American Society of Hematology

Fig. 3.

$Fig. 3. Plasma from thrombocytopenic subjects contained antibody to TPO and circulating TPO-IgG complexes. / (A) TPO levels before (light bars) and after (dark bars) addition of rHuTPO. The TPO concentration was determined before and after the addition of 1500 pg/mL rHuTPO to serum samples from 4 healthy subjects (N1-N4) and from subjects no. 1, 2, and 3. (B) Endogenous TPO circulates as a TPO-IgG complex. The TPO concentration was determined before (−) and after (+) immunoprecipitation with protein A beads, both before (gray bars) and after (black bars) addition of 1500 pg/mL rHuTPO to serum samples from a healthy control subject and subjects no. 1 and 3. The horizontal line indicates the detection limit for the TPO ELISA assay. (C) The immune complexes from subject no. 1 contain full-length, endogenous TPO. Immune complexes were isolated and their TPO content assessed by SDS-PAGE and Western blot as described in “Materials and methods.” Endogenous TPO isolated from plasma from a healthy subject, plasma from an aplastic anemia subject, and conditioned medium from HepG2 cells is shown for comparison. For each sample, 4 equivalent sequential column elution fractions (E1-E4) were analyzed. Lanes containing rHuTPO (T) and molecular weight markers (M) are indicated. The arrow denotes the position of endogenous TPO.$

Plasma from thrombocytopenic subjects contained antibody to TPO and circulating TPO-IgG complexes.

(A) TPO levels before (light bars) and after (dark bars) addition of rHuTPO. The TPO concentration was determined before and after the addition of 1500 pg/mL rHuTPO to serum samples from 4 healthy subjects (N1-N4) and from subjects no. 1, 2, and 3. (B) Endogenous TPO circulates as a TPO-IgG complex. The TPO concentration was determined before (−) and after (+) immunoprecipitation with protein A beads, both before (gray bars) and after (black bars) addition of 1500 pg/mL rHuTPO to serum samples from a healthy control subject and subjects no. 1 and 3. The horizontal line indicates the detection limit for the TPO ELISA assay. (C) The immune complexes from subject no. 1 contain full-length, endogenous TPO. Immune complexes were isolated and their TPO content assessed by SDS-PAGE and Western blot as described in “Materials and methods.” Endogenous TPO isolated from plasma from a healthy subject, plasma from an aplastic anemia subject, and conditioned medium from HepG2 cells is shown for comparison. For each sample, 4 equivalent sequential column elution fractions (E1-E4) were analyzed. Lanes containing rHuTPO (T) and molecular weight markers (M) are indicated. The arrow denotes the position of endogenous TPO.

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