Patients and doctors depend upon lab values. When it comes to Immunoassays, the Industry has not come up with a solution to this problem. Most labs do not screen for Heterophilic Antibodies in Calcitonin Assays and most Oncologists do not follow Procalcitonin for Medullary. Many patients receive labs with a wide range of values and there are no standards on when the labs need to be questioned.
Heterophilic antibodies are an difficult issue: either the are really induced against components which are used by chance in the ELISA (anti-murine-antibodies recognizing the monoclonal antibody at the solid phase in the whole set of opportunities: anti-species, anti-Ig-class. subclass, iditoype, ..., anti-BSA if BSA is used for blocking ...). If you have any idea about the specificity of this antibodies you can either block them by adding the respective antigen into the sample dilution of you can absorb them i.e. by (magnetic) latex particles which are coated with this antigen.
In much more cases you have unspecific antibodies, especially in cancer patients. In many cases they are recognizing their antigen (once again the solid phase proteins) with lower affinities the the specific antibodies used in the assay. Those unspecific reactions can be blocked by increasing the NaCl-concentration of the buffer (normally in PBS: 0.156 mol/L) up to 0.5 mol/L (mostly 0.3 mol/L is sufficient). Also sugars added (glucose, mannose up to 1%) can be helpful. Let the reaction run at room temperature (not at 37 °C).
Hi, Carol,
I agree with Daniel, there are quite a lot of tricks to increasing antibody specificity. One acn use different salt concenjtration, different Ph, and, as a general rule, reduce amount of primary antibody with parallel decrtesing the entropy during antibody incubation. hoever, even these tricks will not give you 100% specificity garantee...
To be 100% sure one can use more precise assay, like WB (in this case one can be sure about at least size of intercating protein). Immunolocalization, from the other side, can give imformation about target localization with tissue, cell and even organelles resolution.. But, of course, WB of immunolocalization are more complicxated assays and can not be performed with may samples simulotaneously. But can be apllied only in the specail case, when resarches havwe a doubt in ELISA results. good luck!
I understand there is a breaking point that the assay cannot be adequately diluted. And handling issues with various labs also add to the issues. Procalcitonin is much more stable and less subject to handling issues. If a Calcitonin sample was in the range of over 5,000 for example or closer to 10,000.
What would also be the interfering factor after a CT with iodine contrast? I have encountered both Heterophilic of unknown origin and a secondary interference in the same person of unknown origin within 24 hrs directly after CT with contrast. This was duplicated for one year every three months. Procalcitonin has now been added to try to get a better look at the trend in Calcitonin for Medullary Thyroid Cancer. The markers are Calcitonin and CEA.
I was using huge concentrations (in the range of 1mg to 2mg per milliliter) purified mouse immunoglobulins (having all mouse isotypes) in serum or analyte dilution buffer in order to block heterophilic or HAMA interferences.
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