Solid Phase vs. Liquid Phase Ligand Capturing

As in real life, often there are different ways to get the same result. Almost the same, I´d better say, because for the bioanalytical ligand binding assay developer it is indeed a difference whether the ligand is captured in the liquid phase, or immobilized on a solid phase (like a microwell plate or magnetic bead).

Well, this guy is crackbrained, you might say, talking about somehow confidential assay development topics on the bioanalytical binding-assay.com blog, so everybody can switch off his mind, and on top of that saves time for his own assay development project. Good point, I would answer, but all the informations I share with you here are already published in the PubMed database. If I hypothesize you can read and write, you will come across this post topic on your own, will you? So what´s the point? It is a difference you develop your own bioanalytical ligand binding assays for research purposes, or you have to develop custom ligand binding assays to support preclinical/clinical drug development of investigative novel drugs (IND). In the latter case strictly compliance to Good Laboratory Practice (GLP) regulations is obligatory, is a must.

Certainly we developed bioanalytical assays for the biotech industry, and stumbled across stones we couldn´t foresee. The devil is in the details. But we won´t tell you.

Now let´s focus on the two ligand capturing strategies I mentioned in the post headline. Either the ligand can be captured in the liquid phase, followed by immobilization of the catcher on microwell plates or magnetic beads. Or the catcher is first immobilized on a solid phase, and the ligand is captured afterwards. Please notice the catcher is pinched in a molecular sandwich.

We believe the immuno qPCR (iqPCR) technology is one of the bioanalytical ligand binding assays which will revolutionize the assay development world. You hardly can find a more sensitive bioanalytical assay with a broader detection window for peptide, hormone, protein detection: the ancient enzyme-linked immuno sorbent assay (ELISA) for many applications is a discontinued model, at least e.g. for antibody detection assays (ADA).

Another application of the immuno qPCR technology is the detection of human pathogens in food samples (1). Staphylococcus aureus enterotoxins are not only one of the main poisons in food, but the methicillin resistant variant (MRSA) is also the main cause of serious bacterial infections in hospitals worldwide. The more sensitive the assay detection limit is, the earlier the pathogen is detected, and can be combatted.

Aspergillus funghi also produce toxigenic substances which even can cause cancer. A recent publication on immuno qPCR (iqPCR) mediated aflatoxin detection demonstrates the capturing of aflatoxin B1 by monoclonal or polyclonal antibodies in solution, followed by complex immobilization on protein G coupled magnetic beads (2). The detection limit is greatly enhanced if aflatoxin is captured in the liquid phase by a monoclonal antibody before immobilization.

The sandwich hybridization assay is a powerful and sensitive bioanalytical assay for the detection of virtually any nucleic acid ligand. Briefly, the ligand can either be captured by a short complementary probe in solution, followed by solid phase immobilization of the capture probe to streptavidin microtiter plates (3). The ligand is then detected by another short complementary probe which also can hybridize to the ligand. Sirna Therapeutics´s Heptazyme drug at that time was hybridized to both probes in a one-step liquid phase capturing, and immobilized afterwards (3), producing reliable results of outstanding bioanalytical assay sensitivity. The ribozyme is a single-stranded nucleic acid drug, and certainly it can improve assay sensitivity if both the capture and detect probe are added to the sample at once.

For the enzyme-linked immuno sorbent assay (ELISA), these solid phase assays are a consequent advancement of the classic radioimmunoassays (RIA), which are a combination of liquid phase ligand capturing with the immunoprecipitation procedure. Antibody performance in sandwich solid phase ELISAs can provide a better assay accuracy, when compared to RIA (4). Additionally, the antibody is more stable for long-term storage when immobilized to microwell-plates.

References:
1. Panneerseelan L & Muriana PM (2009), J Fod Prot 72(12): 2538-46.
2. Babu D & Muriana PM (2011), J Microbiol Methods, doi.org/10.1016/j.mimet.2011.05.002
3. Brown-Augsburger P et al (2004), J Pharm Biomed Anal 34(1): 129-39.
4. Schramm W et al (1987), Clin Chem 33(8): 1331-7.

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