Medium and High Binding Well Plates

Applications & Techniques

Biomat polystyrene medium and high binding well plates are ideal devices to coat biomolecules and to set up ELISA tests for the diagnostic and pharmaceutical fields.

The enzyme-linked immunosorbent assay (ELISA) is one of the most sensitive and reproducible technologies available. The assay you can set up is rapid, simple to perform and easily automated.

Polystyrene is composed of an aliphatic carbon chain with pendant intermittent benzene rings. This provides a very hydrophobic surface and plates of this type are typically referred to as “medium binding”. Thanks to a surface treatment, such as irradiation, it is possible to enhance the polystyrene plates surface binding capacity. These kind of surface treatments break a certain number of benzene rings, yielding carboxyl (COOH) and hydroxyl (OH) groups. The presence of these molecular groups provides an opportunity for hydrophilic interactions. Well plates with such characteristics are referred as “high binding”. The resulting surface is primarily hydrophobic with intermittent carboxylic and hydroxylic groups capable of ionic interactions with positively charged groups on biomolecules.

Risultati immagini per polystyrene structure of microtiter plates

The forces that passively adsorb biomolecules to the surface of medium and high binding well plates are typically:

  • hydrophobic interactions
  • van der Waals forces
  • hydrogen bonding
  • ionic interactions

The following pictures describe the four physical adsorptions that occur when the biomolecule comes in contact with the bottom of the polystyrene plate.

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In order to select the best type of well plates (high-binding or medium-binding) to adsorb the biomolecule it is necessary to know the molecule chemical structure to exploit its interaction with the surface of the well.

The 20 amino acids that make up the primary structure of a protein can be divided into two categories:

  1. Idrophobic: such as glycine, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine that have shown a better binding capacity with medium-binding 96 well plate.
  2. Polar/charged: such as tyrosine, tryptophan, serine, threonine, arginine, histidine, lysine, aspartic acid, glutamic acid that have shown a better binding capacity with high-binding 96 well plates.

Medium and high binding well plates tests

Below are some examples of different molecules coatings on Biomat medium and high binding well plates, in particular using proteins whose amino acid composition is known.

Adsorption of Bovine Serum Albumin

Biotinylated Bovine Serum Albumin at 5 µg/ml was coated on Biomat medium and high binding 96 well plates.

After a post coating step, the plates were evaluated for their biotin activity level. In parallel, microplates treated only with post coating, were tested blank.

BSA-biotin medium and high binding coated wells and uncoated ones were incubated with 100 µl of streptavidin-peroxidase 1 mg/ml diluted 1:70,000 for 30 minutes at room temperature.

After a washing step, the wells were incubated with TMB for 10 minutes at room temperature and blocked with sulphuric acid 1N.

The mO.D. values were read at 450 nm and shown in graph below.

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 Adsorption of Bovine Serum Albumin test results

Results

The results obtained show that biotinylated albumin is more clearly adsorbed on high binding surface than on the medium binding surface. The medium binding surface absorbs about 15% of biotinylated albumin compared to the high binding surface.

In fact, if we analyze the aminoacid composition of the primary structure  of Bovine Serum Albumin we find that 65% is composed of polar/ charged (hydrophilic) amino acids and 35% of hydrophobic amino acids.

The prevalence of hydrophilic amino acids allows a more marked bond on the high binding surface that exploit the ionic interaction and van der Waals forces.

It is also known from the analysis of protein structures that in general hydrophobic residues such as Val, Leu, Ile, Phe, and Met tend to be buried in the interior and polar side chains exposed to solvent.

Adsorption of human IgG

Human IgG at 2.5 µg/ml were coated on Biomat medium and high binding 96 well plates.

After a post coating step, the microplates were evaluated for their IgG binding activity. In parallel, plates treated only with post coating, were tested blank.

Human IgG medium and high binding coated wells and uncoated ones were incubated with 50 µl of αHuman IgG-peroxidase 1 mg/ml diluted 1:400,000 for 1 hour at room temperature.

After a washing step, the wells were incubated with TMB for 10’ at room temperature and blocked with sulphuric acid 1N.

The mO.D. values were read at 450 nm and shown in graph below.

Adsorption of human IgG test results

Results
The results obtained show that human IgG are adsorbed almost equally on medium binding and high binding surface. This occurs because the amino-acid composition of the primary structure of IgG is approximately 50% hydrophobic amino acids and 50% polar/charged (hydrophilic) amino acids. Moreover, in the primary structure of the IgG there are large thickenings of hydrophobic amino-acids and this can justify the bonds to the medium binding surface.

Our test demonstrates that both medium and high binding well plates surfaces adsorb an equivalent amount of antibody.

Another aspect that influences the test sensitivity is the steric position of the IgG after the coating and requires farther analysis. The IgG ideal position requires the Fc part facing the well surface and the F(ab)2 part free to bind the antigen. To choose the most suitable surface for your test you must patiently test both.

Adsorption of Poly-DL-alanine (M.W. 1,000-5,000)

Biotinylated Poly-DL-alanine, at 20 µg/ml, was coated on Biomat medium and high binding 96 well plates. After a post coating step, the plates were evaluated for their biotin activity. In parallel, plates treated only with post-coating, were tested as blank.

Poly-DL-alanine – biotin medium and high binding coated wells and uncoated ones were incubated with 100 µl of Streptavidin-Peroxidase 1 mg/ml diluted 1:60.000 for 30′ at R.T.

After a washing step, the wells were incubated with TMB for 15’ at R.T. and blocked with sulphuric acid 1N.

The mO.D. values were read at 450 nm and shown in graph below.

Adsorption of Poly-DL-alanine test results

Results

The results obtained show that biotinylated Poly-DL-alanine is more clearly adsorbed on medium binding surface than on the high binding surface. The high binding surface absorbs about 45% of biotinylated Poly-DL-alanine compared to the medium binding surface.

In fact, since proline is a non-polar amino acid, this is better absorbed by a totally hydrophobic surface such as medium binding.

The total presence of a hydrophobic amino acid allows a more marked bond for the medium binding surface that exploits the hydrophobic interactions.

In our test the proline polymer served as example of a hydrophobic molecule.

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