| Each well of the microplate is washed twice with wash buffer.* |
Dispense 50µl guinea pig anti-insulin serum per well (reconstituted by Sample diluent 2 ) |
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Dispense 50µl guinea pig anti-insulin serum per well (reconstituted by Sample diluent 1) |
Dispense 45µl of sample diluent 2 and 5µl of sample (or 5µl working insulin standard reconstitued by Sample diluent 2)per well |
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Dispense 50µl sample (or 50µl working insulin standard reconstitued by Sample diluent 1)per well |
| Incubate the microplate overnight at 4 °C |
| Wash the well three times with wash buffer* |
| Dispense 100 µl anti-guinea pig antibody enzyme conjugate per well |
| Incubate the microplate for 3 hours at room temperature |
| Wash the well five times with wash buffer solution* |
| Dispense 100µl enzyme substrate solution per well |
| Incubate microplate for 30 minutes at room temperature while avoiding exposure to light |
| Stop the reaction by adding 50µl of enzyme reaction stop solution per well |
| Immediately measure A492 and subtract A630 values |
| Calculate the insulin concentration by the standard curve |
* Each well should be washed with 300µl of wash buffer. Aspirate the wells completely so all excess solution is removed
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