PTX3 monoclonal antibody (MNB4)

Manuals And Inserts

• Datasheet

Specific Protocol

Sandwich ELISA for PTX3

1. 96 well-ELISA plates (Nunc MaxiSorp 446612) were coated with 100µl of MAb to PTX3 (human) (MNB4) (Prod. No. ALX-804-464), 1μg/ml in 100µl coating buffer (15mM carbonate buffer pH 9.6) and incubated overnight at 4°C.
2. After incubation, the plates were washed three times with 300µl/well of washing buffer (PBS + 0.05% Tween 20), and then 300µl of 5% dry milk in washing buffer were added to block non-specific binding.
3. The plates were incubated for 2 hours at room temperature and then washed three times with washing buffer.
4. 50µl (in duplicate) recombinant human PTX3 standard (Prod. No. ALX-201-124) (75pg/ml to 2.4ng/ml ), cell supernatants or biological fluids diluted in PBS without Ca and Mg + 2% BSA were added and then incubated for 2 hours at 37°C.
5. The plates were washed five times with washing buffer, and then 100µl of PAb to PTX3 (human) (Biotin) (Prod. No. ALX-210-365B) 50ng/ml in washing buffer were added in each well.
6. The plates were incubated for 1 hour at 37°C and then washed five times with 300µl of washing buffer.
7. 100 µl/well streptavidin-horseradish peroxidase diluted 1:4’000 were added and the plates incubated for 1 hour at room temperature.
8. After incubation the plates were washed 5 times with washing buffer and 100µl of TMB substrate solution was added to each well. After incubation for 10 min. at room-temperature, 50μl of stop-solution (1M H₂SO₄) was added. Subsequently, absorbance was read within 30min at 450nm.

Note: Do not use heparin plasma or serum samples with this assay. EDTA samples are recommended. The detection value in serum is generally less than in plasma samples.

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