TMB Chromogen Solution (for ELISA)

The assay can be performed directly with serum and in this case will measure total fructosamine in serum proteins. Alternatively, it is possible to separate albumin from other serum proteins by Affi-Gel blue chromatography and then to perform the colorimetric fructosamine assay. The assay is not sensitive to the labile Schiff’s bases (aldimines) or the free glucose concentration. However, several oxidizable compounds in serum interfere with the assay, in particular bilirubin and uric acid .

A second historically important assay to measure fructosamine is the thiobarbituric acid assay [90,91]. The assay is based on the condensation of 5-hydroxymethylfurfural with thiobarbituric acid under formation of a yellow reaction product. The 5-hydroxymethylfurfural is liberated from fructosamine during mild acid hydrolysis. The assay is subject to serious interference by glucose [93], thus requiring glucose removal by ultrafiltration prior to performing the assay. Thiobarbituric acid also reacts with many other carbonyl compounds and is not commonly used to measure fructosamine anymore. However, the assay is still used to measure the formation of malonyldialdehyde and similar aldehydes resulting from lipid peroxidation [94].

TMB (3,3′, 5,5;-tetramethylbenzidine) chromogen solution may be used as a substrate for horseradish peroxidase (HRP) in ELISAs.

Using TMB
Use this reagent as supplied, no dilution or further preparation is required. In the presence of peroxidase (HRP, POD), TMB will be converted to a blue color that can be read at OD₆₅₀ nm.

Specifications

Product Type	TMB Solution
Substrate Type	HRP (Horseradish Peroxidase) Substrate
Shipping Condition	Wet Ice

Substrate Properties	Chemical Substrate
Substrate	TMB
Detection Method	Colorimetric

Chromogenic Substrates

Chromogenic substrates offer a number of advantages over traditional media based on pH indicators. A color change is seen in a colony after transportation and hydrolysis, and this reduces the potential number of false positives due to mutation by more than 10-fold, as fewer genes are involved in generating a signal.

Rapid Detection Using Chromogenic Substrates

Chromogenic compounds added to conventional or newly devised media are used for the isolation of indicator bacteria. These chromogenic substances are modified either by enzymes (which are typical for the respective bacteria) or by specific bacterial metabolites. After modification, the chromogenic substance changes its color or fluorescence, thus enabling easy detection of colonies displaying the metabolic activity.

In this way, these substances are used to avoid the need for isolation of pure cultures and confirmatory tests. The time required for the determination of different indicator bacteria can be reduced to between 14 to 18 hours. When necessary, the surface antigens can be selected and used as the pathogen recognition element for confirmation.

Glycated Serum Albumin and AGE Receptors

Stefan W. Vetter, in Advances in Clinical Chemistry, 2015

3.2.3 Fructosamine Colorimetric Assay

Several chromogenic substrates have been used to detect fructosamine. These assays are based on the reduction of a chromogenic compound by fructosamine or similar ketoamine compounds (i.e., Amadori products) that can undergo oxidation. Substrates that have been used include MTT (3-(4,5-dimethylthiazol2-yl)-2,4-diphenyltetrazolium bromide) [83] and related tetrazolium compounds. A very frequently used compound is nitroblue tetrazolium (NBT; 2,2′di-p-nitrophenyl-5,5′-diphenyl-(3,3′-dimethoxy)-4,4′-bisphenyleneditetrazolium) [84].

Chromogenic substrates

The chromogenic substrates used in this test are various sugars that are linked to either o-nitrophenol or p-nitrophenol by a glycosidic bond. Hydrolysis of the bond frees the nitrophenol, which is detected by its yellow colour. Nitrophenyl derivatives of a large variety of d and l sugars are commercially available, such as p-nitrophenyl-α-d-glucopyranoside (for α-d-glucosidase), p-nitrophenyl-β-d-glucopyranoside (for β-d-glucosidase), o-nitrophenyl-β-d-galactopyranoside (for β-d-galactosidase), p-nitrophenyl-α-l-rhamnopyranoside (for α-l-rhamnosidase) etc. Bear in mind that hydrolysis of a chromogenic substrate does not necessarily mean that the glycosidase for the natural substrate is present. For instance, Staphylococcus aureus can hydrolyse o-nitrophenyl-β-d-galactopyranoside (ONPG) but not lactose (see Kennedy and Scarborough, 1967). Thus results should be reported as ‘ONPG hydrolysed’, not ‘β-galactosidase present’.

•: (Method of Edberg and Kontnick, 1986) Prepare Sorensen’s phosphate buffer from solutions A and B. Solution A contains 35.6-g Na₂HPO₄·2H₂O per litre of distilled water; solution B contains 27.6-g NaH₂PO₄·H₂O per litre of distilled water. Combine 40.5 ml of solution A with 9.5 ml of solution B to give 50 ml of 0.2-M phosphate buffer and verify that the pH is 7.4. Dilute the stock buffer fourfold to give a 0.05-M working buffer.
•: Dissolve the nitrophenyl glycoside in the working buffer to give a concentration of 0.5 mg/ml. Dispense 0.25-ml portions into a 12 mm×75 mm tube. Inoculate the tube with several colonies of the organism to be tested and incubate in a water bath for 1 h at 35°C. Look for the development of a yellow colour. No colour change should occur in an uninoculated control tube.

AEC Chromogen Kit
abx097196-1Kit	Abbexa	1 Kit	EUR 326.4

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abx097197-1Kit	Abbexa	1 Kit	EUR 326.4

DAB Chromogen, 4ml
NB314D	Innovex	4 ml	EUR 295.2

AEC Chromogen Kit
abx097196-100l	Abbexa	100 µl	EUR 200

AEC Chromogen Kit
abx097196-1ml	Abbexa	1 ml	Ask for price

AEC Chromogen Kit
abx097196-200l	Abbexa	200 µl	Ask for price

DAB Chromogen Kit
abx097197-100l	Abbexa	100 µl	EUR 200

DAB Chromogen Kit
abx097197-1ml	Abbexa	1 ml	Ask for price

DAB Chromogen Kit
abx097197-200l	Abbexa	200 µl	Ask for price

AEC Chromogen Kit
MBS826720-5x60mL	MyBiosource	5x60mL	EUR 745

AEC Chromogen Kit
MBS826720-60mL	MyBiosource	60mL	EUR 185

DAB Chromogen Kit
MBS826735-5x60mL	MyBiosource	5x60mL	EUR 745

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