ELISA microplate Research Article
To effectively immobilize proteins on these types of surfaces, the coupling buffer is a key factor. Amine- free buffers should be utilized to avoid the competitive reaction, and alkaline pH could facilitate the coupling reaction. To find a high performance buffer for immo- bilizing the protein CD86 on these three surfaces, sev- eral buffer systems including 0.1 M phosphate saline buffer at pH 7.4, 0.5 M sodium bicarbonate at pH 8.7 and 0.1M borate buffer at pH 9.5 were investigated. As predicted, higher pH buffer gave higher response due to improved coupling efficiency. 0.1 M borate buffer at pH 9.5 was eventually used for all three surfaces in the study. GBO plates have unmodified polystyrene surface, therefore immobilization on this surface is through pas- sive adsorption. Among the four buffer solutions tested with this plate, 0.5 mol/l sodium bicarbonate was found slightly better than others. Immobilization time was investigated from 0.5 to
4 h with 0.5 h intervals for all the surfaces. In addi- tion, the magnetic beads were checked overnight.
A
3.0 2.5 2.0 1.5 1.0 0.5 0.0
C GBO plate Key term
Immobilization capacity: Maximum amount of ligand that can be immobilized on the surface of one plate well.
The optimal immobilization time for the plates was between 1 to 2 h and 4 h for the magnetic beads. The temperature was investigated at 37°C and room tem- perature. No significant difference was found with these two different settings, and 37°C was used in the study.
Immobilization capacity With maximizing the assay sensitivity as the primary goal, we sought to maximize the amount of capture protein on the various surfaces. For MA plates, the product information showed that MA plate had bind- ing capacity of approximately 125 pmol biotin pentyl- amine (328 Da)/well. Taking this as a reference and assuming the binding behavior of CD86 protein (mass 38 kDa) is similar to that of biotin pentylamine, the
B
200 µl serum, y = 0.0064x + 0.0432, R2
= 0.9925
100 µl serum + 100 µl PBS, y = 0.0031x + 0.003, R2
= 0.9972
0100 200 300 400 500 600 700 800 900 1000 Analyte serum concentration (ng/ml)
3.0 4.0
2.0 1.0 0.0
0 100 200 300 400 500 600 700 800 900 1000 Analyte serum concentration (ng/ml)
Figure 1. Capture capability of CD86 immobilized microplates: (A) unmodified, (B) maleic anhydride-activated and (C) Nunc® Immobilizer™ Amino. Solid lines indicate capture linearly relates to drug concentration, and dashed lines indicate the linearity disappears and capture capacity reached. Data are the average of three measurements. Linear regression equations are included in the diagrams. GBO: Unmodified; MA: Maleic anhydride-activated; NIA: Nunc®
Immobilizer™ Amino; PBS: Phosphate-buffered saline. future science group
www.future-science.com 311 NIA plate
0.30 0.25 0.20 0.15 0.10 0.05 0.00
MA plate
200 µl serum, y = 0.0027x + 0.0015, R2
= 0.9958
100 µl serum + 100 µl PBS, y = 0.0015x + 0.0074, R2
50 µl serum + 150 µl PBS, y = 0.0009x + 0.0037, R2
0100 200 300 400 Analyte serum concentration (ng/ml) 200 µl serum, y = 0.0062x + 0.0408, R2 = 0.9931 50 µl serum, y = 0.0033x + 0.065, R2 = 0.9942
100 µl serum + 100 µl PBS, y = 0.0039x + 0.0348, R2
= 0.9904 = 0.9916 = 0.9909 500
Analyte/IS peak area ratio
Analyte/IS peak area ratio
Analyte/IS peak area ratio
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