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Assay of hemoglobin A 1c using lectin from Aleuria aurantia

View Article: PubMed Central - PubMed

ABSTRACT

Hemoglobin A1c (HbA1c) has an N-terminal fructosyl valine on the β-chain, and this modification is caused by the non-enzymatic glycosylation of hemoglobin (Hb). The relative concentration ratio of HbA1c to total Hb is an important biomarker for the diagnosis of diabetes. HbA1c-binding lectins were screened from 29 sources of lectin, and the lectin from Aleuria aurantia (AAL) was revealed to have higher affinity to HbA1c than to Hb. The concentration of HbA1c was determined by lectin-based enzyme-linked immunosorbent assay (ELISA) using the AAL lectin. Higher reproducibility of the assay was observed at 4 °C than at 25 and 37 °C. This observation is consistent with the known temperature-dependent behavior of lectins. Preincubation of HbA1c with an anti-HbA1c antibody inhibited the binding, suggesting that AAL binds to the N-terminal fructosyl valine epitope of HbA1c. Higher inhibitory effect was observed for 10 mM d-fructose than for the same concentrations of l-fucose, d-fucose, or d-glucose.

No MeSH data available.


Binding between the lectin from Aleuria aurantia (AAL) and HbA1c or Hb. The denatured HbA1c (filled circle) or Hb (open square) were immobilized on the surface of the plate and the amount of biotinylated AAL bound to the protein was assayed by the activity of the specifically bound streptavidin-HRP
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Fig2: Binding between the lectin from Aleuria aurantia (AAL) and HbA1c or Hb. The denatured HbA1c (filled circle) or Hb (open square) were immobilized on the surface of the plate and the amount of biotinylated AAL bound to the protein was assayed by the activity of the specifically bound streptavidin-HRP

Mentions: HbA1c-binding lectins were screened from 30 sources using lectin-based ELISA, as described in the “Materials and methods” section. Among the screened lectins, eight (AAL, DBA, ECA, HHA, LCA, Lotus, MPA, and UEA-I) were found to bind to HbA1c, although they showed similar or higher binding-affinity to Hb except for AAL (Fig. 1). AAL was selected for further experiments as it was found to bind with HbA1c, but not with Hb. The binding Fig. 2 shows the binding between AAL and HbA1c and between AAL and Hb. The results show that AAL binding increased as the HbA1c concentration increased. However, only a small increase in binding was observed when the Hb concentration increased. The neutralization before the addition of the ELISA plates increased the values of ELISA, because high pH buffer can increase the solubility of the proteins and makes the proteins unprotonated which helps binding to a positively charged ELISA plates.Fig. 1


Assay of hemoglobin A 1c using lectin from Aleuria aurantia
Binding between the lectin from Aleuria aurantia (AAL) and HbA1c or Hb. The denatured HbA1c (filled circle) or Hb (open square) were immobilized on the surface of the plate and the amount of biotinylated AAL bound to the protein was assayed by the activity of the specifically bound streptavidin-HRP
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC5120163&req=5

Fig2: Binding between the lectin from Aleuria aurantia (AAL) and HbA1c or Hb. The denatured HbA1c (filled circle) or Hb (open square) were immobilized on the surface of the plate and the amount of biotinylated AAL bound to the protein was assayed by the activity of the specifically bound streptavidin-HRP
Mentions: HbA1c-binding lectins were screened from 30 sources using lectin-based ELISA, as described in the “Materials and methods” section. Among the screened lectins, eight (AAL, DBA, ECA, HHA, LCA, Lotus, MPA, and UEA-I) were found to bind to HbA1c, although they showed similar or higher binding-affinity to Hb except for AAL (Fig. 1). AAL was selected for further experiments as it was found to bind with HbA1c, but not with Hb. The binding Fig. 2 shows the binding between AAL and HbA1c and between AAL and Hb. The results show that AAL binding increased as the HbA1c concentration increased. However, only a small increase in binding was observed when the Hb concentration increased. The neutralization before the addition of the ELISA plates increased the values of ELISA, because high pH buffer can increase the solubility of the proteins and makes the proteins unprotonated which helps binding to a positively charged ELISA plates.Fig. 1

View Article: PubMed Central - PubMed

ABSTRACT

Hemoglobin A1c (HbA1c) has an N-terminal fructosyl valine on the β-chain, and this modification is caused by the non-enzymatic glycosylation of hemoglobin (Hb). The relative concentration ratio of HbA1c to total Hb is an important biomarker for the diagnosis of diabetes. HbA1c-binding lectins were screened from 29 sources of lectin, and the lectin from Aleuria aurantia (AAL) was revealed to have higher affinity to HbA1c than to Hb. The concentration of HbA1c was determined by lectin-based enzyme-linked immunosorbent assay (ELISA) using the AAL lectin. Higher reproducibility of the assay was observed at 4 °C than at 25 and 37 °C. This observation is consistent with the known temperature-dependent behavior of lectins. Preincubation of HbA1c with an anti-HbA1c antibody inhibited the binding, suggesting that AAL binds to the N-terminal fructosyl valine epitope of HbA1c. Higher inhibitory effect was observed for 10 mM d-fructose than for the same concentrations of l-fucose, d-fucose, or d-glucose.

No MeSH data available.