Figure 1.
SDS-PAGE and Western blot analysis of recombinant human Trx1.
A) SDS-PAGE analysis: Lanes were loaded with 5 µg of Trx1 from R&D (1) or Trx1 from IMCO (2). Trx1 samples to the right had first been treated with DTT for 30 min. The samples were separated under non-reducing conditions on the gel. B) Western blot analysis: Lanes were loaded with either 1 µg of Trx1 from R&D (1) or Trx1 from IMCO (2), treated or not with DTT as above, After separation in SDS-PAGE as above and transfer to nitrocellulose membrane, Trx1 was detected by mAb 2G11. The experiments were repeated with consistent results.
Figure 2.
ELISA analysis of two different recombinant Trx1 by capture ELISA.
Antibody combinations used for capture/detection were pAb/pAb (A) and mAb/pAb (B) and mAb/mAb (C). Trx1 standards were from IMCO and R&D. Both Trx1 standards were also analysed after being reduced by DTT. Data shown is from one of three experiments with consistent results.
Figure 3.
Heterophilic antibody (HA) interference in the different capture ELISA setups.
Impact of HA on the detection of Trx1 in two human plasma samples. Two plasmas were diluted 5-fold in incubation buffer or HA blocking buffer and analyzed by Trx1 ELISA. A: analysis with pAb/pAb system where the capture pAb also was replaced by an irrelevant goat pAb. B: analysis with mAb/pAb system where the capture mAb also was replaced by an irrelevant mouse mAb. The experiment was repeated with similar results.
Figure 4.
Analysis of the level of Trx1 in plasma samples by capture ELISA.
Eighteen plasma samples from healthy blood donors were analysed for the level of Trx1 using the ELISA formats, pAb/pAb (A) and mAb/pAb (B), in the presence of either incubation buffer or a HA blocking buffer. One of two experiments with similar results is shown.
Figure 5.
Agreement of the level of Trx1 in plasma samples by pAb/pAb and mAb/pAb capture ELISA.
The agreement between the two ELISAs used for analysis of eighteen plasma samples from healthy blood donors and diluted in incubation buffer (A) or HA blocking buffer (B) was assessed with Bland-Altman analysis. One of two experiments with similar results is shown. ––– Bias •••••••••••• CI Bias (95%) – – – – CI (95%).
Figure 6.
Agreement of the level of Trx1 in CSF samples by pAb/pAb and mAb/pAb capture ELISA.
Fifty-seven CSF samples from patients with diverse diagnosis were analysed for the level of Trx1 using the ELISA formats pAb/pAb and mAb/pAb in the presence of either incubation buffer or a HA blocking buffer. The agreement between the two ELISA formats used for analysis of samples diluted in incubation buffer (A) or HA blocking buffer (B) was assessed with Bland-Altman analysis. One of two experiments with similar results is shown. ––– Bias •••••••••••• CI Bias (95%) – – – – CI (95%).