Preparation method of conjugate and relative kit
A combination and kit technology, applied in the field of immunoassay, can solve the problems of complex operation, affecting the sensitivity of immunoassay, alkaline phosphatase marker activity and unsatisfactory labeling rate, etc.
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Embodiment 1
[0067] Dissolve sodium acetate (sodium acetate) in 800 μl 30mM MES buffer (pH 6.0), add EDC and NHS, add buffer to 1000 μl, the final concentration of sodium acetate is 1.8mol / L, the final concentration of EDC is 18mmol / L, NHS The final concentration is 18mmol / L. After reacting at 22° C. for 30 minutes, 2 mg (18 nmol) of alkaline phosphatase and 2 ml of 0.1 M phosphate buffer (pH 7.2) were added. After reacting at 22°C for 3 hours, use 15ml of 30KD molecular weight cut-off ultrafiltration tube (Millipore Company), use 30mM MES (pH 6.0) buffer as the replacement buffer, and perform ultrafiltration 3 times to remove free sodium acetate, EDC, and NHS and reaction by-products to obtain an amino-blocked alkaline phosphatase solution.
[0068] Add EDC (final concentration 10mmol / L) and NHS (final concentration 20mmol / L) to 1ml 10mmol / L amino-blocked alkaline phosphatase solution. After reacting at 22° C. for 30 minutes, mercaptoethanol with a final concentration of 10 mM was added...
Embodiment 2
[0071] Dissolve acetic acid in 2000 μl 30mM MES buffer (pH 5.0), adjust the pH value to 5.0, add EDC and NHS, add 2mg (18nmol) alkaline phosphatase, add 30mMMES buffer (pH 5.0) to 3000μl, the final acetic acid The concentration is 0.06mol / L, the final concentration of EDC is 24mmol / L, the final concentration of NHS is 6mmol / L, and the final concentration of alkaline phosphatase is 6μmol / L. After reacting for 3 hours at 22°C, use 15ml of 30KD molecular weight cut-off ultrafiltration tube (Millipore Company), use 30mM MES buffer (pH 6.0) as the replacement buffer, and ultrafilter three times to remove free acetic acid, EDC, NHS and Reaction by-products to obtain amino-blocked alkaline phosphatase solution.
[0072] Add EDC (5 mmol / L final concentration) and NHS (25 mmol / L final concentration) to 1 ml of 10 mmol / L amino-blocked alkaline phosphatase solution. After reacting at 22° C. for 60 minutes, mercaptoethanol with a final concentration of 10 mM was added to obtain a carboxy...
Embodiment 3
[0075] Dissolve formic acid in 800 μl 30mM MES buffer (pH 6.0), adjust the pH value to 6.0, add EDC and NHS, add buffer to 1000 μl, the final concentration of formic acid is 13.5mmol / L, the final concentration of EDC is 0.36mmol / L, The final concentration of NHS was 1.8mmol / L. After reacting at 37° C. for 5 minutes, 2 mg (18 nmol) of alkaline phosphatase and 2 ml of 0.1 M phosphate buffer (pH 7.2) were added. After reacting at 4°C for 20 hours, use 15ml of 30KD molecular weight cut-off ultrafiltration tube (Millipore Company), use 30mM MES buffer (pH 6.0) as the replacement buffer, and ultrafilter three times to remove free formic acid, EDC, NHS and Reaction by-products to obtain amino-blocked alkaline phosphatase solution.
[0076] Add EDC (final concentration 10mmol / L) and NHS (final concentration 20mmol / L) to 1ml 10mmol / L amino-blocked alkaline phosphatase solution. After reacting at 37° C. for 10 minutes, mercaptoethanol with a final concentration of 10 mM was added to o...
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