High Binding Capacity Surfaces for Immunological Assays

HB 8

A hydrophilic surface suitable for passive adsorption of proteins with different grades of hydrophilicity

Features

Assays in which the adsorbed molecule exceeds (up to 400ng/cm2) the complementary molecules that have to bedetected.

Furthermore this surface is highly selective and shows high affinity towards the adsorption of molecules also whenthose are present in very small amounts (<50 ng/cm2) allowing to obtain the maximum sensitivity of the test.

Application

Protein - Antibody Anti-IgM adsorbed onto solid phase used in IgM capture assays
Lipo-protein - Rubella antigen adsorbed onto solid phase used in IgG assays
ELISA (competitive) tests for Steroid Hormones and TSH

Performances

A test simulating a competitive method shows the performance of this surface.

Principle

A limited amount of biotinylated albumin, coated on the well surface, was allowed to react with a constant amount ofstreptavidin peroxidase along with various amounts of unlabelled streptavidin used as standard solutions. Unboundreagents were rinsed away

After incubation with TMB and stopping by adding Sulphuric Acid, the colour intensity was read at 450 nm.Calculation of results

The enzymatic activity, present in the well, is inversely proportional to the concentration of unlabelled streptavidinpresent in the standard solution.

Table 1 shows the records of the absorbance at 450 nm for each point of standard solution.

  HB 8 B standard/B
Max x 100
Competitor B standard/B
Max x 100
B Max 1327 100 1287 100
B 5 ng/ml 1093 82.4 1129 87.7
B 10 ng/ml 921 69.4 898 69.8
B 25 ng/ml 644 48.5 627 48.7
B 50 ng/ml 424 31.9 421 32.7
B 100 ng/ml 267 20.1 264 20.5
B 200 ng/ml 131 9.9 170 13.2

The maximum binding reactivity (B Max) is represented by the absorbance derived from streptavidin-peroxidase in thepresence of 0 ng of unlabelled streptavidin.

The presence of unlabelled streptavidin in the standard solutions is expressed using a percentage ratio between therelative absorbance of that standard solution (B standard concentration) and the absorbance derived from streptavidinperoxidasein the presence of 0 ng of unlabelled streptavidin.