A novel assay for the detection of anti-human platelet antigen antibodies (HPA-1a) based on peptide aptamer technology

Abstract

Background: Neonatal alloimmune thrombocytopenia is mostly due to the presence of maternal antibodies against the fetal platelet antigen HPA-1a on the platelet integrin GPIIb-IIIa. Accurate detection of anti-HPA-1a antibodies in the mother is, therefore, critical. Current diagnostic assays rely on the availability of pools of human platelets that vary according to donors and blood centers. There is still no satisfactory standardization of these assays.

Design and methods: Peptide aptamer was used to detect and identify HPA-1a-specific antibodies in human serum that do not require human platelets. A peptide aptamer library was screened using an anti-HPA-1a human monoclonal antibody as a bait to isolate an aptamer that mimics the human platelet antigen HPA-1a.

Results: This is the first report in platelet immunology of the use of a peptide aptamer for diagnostic purposes. This assay gives better results than the MAIPA currently in use, detecting around 90% of the expected alloantibodies.

Conclusions: This assay could help define a standard for the quantitation of anti-HPA antibodies. This report also demonstrates that peptide aptamers can potentially detect a variety of biomarkers in body fluids; this is of particular interest for diagnostic purposes.

Figure 1.

Isolation of a peptide aptamer that binds specifically to the anti-HPA-1a monoclonal antibody. Western blotting analysis of whole protein extracts from bacterial clones (lanes 1–4) selected following FliTrx^® screening steps. Membranes were probed with the Camtran-B2 anti-HPA-1a monoclonal antibody. Positive clones are characterized by 63 kD band (lanes 2 and 3). A sample from HPA-1a platelets was used as a positive control (right lane, +).

Figure 2.

Characterization and verification of peptide aptamer. (A) Sequence of the Trx-HPA-1a protein. The protein contains a twelve aminoacid-long sequence cloned in frame into the active site of thioredoxin, as well as a poly-histidine tail in C-terminus. All sequenced positive clones exhibited the same twelve aminoacids. (B) Analysis of the purified Trx-HPA-1a protein. (1) Detection of the recombinant protein by SDS-PAGE (Coomassie blue staining). Protein (20 μg) purified by anion exchange chromatography was run on a 20% polyacrylamide gel. Molecular weight markers are shown on the left. (2) Electrospray mass spectrum of the Trx-HPA-1a protein. A major peak at the expected size (14637 Da) is detected. The minor peak (14373.5 Da) corresponds to Trx-HPA-1a lacking two methionine residues at the N-terminus. (3) MALDI-TOF spectrum obtained after tryptic digestion of Trx-HPA-1a. Black arrow (➡) corresponds to peaks at the expected size. Together these peaks match 80% of the Trx-HPA-1a sequence.

Figure 3.

Characterization of the interaction between Trx-HPA-1a protein and anti-HPA-1a antibodies. Two human serum samples, with (1, 3, 5, 7) or without (2, 4, 6, 8) HPA-1a-specific alloantibodies were incubated with purified Trx-HPA-1a protein and immunoprecipitated with protein A-sepharose. Immunoprecipitates were analyzed by Western blotting using anti-His antibody to detect Trx-HPA-1a. Buffers used for immunoprecipitation were PBS (lanes 1, 2, 9, 10), PBS-Tween 20 (0.01%) (lanes 3, 4), PBS-NP40 (0.1%) (lanes 5, 6), PBS-Triton X-100 (0.5%) (lanes 7, 8). Negative controls without Trx-HPA-1a (9) or without protein A-sepharose (10) are shown. A signal corresponding to the apparent molecular weight of Trx-HPA-1a (16,500 Da) is clearly detected in lanes 1–3, 5 and 7; Black arrow (➡). In the absence of detergent (lanes 1, 2) the Trx-HPA-1a aptamer is immunoprecipitated, irrespective of the presence of anti-HPA-1a antibodies, while in the presence of detergent (lanes 3–8) the aptamer is preferentially immunoprecipitated when incubated with the serum that contains HPA-1a-specific antibodies. Additional bands in the upper part of the blot correspond to immunoglobulins that are detected in a non-specific manner.

Figure 4.

Detection of anti-HPA-1a antibodies in human serum using the Trx-HPA-1a protein (Immunocapture assay). The following human serums were used: 2 different samples from patients with NAIT babies, containing anti-platelet HPA-1a alloantibodies (rows 1, 2), one sample containing anti-platelet HPA-1b alloantibody (row 3), one negative control (row 4). Wells were coated with 50 ng (column A) or 100 ng (column B) of Trx-HPA-1a protein. Interaction between Trx-HPA-1a and alloantibodies is only detected with sera containing anti-HPA-1a immunoglobulins (row 1, 2).

Figure 5.

Detection of anti-HPA-1a antibodies in human serum using the Trx-HPA-1a protein (ELISA assay). (A) Time course analysis. ELISA was carried out with Trx-HPA-1a coated onto microwell plates (100 ng per well). Human serum with anti-HPA-1a alloantibodies (positive control), as previously determined by MAIPA, was used at different dilutions as indicated (0.7; 0.33; 0.10). (B) End point measurement. ELISA was carried out with various amounts of coated Trx-HPA-1a. The colorimetric reaction was arrested after 20 min incubation. Optical density (OD_20min) as a function of the amount of coated aptamer is shown. Results obtained with two concentrations of serum (positive control) are shown. (C) Typical kinetics obtained with a sample from an anonymous donor previously tested MAIPA negative (negative control). The curve obtained with the standard positive control routinely in use at the EFS-Lyon is shown for comparison. In this experiment, the samples are used at 1:3 dilution, 100ng Trx-HPA-1a protein were coated in each well.

Figure 6.

Depletion assay. Analysis of the MAIPA signal intensity as a function of the amount of Trx-HPA-1a protein. Depletion was carried out on the serum of 2 mothers whose infants were diagnosed with NAIT (“Positive control” used in EFS and “NAIT”), as well as the monoclonal anti-HPA-1a antibody (“monoclonal Ab”). The signal decreases as a function of the amount of Trx-HPA-1a, showing the neutralization of anti-HPA-1a antibodies.