FUNCTIONAL FEATURES OF STREPTAVIDIN COATED PLATES

The following parameters were analysed

1. binding capacity towards biotin
2. specificity towards biotin
3. binding capacity towards biotinylated IgG
4. uniformity
5. stability tests:
   1. endurance under strong chemical contacts
   2. shelf life at 37°C
   3. temperature stress (transport simulation)
   4. long storage

1. Binding capacity towards biotin

Streptavidin coated wells (and BSA saturated control wells) were incubated with a calibrated biotin solution.

Subsequently, aliquots of this solution, concomitantly with biotin standards, were mixed with biotinylated peroxidase and transferred into new empty streptavidin coated wells. From the amount of enzyme bound to the solid phase , the biotin content of the samples was calculated. This value was compared with the amount of biotin originally added; from the difference (corrected for-non specific binding of biotin to the control wells), the binding capacity of the surface for biotin was derived.

Streptavidin coated surfaces

2. Specificity towards biotin (method 2)

Streptavidin coated wells were incubated with solutions (from 10 to 0.12 ng/ml) of biotinylated peroxidase and unbiotinylated peroxidase (blanks) for 30' RT
After a washing step, the wells were incubated with TMB and blocked with sulphuric acid 1N
The OD values were read at 450 nm

Streptavidin coated surfaces

3. Binding capacity towards biotinylated IgG (method 1)

Streptavidin coated wells were incubated with solutions (from 500 to 0 ng/ml) of biotinylated IgG for 30' RT
After a washing step, the wells were incubated with AHIgG-Pod for 30' RT, again washed and incubated with TMB and blocked with sulphuric acid 1N
The OD values were read at 450 nm

Streptavidin coated surfaces

4. Uniformity of biotin binding

Test conditions:

• A 96 wells plate was incubated with a biotinylated peroxidase solution.
• After a washing step, the plate was incubated with the TMB, then the reaction was stopped adding sulphuric acid 1N
• The optical density was determined at 450 nm and used for calculating the CV%

Streptavidin coated surfaces

5. Stability tests:

5.1. Endurance under strong chemical contacts

Endurance under strong chemical contacts was determined following method 1 (Biotinylated IgG from 500 to 0 ng/ml) where the first washing step was substituted by the following incubations

Results

Urea 8M

5.2. Shelf life at 37°C

Streptavidin coated wells maintained for 15 days at 37°C in comparison with standard stored at 4°C, were analysed with method 1(biotinylated IgG =100 ng/ml)

results

5.3. Temperature stress (transport simulation)

Stability was checked under different temperatures as those which may occur during transport.
Method 2 was used for comparing streptavidin coated plates subjected to the following conditions:

Results

5.4. Long storage

Streptavidin coated wells maintained for 30 months in a warehouse without air conditioning (temperature range from 10°C to 40 °C) in comparison with samples stored at 4°C(standard condition), were analysed with method 2.
The results show the exceptional stability of the coating.

Results