Comparison of different types of High Binding capacity polystyrene strips

In order to check the performances of different polystyrene strips' surfaces we performed an extensive study comparing biomat's High Binding Capacity (HB8) strips with one of the most used High Binding Capacity type of strips currently available on the market.

To ensure the validity of results the test was performed with methods as near as possible to the standard methods which, in our knowledge, are used by manufacturers when preparing polystyrene strips as solid phase to set up diagnostic kits (e.g. torch ELISA kits).

The kind of molecules used for testing were both IgM and IgG for determination of Rubella, Cytomegalovirus and Toxoplasma.

Materials and methods

I Preparation of plates

Antigens for Rubella, Cytomegalovirus, Rabbit IgG to Human IgM (DAKO A426) were diluted in carbonatebicarbonate buffer 0,1 M pH 9,6 and both samples of strips were coated at the same time. The coating was performed at 4° C.

After a washing step the plates were saturated with PBS 0,1 M pH 7,2 containing 1% Bovine Serum Albumin and incubated overnight at 4°C

After a further washing step the plates were dried at 37°C for two hours, then sealed under vacuum and stored at 4°C until use.

All the sera used in this test came form hospital laboratories and were certified to be positive or negative using the commercial kits manufactured by: Behring; Biomerieux-Vidas; Sorin Biomedica.

II IgG assay

The scheme for performing the IgG assays was the following:

  1. 100µl diluted samples and calibrators were incubated for 30 min. at room temperature in each type of antigencoated wells
  2. a washing step with 0.1M PBS pH 7.2 + 0.05% Tween® 20 was performed
  3. 100µl/well of purified goat-anti Human Fc IgG Peroxidase were added and incubated for 30 min at room temperature
  4. a further washing step as that at point 2. was performed
  5. 100µl/well of substrate (TMB) were added and incubated for 15 min at room temperature
  6. the reaction was stopped by adding 100 µl of sulphuric acid
  7. reading at 450 nm was then performed

III IgM capture assay

The scheme for performing the IgM assays was the following:

  1. 100µl diluted samples and calibrators were incubated for 1 hour at room temperature in the common anti-IgM coated wells
  2. a washing step with with 0.1M PBS pH 7.2 + 0.05% Tween® 20 was performed
  3. 100µl/well of a complex of the appropriate biotinylated purified antigen and streptavidin-peroxidase was added and incubated for 1 hour at room temperature
  4. a further washing step as that at point 2. was performed
  5. 100µl/well of substrate (TMB) were added and incubated for 30 min at room temperature
  6. the reaction was stopped by adding 100 µl of sulphuric acid
  7. reading at 450 nm was then performed

Analysis of Data

The data obtained from the two types of samples of microplates were processed in the following way:

NEGATIVE SAMPLES POSITIVE SAMPLES
a. min. O.D. observed for each type of strip a. coefficient of correlation
b. Max. O.D. observed for each type of strip b. linear regression calculated as
y=a+bx (1)
with a confidence level of 95%
x values were those obtained with competitor's samples
y values were those obtained with biomat's HB samples
c. mean of Standard Deviations

the results are exposed in the following tables

The above results are confirmed by the correspondence with the clinical data obtained by the hospital laboratory

Discussion of Results

The analysis of the data exposed in tables A and B shows:

Methods of Analysis

The increase of binding capacity of High Binding (HB) strips is checked through the two different methods of analysis described here in which Medium (MB) and High Binding (HB) and commercial comparison (Comp HB) strips are compared.

Method 12

Method 12 is a sandwich method with Rabbit AHIgG (primary antibody)- HIgG(secondary antibody) -AHIgG/POD conjugate

  1. dispense 100 µl/well of 0.15 µg/ml Rabbit Anti-HIgG in 0.1M Carbonate Buffer pH 9.6, incubate 3 hrs. at 37°C and then overnight at 4°C
  2. wash 2 times with 0.1M PBS pH 7.2 + 0.05%Tween® 20
  3. dispense 150 µl/well of BSA 1% in 0.1M PBS pH 7.2 and incubate 30' at 37°C for blocking the remaining active sites
  4. wash 3 times with 0.1M PBS pH 7.2+ 0.05% Tween® 20
  5. dispense 100 µl/well of HIgG (concentration from 20 to 0.009 µg/ml) and incubate 45' at 37°C
  6. wash 3 times with 0.1M PBS pH 7.2 + 0.05% Tween® 20
  7. dispense 100 µl/well of Goat Anti-HIgG-POD conjugate and incubate 45' at 37°C. Dilution factor 1/10.000
  8. wash 3 times with 0.1M PBS pH 7.2 + 0.05% Tween® 20
  9. dispense 100 µl/well of TMB
  10. after 30' stop the reaction with H2SO4 1 N
  11. reading is made at 450 nm

Method 14

Method 14 exploits the sensitivity of Streptavidin against the molecule of Biotin which is bound on the surface with the secondary Antibody.

Method 14 is currently used for Quality Control tests

Coating

  1. dispense 100µl/well of 5 µg/ml of Rabbit Anti-HIgM in 0.1M Carbonate Buffer pH 9.6 and incubate overnight at 4°C.
  2. wash 4 times with 0.1 M PBS pH 7.2
  3. dispense 150 µl/well of BSA 1% in 0.1M PBS pH 7.2 and incubate overnight at 4°C for blocking the remaining active sites
  4. decant the solution, essicate the plate and store at 4°C until use

Test

  1. dispense 100 µl/well of Biotinylated Goat Anti-Rabbit IgG (concentration from 62 to 0.7 ng/ml) and incubate 1 hour at room temperature
  2. wash 4 times with PBS pH 7.2 + 0.05% Tween® 20
  3. dispense 100 µl/well of 160 ng/ml of Streptavidin-POD and incubate 1 hour at room temperature
  4. wash 4 times with PBS pH 7.2 + 0.05% Tween® 20
  5. dispense 100 µl/well of TMB
  6. after 30' stop the reaction with H2SO4 1 N
  7. reading is made at 450 nm