TMB we have decided to add our TMB product line with s(HS)TMB
Weakener, which is essentially the same basic formulation as s(HS)TMB containing no
TMB and hydrogen peroxide. Now users who wish to reduce signals in their system when
switching over to our s(HS)TMB can adjust performance to previous
level by simply mixing s(HS)TMB with Weakener. This blank optimization TMB diluent
offers a possibility of achieving desired OD values while maintaining overall stability of
prepared mixture at a level of undiluted s(HS)TMB.

s(HS)TMB Weakener, ready-to-use       

esTMB is a newer, enhanced product that gives 30-to-40%
larger signals and respectively higher sensitivity by the same clean background.

es(HS)TMB, enhanced stable soluble 1-component TMB
Substrate for High Sensitivity ELISA applications
100% aqueous formula,

epTMB is a newer, enhanced product that gives 20-to-30%
larger signals and respectively higher sensitivity by the same clean
background (compared to the discontinued pTMB)

NOTE: this product is not applicable in blotting. For HS blotting and ELISPOT
order epTMB-mA item

ep(HS)TMB, stable precipitating 1-component TMB
Substrate for High Sensitivity 3D-(Immuno)Filtration
100% aqueous formula, ready-to-use    

ep(HS)TMB-mA is a novel, one component stable enhanced precipitating TMB Substrate especially
designed for the microArray applications. It produces smaller, clearly shaped colored 1e-oxidized
TMB precipitate at the sites of the HRP activity. Colored product features higher density and
perfect adhesion and grip on the porous (3D) and non-porous (2D) surfaces of the different
solid phase materials used as reactive matrices/supports in the diverse heterogeneous binding
assays of the modern MicroChip format.
ep(HS)TMB-mA is a High Sensitivity product applicable in:

- Microchips / planar multiplex arrays
- fine micro-blotting
- ELISPOT assays

ep(HS)TMB, enhanced stable precipitating 1-component TMB
Substrate for High Sensitivity microArray applications, 100% aqueous formula,

Stop/Elution Reagent (#SER)

Chemical purity of acid used in stopping enzymatic reaction with TMB substrate is
important factor that also influences overall performance of the ELISA test. Divalent Fe++,
Ni++, Co++, Cd++, Cr++, Cu++, Mn++, Mg++ cations, if present in stop reagent, may provoke
huge backgrounds as a result of post-enzymatic (when HRP is already dead) catalytic
reaction where elevated blank signals become visible also through larger negative control
and/or cut-off values, even if measured shortly after stopping with dirty acid reagent. That
much evident picture is an extreme. None less, in practice, a possible impact of
Fe++/Ni++/Co++-associated post-enzymatic reaction in actually observed backgrounds is just
a matter of quantitative dependence. It might be quite possible that a couple of decades of
microOD units in your ELISA system are due to not optimal stopping reagent. In
colorimetric test, each 10 micro OD units in lower OD range, i.e. with blanks, negative
controls and cut-offs, are often critical.

Adding TMB substrate formulation with strong Fe++/Ni++/Co++-sequestering agents, as
Diethylenetriamine penta-methylenephosphonic acid(DTPMPA) and other phosphonates
(multivalent derivatives of methylene phosphonic acid), also known as effective peroxide
stabilizers, can compensate for metal ion contamination of Stop Reagent. This however
weakens substrate developmental strength in reaction with peroxidase label through
interference with iron in Protohematin IX and therefore is not rational method compared to
exclusion metal ions in Stop Reagent.

Our Stop/Elution Reagent is essentially Ultra-pure 8% Sulfuric Acid containing no
detectable bivalent metal ions. Therefore post-enzymatic oxidizing of TMB after stopping
substrate reaction is absolutely excluded. #SER guarantees stability of developed color
reaction with s(HS)TMB during three hours at room temperature, not protected from light
and air, providing for a possibility of dependable later measuring ELISA plates which is
practicable in running larger series involving many tests. This adds security and
convenience in working with intensive test flows and on the other hand improves
robustness of the each individual test in labor intensive series.

Besides, #SER has been extra validated as eluting reagent optimal for 3D-ImmunoFiltratio
tests with PolyHRP/p(HS)TMB detection. Oxidized in enzymatic reaction with HRP label
cationic TMB product will be in situ precipitated by polyanionic polyelectrolyte polymer
present in p(HS)TMB formulation with formation of dark blue electrostatic complex
deposited onto the surface of porous 3D device filter matrix. Precipitated TMB product can
be eluted from device filters by applying strong acid Elution Reagent #SER which destroys
electrostatic complex and simultaneously, within same elution event, further converts blue
TMB product into super-oxidized final yellow product. Eluted/stopped product remains
soluble and can be measured exactly as in conventional ELISA test in standard ELISA
reader at 450/620 nm. This creates option of complementary read-out in 3D-
ImmunoFiltration test. Said option might be practicable when running larger series arranged
in compatible with 8x12 microwell ELISA plate geometry format with elution directly in
standard serocluster 8x12 microplates and measuring in common ELISA reader. #SER
elutes in a minute, and OD readout values in eluted substrate product remain stable within
3 hours after elution.

Stop/Elution Reagent for stopping
s(HS)TMB in ELISA and eluting
p(HS)TMB in 3D ImunoFiltration assay