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M. Montesinos-Rongen et al.
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Figure 2. Recognition pattern of recombinant antibodies (recAb) derived from naïve B cell receptor (nBCR) and tumor cell B-cell receptor (tBCR) receptor of primary lymphoma of the central nervous system (PCNSL). (A) BoxPlot diagram of ProtoArray analysis showing numbers of proteins recognized by at least one nBCR-derived recAb or tBCR-derived recAb. Numbers of proteins recognized exclusively by either nBCR or tBCR in individual PCNSL were used for calculation. Statistical significance was determined by exact Wilcoxon signed rank test. (B) Number of proteins on the ProtoArray that are recognized by recombinant antibodies derived from nBCR and tBCR are shown for individual PCNSL cases. In addition to proteins recognized by either nBCR- or tBCR-derived recAbs, the number of shared target proteins is depict- ed. (C) BoxPlot diagram shows the numbers of proteins known to be expressed in the central nervous system (CNS) that were recognized by at least one nBCR and tBCR recAb. Proteins known to be expressed in the CNS recognized exclusively by either nBCR or tBCR in individual PCNSL were used for calculation. Statistical sig- nificance was determined by exact Wilcoxon signed rank test. (D) The number of proteins on the ProtoArray known to be expressed in the CNS that are recognized by recombinant antibodies derived from nBCR and tBCR is shown for individual PCNSL cases. In addition to proteins recognized by either nBCR- or tBCR-derived recombinant antibodies, the number of shared target proteins is depicted.
While a lack of information on protein characteristics on the ProtoArray and within human brain samples regarding isoform(s) and post-translational modifications hampers direct comparison of the proteins identified in these two assays, both techniques led to the conclusion that tBCR recognize significantly increased numbers of CNS pro- teins and identified proteins recognized exclusively by the tBCR. Among the proteins differentially recognized by the tBCR, but not the nBCR, were ribosomal proteins, his- tones, and proteins involved in mitochondrial and glial cell metabolism (laminin subunit β-1, annexin A7, protein S100-A9) (Online Supplementary Table S6).
PCNSL lysates was limited due to tissue scarcity with low protein content; nevertheless, there was a tendency towards increased numbers of proteins recognized by tBCR compared to nBCR, which, however, did not reach statistical significance (Online Supplementary Figure S5). RecAb binding in dot blots confirmed that western blot conditions were too stringent (Online Supplementary Figures S6 and S7).
Immunofluorescence studies confirm binding of recombinant antibodies derived from naïve and tumor B-cell receptor of proteins recognized on the ProtoArray
Immunoprecipitation of the nBCR and tBCR with
To confirm the ProtoArray data by an independent
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haematologica | 2021; 106(3)