Common enzymes and substrates in ELISA detection

The most commonly used chromogen substrates for HRP are o-phenylenediamine (OPD), 2,2'-azine- (3-acetylphenylthiazolesulfonic acid-6) [2,2'-azino-di (3-ethylben2thiazolinesulphonicacid -6), ABTS] (heterocyclic azine), tetramethylbenzidine (TMB) and 4-aminoantipyrine: phenol coupling substrate equivalent. The above-mentioned chromogen substrates are mainly reacted by HRP in two forms: â‘  oxidation of redox substrates, such as OPD, ABTS, etc .; â‘¡ the oxidation coupling of an amino aromatic agent to another aromatic compound, such as 4- Aminoantipyrine: phenol, etc.

(1) Redox chromogen substrate

OPD is considered to be one of the most sensitive chromogen substrates for HRP. It is composed of hydrogen peroxide (H202) under the action of HRP.

It is oxidized and polymerized into 2,2'-diaminoazobenzene (DAB). At around pH5.0, DAB has a wide range of maximum absorption at a wavelength of 450nm. When the pH value drops to 1.0, the maximum absorption wavelength moves to 492nm, and at the same time, the molar extinction coefficient becomes larger and the color development becomes deeper (Figure 4-2) . Therefore, strong acids such as sulfuric acid or hydrochloric acid are commonly used to terminate the reaction. In actual work, after terminating the reaction with strong acid, especially hydrochloric acid, the color development is not stable, and the color often darkens with time. This is due to the remaining hydrogen peroxide that continues to oxidize OPD after the reaction to produce non-enzymatic DAB. Someone added a reducing agent such as sodium sulfite to the strong acid reaction termination solution. The reducing agent can quickly and completely reduce the remaining hydrogen peroxide to prevent the non-enzymatic oxidation of OPD. The result is stable color development, which does not change within tens of hours and does not need Avoid light. The disadvantage of OPD is that it has a mutagenic effect on the body. Due to the instability of OPD, in current commercial kits, OPD is supplied as tablets or powders, and then dissolved in the corresponding buffer before use.

In the ELISA measurement, the specific formulation of the OPD chromogen substrate is â‘  color substrate solution: containing 20 mmol / L OPD and 12 mmo! / L in 0.1mol / L citrate buffer (pH5.0) H202, or 10 mmol / L OPD and 5.5 mmol / L H202; â‘¡ Stop solution: 2 mol / L sulfuric acid (containing 0.1 mol / L sodium sulfite); â‘¢ Measurement wavelength: 492 nm.

TMB is a new HRP chromogen substrate superior to OPD. The oxidation product bifenone has a maximum extinction coefficient at a wavelength of 450 nm. If the amount of HRP is small and the hydrogen peroxide solution and TMB are excessive, blue cation roots are formed. By lowering the pH, the blue cationic root can be converted into yellow bifenone (Figure 4-3). The use of sulfuric acid as a terminator can stabilize the product for 90 minutes, but then the background color continues to deepen. Some people in China believe that the use of 1% SDS as a terminator can keep the bright blue color unchanged for 24 hours. The positive and negative contrasts are very obvious, but In our laboratory, it is found that 1% SDS has a strong fading effect on the above color development, and it is still ideal to use sulfuric acid as a terminator. In ELISA, after the color of the substrate reaction is developed, the colorimetric measurement result at the wavelength of 450 nm with the maximum absorption is often selected. In order to improve the sensitivity of ELISA measurement using TMB as a substrate, 'Madersbacher and Berger et al. Chose the colorimetric measurement at a wavelength of 405 nm when the color development was exponentially deepened. They first measured the values ​​of a series of standards with known concentrations at 450nm and 405nm, and confirmed that the ratio of A450nm / A405nm was 3.2, and then measured the OD value obtained by using a sample with a low concentration of the test substance at a wavelength of 405nm and multiplied by a constant of 3.2 , Which is the true value of the specimen to be tested at a wavelength of 450nm. This dual-wavelength measurement method can increase the ELISA measurement range by 3 times. Although the color development reaction of TMB is the same as the redox reaction of OPD, the reaction of the former is reversible. If the reducing agent (vitamin C, sodium sulfite, SDS, etc.) is present in the termination solution, the color development of the reaction can quickly disappear. Although TMB has relatively low solubility, due to its high detection sensitivity and no mutagenic effect, it has basically replaced OPD and has become the most common chromogen substrate for HRP.

In commercial ELISA kits, especially domestic products, TMB chromogen substrates are often prepared with two liquid reagents A and B, one of which is a solution containing a certain concentration of hydrogen peroxide, and the other is a TMB solution. In view of the relatively unstable characteristics of hydrogen peroxide and TMB in the solution, when we use the ELISA kit, if we find that the color of substrate A and (or) B appears, or take a drop of each and mix the color, it means that The substrate solution of this kit has deteriorated or has been contaminated and must be discarded.

In the ELISA measurement, the specific formulation of TMB chromogen substrate is â‘ color substrate solution: first dissolve TMB in dimethyl sulfoxide at a concentration of 0.1 mol / L, and then dissolve it in 1 mmol / L and H202 Dissolve in 0.2mol / L sodium acetate / citrate buffer solution (pH4.0) at a concentration of 3.0 mmol / L, and then apply. â‘¡ Stop solution: 2 mol / L sulfuric acid. â‘¢Determination wavelength: 450nm.

ABTS is also a highly sensitive substrate for HRP. In the presence of H: O :, the ammonia salt of ABTS turns into a cationic root that is prone to disproportionation (Figure 4-4). The cationic root is green, which is more suitable for visible light measurement than the yellow oxidation product of OPD (measurement wavelength = 414 nm). The above color rendering is also unstable, 10 mmol

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