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Circulating Levels of sFlt1 Splice Variants as Predictive Markers for the Development of Preeclampsia.

Souders CA, Maynard SE, Yan J, Wang Y, Boatright NK, Sedan J, Balyozian D, Cheslock PS, Molrine DC, Simas TA - Int J Mol Sci (2015)

Bottom Line: Here we describe the development and use of diagnostic monoclonal antibodies specific to the two main splice variants of sFlt1, sFlt1-1 and sFlt1-14.These antibodies were selected for their sensitivity and specificity to their respective sFlt1 isoform in a capture ELISA format.It may be possible to improve current diagnostic platforms if more specific antibodies are utilized.

View Article: PubMed Central - PubMed

Affiliation: MassBiologics of the University of Massachusetts Medical School, Boston, MA 02126, USA. Colby.Souders@UMassMed.edu.

ABSTRACT
Angiogenic biomarkers, including soluble fms-like tyrosine kinase 1 (sFlt1), are thought to be predictors of preeclampsia onset; however, improvement is needed before a widespread diagnostic test can be utilized. Here we describe the development and use of diagnostic monoclonal antibodies specific to the two main splice variants of sFlt1, sFlt1-1 and sFlt1-14. These antibodies were selected for their sensitivity and specificity to their respective sFlt1 isoform in a capture ELISA format. Data from this pilot study suggest that sFlt1-1 may be more predictive of preeclampsia than total sFlt1. It may be possible to improve current diagnostic platforms if more specific antibodies are utilized.

No MeSH data available.


Related in: MedlinePlus

C-terminal peptide sequences used for generation of mouse mAbs specific to sFlt1-1 (solid line) and sFlt1-14 (dashed line). (A) A keyhole limpet hemocyanin (KLH) fusion protein preceded each sFlt1 peptide antigen used for immunizations while a Thioredoxin (TRX) or glutathione S-transferase (GST) tag preceded the sFlt1-14 and sFlt1-1 peptides, respectively, used for screening ELISAs. Full-length sFlt1-1 containing a C-terminal his tag was used to generate all other mouse mAbs and HuMAbs that recognize total sFlt1; (B) Schematic of sFlt1-14 and sFlt1-1 proteins identifying the unique epitopes antibodies were directed against.
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ijms-16-12436-f001: C-terminal peptide sequences used for generation of mouse mAbs specific to sFlt1-1 (solid line) and sFlt1-14 (dashed line). (A) A keyhole limpet hemocyanin (KLH) fusion protein preceded each sFlt1 peptide antigen used for immunizations while a Thioredoxin (TRX) or glutathione S-transferase (GST) tag preceded the sFlt1-14 and sFlt1-1 peptides, respectively, used for screening ELISAs. Full-length sFlt1-1 containing a C-terminal his tag was used to generate all other mouse mAbs and HuMAbs that recognize total sFlt1; (B) Schematic of sFlt1-14 and sFlt1-1 proteins identifying the unique epitopes antibodies were directed against.

Mentions: CD-1 wild type mice or HuMAb mice were immunized with full-length sFlt1-1, sFlt1-C or 14-pep1 (Figure 1). Sera responses and subsequent hybridoma cultures following splenic fusion were monitored by ELISA against full-length sFlt1-1 and sFlt1-14 (Figure 1B) or isoform-specific peptides GST-1C and TRX-Exon14 (Figure 1A). Of the 359 total positive murine clones, several had specific activity only to sFlt1-1 (Figure 2A) or sFlt1-14 (Figure 2B), as shown by ELISA. Mouse mAb 10ugR#9 was selected for its ability to bind both full length sFlt1-1 and sFlt1-14 (Figure 3A,B), but not the isoform-specific peptides GST-1C or TRX-Exon14 (Figure 2). Of the isoform-specific clones shown in Figure 2, mouse mAb 1CKLH18 and Ex14-1 were selected for their specific binding properties to sFlt1-1 and sFlt1-14, respectively (Figure 3A,B).


