Generation and characterization of monoclonal antibodies against the N-terminus of alpha-2-antiplasmin

Around 70% of circulating alpha-2-antiplasmin (α2AP), the main natural plasmin inhibitor, is N-terminally cleaved between residues Pro12 and Asn13 by antiplasmin-cleaving enzyme. This converts native Met-α2AP into the more potent fibrinolysis inhibitor Asn-α2AP. The Arg6Trp (R6W) polymorphism affects the N-terminal cleavage rate of Met-α2AP in a purified system, with ~8-fold faster conversion of Met(R6)-α2AP than Met(W6)-α2AP. To date, assays to determine N-terminally intact Met-α2AP in plasma have been limited to an ELISA that only measures Met(R6)-α2AP. The aim of this study was to generate and characterize monoclonal antibodies (mAbs) against Met(R6)-α2AP, Met(W6)-α2AP and all α2AP forms (total-α2AP) in order to develop specific Met(R6)-α2AP and Met(W6)-α2AP ELISAs. Recombinant Met(R6)-α2AP, Met(W6)-α2AP and Asn-α2AP were expressed in Drosophila S2 cells. Using hybridoma technology, a panel of 25 mAbs was generated against a mixture of recombinant Met(R6)-α2AP and Met(W6)-α2AP. All mAbs were evaluated for their specific reactivity using the three recombinant α2APs in one-site non-competitive ELISAs. Three mAbs were selected to develop sandwich-type ELISAs. MA-AP37E2 and MA-AP34C4 were selected for their specific reactivity against Met(R6)-α2AP and Met(W6)-α2AP, respectively, and used for coating. MA-AP15D7 was selected for its reactivity against total-α2AP and used for detection. With the novel ELISAs we determined Met(R6)-α2AP and Met(W6)-α2AP levels in plasma samples and we showed that Met(R6)-α2AP was converted faster into Asn-α2AP than Met(W6)-α2AP in a plasma milieu. In conclusion, we developed two specific ELISAs for Met(R6)-α2AP and Met(W6)-α2AP, respectively, in plasma. This will enable us to determine N-terminal heterogeneity of α2AP in plasma samples

Hito R124H henigata TGFBI idenshi o mochiita karyujo kakumaku jisutorofi 2gata moderu mausu sakusei

Around 70% of circulating alpha-2-antiplasmin (α2AP), the main natural plasmin inhibitor, is N-terminally cleaved between residues Pro12 and Asn13 by antiplasmin-cleaving enzyme. This converts native Met-α2AP into the more potent fibrinolysis inhibitor Asn-α2AP. The Arg6Trp (R6W) polymorphism affects the N-terminal cleavage rate of Met-α2AP in a purified system, with ~8-fold faster conversion of Met(R6)-α2AP than Met(W6)-α2AP. To date, assays to determine N-terminally intact Met-α2AP in plasma have been limited to an ELISA that only measures Met(R6)-α2AP. The aim of this study was to generate and characterize monoclonal antibodies (mAbs) against Met(R6)-α2AP, Met(W6)-α2AP and all α2AP forms (total-α2AP) in order to develop specific Met(R6)-α2AP and Met(W6)-α2AP ELISAs. Recombinant Met(R6)-α2AP, Met(W6)-α2AP and Asn-α2AP were expressed in Drosophila S2 cells. Using hybridoma technology, a panel of 25 mAbs was generated against a mixture of recombinant Met(R6)-α2AP and Met(W6)-α2AP. All mAbs were evaluated for their specific reactivity using the three recombinant α2APs in one-site non-competitive ELISAs. Three mAbs were selected to develop sandwich-type ELISAs. MA-AP37E2 and MA-AP34C4 were selected for their specific reactivity against Met(R6)-α2AP and Met(W6)-α2AP, respectively, and used for coating. MA-AP15D7 was selected for its reactivity against total-α2AP and used for detection. With the novel ELISAs we determined Met(R6)-α2AP and Met(W6)-α2AP levels in plasma samples and we showed that Met(R6)-α2AP was converted faster into Asn-α2AP than Met(W6)-α2AP in a plasma milieu. In conclusion, we developed two specific ELISAs for Met(R6)-α2AP and Met(W6)-α2AP, respectively, in plasma. This will enable us to determine N-terminal heterogeneity of α2AP in plasma samples

Generation and characterization of monoclonal antibodies against the N-terminus of alpha-2-antiplasmin - Fig 3

<p><b>Reactivity of recombinant α2AP in the (A) Met(R6)-α2AP ELISA and (B) Met(W6)-α2AP ELISA.</b> 96-well microtiter plates were coated with MA-AP37E2 or MA-AP34C4 (2 μg/ml) and serial dilutions (1000–15.6 ng/ml) of recombinant Met(R6)-α2AP (■), Met(W6)-α2AP (●) or Asn-α2AP (▲) were added. HRP-conjugated MA-AP15D7 was used as a detection antibody. Representative experiments out of three similar experiments are shown.</p

Evaluation of Met-α2AP to Asn-α2AP conversion in normal pooled plasma incubated at 29°C for 72 hours.

<p>Percentage of Met(R6)-α2AP (■) and Met(W6)-α2AP (●) as determined by the novel Met(R6)-α2AP and Met(W6)-α2AP sandwich-type ELISAs. The variability of assays performed in duplicate is shown with error bars.</p

Generation and characterization of monoclonal antibodies against the N-terminus of alpha-2-antiplasmin - Fig 2

<p><b>Reactivity of (A) MA-AP37E2, (B) MA-AP34C4 and (C) MA-AP15D7 with recombinant α2AP on Western blot.</b> Lane 1: Met(R6)-α2AP (300 ng), lane 2: Met(W6)-α2AP (300 ng), lane 3: Asn-α2AP (300 ng). The migration distances of molecular weight marker proteins are indicated on the left.</p

Generation and characterization of monoclonal antibodies against the N-terminus of alpha-2-antiplasmin - Fig 4

<p><b>Reactivity of plasma α2AP in the (A) Met(R6)-α2AP ELISA and (B) Met(W6)-α2AP ELISA.</b> 96-well microtiter plates were coated with MA-AP37E2 or MA-AP34C4 (2 μg/ml) and varying plasma concentrations (0–2.5% for MA-AP37E2 and 0–20% for MA-AP34C4) of R6 pooled plasma (■), W6 pooled plasma (▲), normal pooled plasma (●) or α2AP-depleted plasma (▼) were tested. Representative experiments out of three similar experiments are shown. Standard deviations of assays performed in triplicate are shown as error bars.</p

Variation of Met(R6)-α2AP and Met(W6)-α2AP in R6 and W6 homozygote individuals.

<p>Concentrations of Met(R6)-α2AP (●<i>)</i> as determined by the Met(R6)-α2AP ELISA and concentrations of Met(W6)-α2AP (■) as determined by the Met(W6)-α2AP ELISA. Met(R6)-α2AP levels in 18 R6 homozygote individuals (RR) ranged from 9 to 19 μg/ml with a mean ± sd of 13 ± 3 μg/ml (CV = 24%). Met(W6)-α2AP levels in 17 W6 homozygote individuals (WW) ranged from 24 to 54 μg/ml with a mean ± sd of 36 ± 8 μg/ml (CV = 22%). Bars represent the mean Met-α2AP level.</p