Serum Antibody Repertoire Profiling Using In Silico Antigen Screen

<div><p>Serum antibodies are valuable source of information on the health state of an organism. The profiles of serum antibody reactivity can be generated by using a high throughput sequencing of peptide-coding DNA from combinatorial random peptide phage display libraries selected for binding to serum antibodies. Here we demonstrate that the targets of immune response, which are recognized by serum antibodies directed against sequential epitopes, can be identified using the serum antibody repertoire profiles generated by high throughput sequencing. We developed an algorithm to filter the results of the protein database BLAST search for selected peptides to distinguish real antigens recognized by serum antibodies from irrelevant proteins retrieved randomly. When we used this algorithm to analyze serum antibodies from mice immunized with human protein, we were able to identify the protein used for immunizations among the top candidate antigens. When we analyzed human serum sample from the metastatic melanoma patient, the recombinant protein, corresponding to the top candidate from the list generated using the algorithm, was recognized by antibodies from metastatic melanoma serum on the western blot, thus confirming that the method can identify autoantigens recognized by serum antibodies. We demonstrated also that our unbiased method of looking at the repertoire of serum antibodies reveals quantitative information on the epitope composition of the targets of immune response. A method for deciphering information contained in the serum antibody repertoire profiles may help to identify autoantibodies that can be used for diagnosing and monitoring autoimmune diseases or malignancies.</p></div

Epitopes predicted by the SVMTriP inside the PAP protein.

<p>The table shows the hypothetical epitopes predicted by the SVMTriP software on the PAP protein. In bold are the sequences that produce matches to the peptides recognized by serum antibodies of the Pap3 serum.</p

Epitope mapping for human PAP-specific antibodies from four immune sera samples

<p>The reactivity of 4 mouse PAP-specific immune sera (PAP1, PAP2, PAP3 and PAP4) was tested by ELISA using a library of overlapping 20-mer peptides that span the entire amino acid sequence of human PAP. Base1 and base 2 samples derived from naïve mice served as negative controls. The results for the PAP protein region corresponding to amino acids 193–362 are shown.</p

Top candidate antigens selected by the SAS for the melanoma patient serum.

<p>The table shows the top candidate antigens selected for the antibodies of the melanoma patient serum by sorting the data generated by processing the results of BLAST database search for the corresponding peptide sequences.</p

Epitope profile of the PAP protein generated by PAP1 mouse antiserum.

<p>(<b>A</b>) The NP_001090.2 PAP variant amino acid sequence and the 40 matching peptides with the highest match scores generated by bl2seq program are shown. The sequences of the protein matching to peptides and the corresponding peptide sequences that match to protein are underlined. <b>B</b>. The graphic shows the distribution of the total score of the peptide matching over the sequence of the PAP protein.</p

Candidate antigens for mouse sera.

<p>The table shows the top candidate antigens selected for the antibodies of the PAP1, PAP2 and PAP3 antisera by sorting the data generated by processing the results of BLAST database search for the corresponding peptide sequences.</p

Epitope profile of the CPA3 protein generated by the serum from a melanoma patient.

<p>(<b>A</b>). The CPA3 amino acid sequence and the 36 matching peptides with the highest match scores generated by bl2seq program are shown. The sequences of the protein matching to peptides and the corresponding peptide sequences that match to protein are underlined. (<b>B</b>). Western blot analysis of the 293T cell lysates overexpressing the recombinant CPA3 protein or transfected with empty vector. The membranes were incubated with serum from either melanoma patient (left panel) or healthy donor (right panel). The reaction was developed as described in the “Materials and Methods” section.</p

Motifs identified by MEME software for the top candidate antigens selected for the PAP1 and PAP3 antisera.

<p>Motifs for the antigens are related to the motifs identified for the corresponding top 500 the most abundant peptides.</p

Motifs identified by MEME software for the 500 peptide lists for the PAP1, PAP, PAP3 and PAP4 antisera.

<p>Motifs identified by MEME software for the 500 peptide lists for the PAP1, PAP, PAP3 and PAP4 antisera.</p