Circulating Levels of sFlt1 Splice Variants as Predictive Markers for the Development of Preeclampsia.

Souders CA, Maynard SE, Yan J, Wang Y, Boatright NK, Sedan J, Balyozian D, Cheslock PS, Molrine DC, Simas TA - Int J Mol Sci (2015)

C-terminal peptide sequences used for generation of mouse mAbs specific to sFlt1-1 (solid line) and sFlt1-14 (dashed line). (A) A keyhole limpet hemocyanin (KLH) fusion protein preceded each sFlt1 peptide antigen used for immunizations while a Thioredoxin (TRX) or glutathione S-transferase (GST) tag preceded the sFlt1-14 and sFlt1-1 peptides, respectively, used for screening ELISAs. Full-length sFlt1-1 containing a C-terminal his tag was used to generate all other mouse mAbs and HuMAbs that recognize total sFlt1; (B) Schematic of sFlt1-14 and sFlt1-1 proteins identifying the unique epitopes antibodies were directed against.
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Related In: Results  -  Collection

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ijms-16-12436-f001: C-terminal peptide sequences used for generation of mouse mAbs specific to sFlt1-1 (solid line) and sFlt1-14 (dashed line). (A) A keyhole limpet hemocyanin (KLH) fusion protein preceded each sFlt1 peptide antigen used for immunizations while a Thioredoxin (TRX) or glutathione S-transferase (GST) tag preceded the sFlt1-14 and sFlt1-1 peptides, respectively, used for screening ELISAs. Full-length sFlt1-1 containing a C-terminal his tag was used to generate all other mouse mAbs and HuMAbs that recognize total sFlt1; (B) Schematic of sFlt1-14 and sFlt1-1 proteins identifying the unique epitopes antibodies were directed against.
Mentions: CD-1 wild type mice or HuMAb mice were immunized with full-length sFlt1-1, sFlt1-C or 14-pep1 (Figure 1). Sera responses and subsequent hybridoma cultures following splenic fusion were monitored by ELISA against full-length sFlt1-1 and sFlt1-14 (Figure 1B) or isoform-specific peptides GST-1C and TRX-Exon14 (Figure 1A). Of the 359 total positive murine clones, several had specific activity only to sFlt1-1 (Figure 2A) or sFlt1-14 (Figure 2B), as shown by ELISA. Mouse mAb 10ugR#9 was selected for its ability to bind both full length sFlt1-1 and sFlt1-14 (Figure 3A,B), but not the isoform-specific peptides GST-1C or TRX-Exon14 (Figure 2). Of the isoform-specific clones shown in Figure 2, mouse mAb 1CKLH18 and Ex14-1 were selected for their specific binding properties to sFlt1-1 and sFlt1-14, respectively (Figure 3A,B).

Bottom Line: Here we describe the development and use of diagnostic monoclonal antibodies specific to the two main splice variants of sFlt1, sFlt1-1 and sFlt1-14.These antibodies were selected for their sensitivity and specificity to their respective sFlt1 isoform in a capture ELISA format.It may be possible to improve current diagnostic platforms if more specific antibodies are utilized.

View Article: PubMed Central - PubMed

Affiliation: MassBiologics of the University of Massachusetts Medical School, Boston, MA 02126, USA. Colby.Souders@UMassMed.edu.

ABSTRACT
Angiogenic biomarkers, including soluble fms-like tyrosine kinase 1 (sFlt1), are thought to be predictors of preeclampsia onset; however, improvement is needed before a widespread diagnostic test can be utilized. Here we describe the development and use of diagnostic monoclonal antibodies specific to the two main splice variants of sFlt1, sFlt1-1 and sFlt1-14. These antibodies were selected for their sensitivity and specificity to their respective sFlt1 isoform in a capture ELISA format. Data from this pilot study suggest that sFlt1-1 may be more predictive of preeclampsia than total sFlt1. It may be possible to improve current diagnostic platforms if more specific antibodies are utilized.

No MeSH data available.


Related in: MedlinePlus