ELISA reagents and enzymes

ELISA enzyme should be used to meet the following requirements: high purity, catalytic conversion rate, specificity and strong, stable nature
Set, rich source, inexpensive, prepared conjugate to retain it after the active part and catalytic ability. The best
Specimen does not exist in the same subjects enzymes. In addition, it is easy to prepare the corresponding substrate and preservation, inexpensive, non-ferrous production
Easy to measure such things.
In ELISA, the commonly used enzyme horseradish peroxidase (horseradish peroxidase, HRP) and alkaline phosphate
Acid enzyme (alkaline phosohatase, AP). ELISA reagents in a few commodities, the application of the enzyme glucose oxidase still
Enzyme, β-D-galactosidase and urease enzymes.
Made ELISA reagents are generally prepared with the HRP conjugate. HRP is a glycoprotein, is about 18% sugar, divided
Molecular weight of 44,000, is a complex enzyme, the main enzyme (enzyme protein) and cofactor (heme) combination is a
Kinds of porphyrin proteins. The main enzyme colorless glycoprotein in 275nm wavelength of maximum absorption, the iron cofactor is dark brown porphyrin
Ring in the 403nm wavelength of the highest peaks. The purity of HRP with RZ (Reinheit Zahl, German, meaning purity
Number) that the 403nm absorbance and 280nm absorbance ratio, high purity of HRP RZ ≥? .0.
HRP ELISA in addition to meeting the above requirements marker enzyme, more affordable and more stable nature of the characteristics. Value
Of note is that in the selection of enzymes, in addition to its purity RZ, but also should be noted that enzyme activity. Enzymes such as the preservation of high purity does not
When, energy will be reduced. Enzyme activity in enzyme units contained, the role of substrate available products generated after
Determination of the amount tested.
Many foreign countries use alkaline phosphatase ELISA reagents (AP) as a marker enzyme. There are two common sources of the AP, respectively,
From E. coli and calf intestinal membrane extract. Biochemical characteristics of enzymes from different sources slightly different features, extracted from E. coli
The AP molecular weight of 80,000, the most suitable enzyme at pH 8.0; extract with calf intestinal membrane molecular weight of the AP
100000, at pH 9.6. In the ELISA, AP sensitivity of the system is generally higher than HRP system, compared with the blank value
Low, but the AP is expensive to prepare conjugates with low income rate than HRP.
3.2.2 antigen and antibody
Preparation of antibody conjugates generally are used when high purity of the IgG, in order to avoid linking with the enzyme protein, other miscellaneous dry
Interference. Best pure antibody affinity chromatography, so are all conjugate with specific immune activity in the high thin
Address the degree of reaction, the end results of the pale. As with F (ab ') 2 is marked, more samples can avoid RF interference.
ELISA antigen in the ELISA using small model, the total requirement is antigen must be of high purity.
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