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Table 1 Recovery of EGFP in EGFP-spiked plasma using EGFP as a flow-through protein indicator

From: Online monitoring of immunoaffinity-based depletion of high-abundance blood proteins by UV spectrophotometry using enhanced green fluorescence protein and FITC-labeled human serum albumin

Run

O.D 488 nm a)

EGFP conc. (mg/ml) b)

Recovery (%) c)

Retention Time (min)

Peak Area

O.D 488 nm d)

Retention Time (min)

Peak Area

Unbound fraction

Bound fraction

20

0.318

0.288

96.108

17.204

41318386

0.042

35.119

21663431

40

0.31

0.28

93.291

17.198

41864592

0.042

35.132

20405735

60

0.316

0.286

95.404

17.13

46612910

0.043

35.003

27522817

80

0.314

0.284

94.7

17.234

48029530

0.043

35.087

40458786

100

0.313

0.283

94.348

17.116

47410534

0.042

34.888

29504690

120

0.302

0.271

90.474

17.117

42958986

0.042

34.9

24795107

140

0.309

0.279

92.939

17.157

44253260

0.042

34.918

23585379

160

0.312

0.282

93.995

17.143

37695839

0.043

34.889

21280293

180

0.314

0.284

94.7

17.119

38865664

0.042

34.906

21914998

200

0.317

0.287

95.756

17.166

39476099

0.042

34.916

25579936

Average

0.313

0.283

94.172

17.158

42848580

0.042

34.976

25671117.2

S.D.

0.0047

0.0049

1.6456

0.042

3665255.437

0.0005

0.1

5954454.913

C.V.(%)

1.502

1.731

1.747

0.245

8.554

1.19

0.286

23.195

  1. a) The unbound fractions were concentrated to 1 ml, for which the UV absorbance was measured at 488 nm.
  2. b) EGFP concentration was calculated using the standard curve, as shown in Figure 3H.
  3. c) EGFP recovery (%) = EGFP concentration/0.3 × 100.
  4. d) The bound fractions were concentrated to 1 ml, for which the UV absorbance was measured at 488 nm.
  5. EGFP-spiked plasma (300 μg of EGFP and 2.62 mg of plasma in 100 μl) was depleted every 20th run from runs 20 to 200. Ten chromatograms were analyzed to calculate retention time and peak area. At the same time, unbound fraction and bound fraction from 10 independent depletions were concentrated to 1.0 ml, and OD488 nm was measured to calculate recovery. Concentrations of EGFP in the unbound fraction and bound fraction were calculated according to the standard curve, generated in Figure 3. The coefficient of variation (C.V. (%)) was calculated from the standard deviation, divided by the average and multiplied by 100. There was fluctuation in the 4th run, causing increase in C.V. (%) in the bound fraction peak area.