Research Article Yang, Kernstock, Simmons & Alak


with the other surfaces, and therefore a narrower linear range, its LLOQ was the lowest. This implies that it provided stronger surface binding to the capture pro- tein via the chemical bonds, or its chemical coating is more stable, resulting in less loss of the immuno- complex during washing. Examining the chromato- grams at LLOQ level (Figure 2A–D) also revealed both microplate and magnetic beads platforms have efficient sample cleanup. The noise peaks were generally below 20% of LLOQ. Inter- and intra-batch precision and accuracy with


the microplates and magnetic beads for QCs are sum- marized in Table 3. All results meet LBA validation acceptance criteria of relative error within ± 20% (± 25% at the LLOQ and ULOQ) and relative deviation within 20% (25% at the LLOQ and ULOQ). Overall, the microplates gave better accuracy and precision. Matrix interference, or assay selectivity, was exam-


ined with all these sample preparation platforms. At the retention time of the analyte or IS, no quantifiable peak was observed from blank human serum samples.


A


40 35 30


25 20 15 10 5 0


0.50..5 0 C 150 100 5.1 50 7 07.7 .70 0.60 2.79 3.20 0.60 2.79 3.20 0 0


24 68 Time (min)


8.81 8.8 8 1 5.15 6.40 6.40 50 2.04 2.04 2.72 4.38 2.72 4.38


4.66 6.75 5.68


4.66 6.75 9.97 5.68 9.97 7.18 7.847.84 7.967.96


The typical chromatograms are shown in Figure 3. Nonspecific binding with these sample preparation platforms was also investigated. In this experiment, capture protein, CD86, was replaced with PBS buffer and serum samples containing 1000 ng/ml of ASP2409 were applied. As shown in Figure 4, when capture pro- tein was absent, no analyte peak was observed even with high analyte concentration samples, proving the approaches had high selectivity with no occurrence of nonspecific binding.


Conclusion An unmodified ELISA microplate represented by GBO provided a comparable linear range and an even lower LLOQ compared with magnetic beads. When a chemically activated microplate is used, the plate per- formance is altered by the surface chemistry. The MA plate had a narrower linear range but lower LLOQ than both the NIA plate and magnetic beads. The lower LLOQ is likely due to its strongly bound cap- ture protein on the surface. ELISA microplate plat-


B


1000 2000 3000 4000 5000


0


2 46 8 Time (min)


4.22 D


1000 2000 3000 4000


0


02 46 8 Time (min)


Figure 4. Typical multiple reaction monitoring chromatograms. (A&C): analyte channel; (B&D): IS channel of 1000 ng/ml ASP2409 in human serum when CD86 was absent with unmodified plate (A&B) and magnetic beads (C&D).


316 Bioanalysis (2015) 7(3) future science group


02 46 8 Time (min)


6.38 6.49 6.49


Intensity (cps)


Intensity (cps)


Intensity (cps)


Intensity (cps)


Page 1  |  Page 2  |  Page 3  |  Page 4  |  Page 5  |  Page 6  |  Page 7  |  Page 8  |  Page 9  |  Page 10  |  Page 11  |  Page 12  |  Page 13  |  Page 14  |  Page 15  |  Page 16  |  Page 17  |  Page 18  |  Page 19  |  Page 20  |  Page 21  |  Page 22  |  Page 23  |  Page 24  |  Page 25  |  Page 26  |  Page 27  |  Page 28  |  Page 29  |  Page 30  |  Page 31  |  Page 32  |  Page 33  |  Page 34  |  Page 35  |  Page 36  |  Page 37  |  Page 38  |  Page 39  |  Page 40  |  Page 41  |  Page 42  |  Page 43  |  Page 44  |  Page 45  |  Page 46  |  Page 47  |  Page 48  |  Page 49  |  Page 50  |  Page 51  |  Page 52  |  Page 53  |  Page 54  |  Page 55  |  Page 56  |  Page 57  |  Page 58  |  Page 59  |  Page 60  |  Page 61  |  Page 62  |  Page 63  |  Page 64  |  Page 65  |  Page 66  |  Page 67  |  Page 68  |  Page 69  |  Page 70  |  Page 71  |  Page 72  |  Page 73  |  Page 74  |  Page 75  |  Page 76  |  Page 77  |  Page 78  |  Page 79  |  Page 80  |  Page 81  |  Page 82  |  Page 83  |  Page 84  |  Page 85  |  Page 86  |  Page 87  |  Page 88  |  Page 89  |  Page 90  |  Page 91  |  Page 92  |  Page 93  |  Page 94  |  Page 95  |  Page 96  |  Page 97  |  Page 98  |  Page 99  |  Page 100  |  Page 101  |  Page 102  |  Page 103  |  Page 104  |  Page 105  |  Page 106  |  Page 107  |  Page 108  |  Page 109  |  Page 110  |  Page 111  |  Page 112  |  Page 113  |  Page 114  |  Page 115  |  Page 116  |  Page 117  |  Page 118  |  Page 119  |  Page 120  |  Page 121  |  Page 122  |  Page 123  |  Page 124  |  Page 125  |  Page 126  |  Page 127  |  Page 128  |  Page 129  |  Page 130  |  Page 131  |  Page 132  |  Page 133  |  Page 134  |  Page 135  |  Page 136  |  Page 137  |  Page 138  |  Page 139  |  Page 140  |  Page 141  |  Page 142  |  Page 143  |  Page 144  |  Page 145  |  Page 146  |  Page 147  |  Page 148  |  Page 149  |  Page 150  |  Page 151  |  Page 152  |  Page 153  |  Page 154