1.Preparation of Standard
Prepare standard is a very difficult and complicated step during the labeled immunoassays. Following are talking about how to produce standard for laboratory and business.
Matrix Preparation. Matrix of the standard should be as same as tested sample. The serum without under-test materials should be used for produce some standards which can determine specific substance in serum. This can supply a suitable medium environment for reaction. Because zero-serum is difficult to made, some suitable buffer can be use for standards’ production. Adding carrier protein (usually is 1%~2% Albumin Bovine) in buffer to make the medium environment similar with the sample.
Methods for zero-serum production
- Adsorption: Using carbon to remove some small molecules in serum, such as T3, T4. Although the process is very simple, clear of tested materials absolutely is difficult. Furthermore, macromolecular substances cannot be made by this way.
- Repeated freeze-thaw method: Low-value serum can be chosen for macromolecular substances production. Repeated freeze-thaw can make the materials inactive. This method can lead some protein hormone out of activity. But is still difficult to get the real zero-serum.
Affinity chromatography
Use specific antibody to produce Affinity Column. This can absorb tested antigen. This method can get a high quality zero-serum but it’s complicated and need a high cost.
How to choose pure standards. The standards should choose the materials with high CP and immune pure. The immune pure is more important than CP. The material which has cross-reactive with tested materials should not be included in standard.
Produce Standards
Produce high concentrated standards by using zero-serum. According to the laboratory’s requirement, produce series standards which composed by different concentrations.
2. Standard’s Identification
a) Identification of immune activity
Select high specificity antiserum to make dose-response curve of the recognized standards (such as WHO international reference materials, national standards). Then, observe the parallel nature of the two curves. If the two curves are paralleled, it means there is same appetency between two substances. Both substances are homogeneity materials. On the other hand, if the two curves can not be paralleled, there is heterogeneity of the two substances. So, this standard can not be use.
Calibrate the concentration
Using immune activity standard with organization stands make dose-response curves at same concentration. At this time, two curves should be fundamental overlap. If these two curves parallel but not overlap, take two dose-response curves’ corresponding dose at 50% combining site (ED50) to calculate their conversion factor. Then, adjust the concentration of standard until the two curves basically overlap.
3. Standard Measurement
A lot of small molecule antigen or hapten has been able to get pure or synthetic. Most of them have the ability to be use as standard. Their measurement usually displays by weight. Such as mg/ml, ng/ml, pg/ml and so on. At present, WHO recommend to use mass units instead unit of weight. For example, mol/L, mmol/L and nmol/L. It’s complicated for macromolecular active substances. The absolutely pure is difficult to get lead to measurement direct use the weight or quality. Therefore, the biological activity is usually chosen as measurement unit, such as IU/L, mIU/L and so on.
