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.

results    12 pmol/well (200 µl volume)


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


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


4. Uniformity of biotin binding

Test conditions:

specificity
CV <5%


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

chemical compound conditions
SDS 0.1% 0.6M NaCl 56°C 2h
30% Formamide in 0.6M NaCl 37°C 2h
0.1% Tween® 20 in 0.1M PBS 37°C 2h
0.05M NaOH RT 20'
0.2M NaOH RT 20'
standard -0.1% Tween® 20 in 0.1M PBS 4 x
Urea 8M 60'-30'-15'-5'

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

temperature 4 ° C 37°C
OD 2568 2667
CV% 3.3 3.6

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:

plate n. conditions time
1 4° C 10 days
2 22°-23° C 10 days
3 37° C
22°-23° C
- 20° C
37° C
22°-23° C
3 days
12 h.
10 h.
3 days
3 days

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

temperature 4 ° C RT
OD 1890 1950
CV% 1.8 2.3