A Sensitive Method for the Measurement of Glycosylated Plasma Proteins Using Affinity Chromatography

Abstract

SUMMARY.We describe a simple, sensrnve affinity technique for the routine measurement of glycosylated plasma proteins in clinical laboratories. The commercially available phenylboronic acid gel used for the chromatography has recently been marketed as a kit for this purpose (Glycogel Test Kit. Pierce Chemical Co). The manufacturers of this kit recommend loading 200 fl.l neat plasma to each I rnl gel column. This high loading is to enable the direct measurement of protein in the bound and unbound fractions at 280 nm. This loading is consistent with 10-15 mg protein being added per ml gel. Our results show that protein levels greater than 2 mg per ml gel overload the column. Therefore we used a modification of the more sensitive Bradford procedure to measure protein. The method discriminates between normals (6'29 ± 1'87%) and diabetic patients (12·62 ± 3'36~~) and has good precision (CV 4-6 %). The results obtained correlate with the colorimetric method using thiobarbituric acid (r C~O' 70) and with glycosylated haemoglobin (r = O' 82).The measurement of glycosylated haemoglobin is gaining increasing use in the management of patients with diabetes." Other proteins are also glycosylated non-enzymically, and it has been shown that both glycosylated albumin and glycosylated plasma proteins respond more quickly than glycosylated haemoglobin to improvement in glycaernic control.f 3The original method for measuring glycosylated albumin.! which involves isolation of the pure protein followed by estimation of the glycosylated fraction, is laborious. Glycosylated plasma proteins can be estimated directly by a colorimetric method" or after protein precipitation." For accurate results, these methods also require the estimation of protein, the removal of glucose," and correction for background colour" on each sample. A specific method for quantitation of the lysine-bound glucose in serum albumin and other glycosylated proteins is also available." This requires isolation of pure protein, followed by hydrolysis and then analysis by HPLC.None of the available methods for measurement of glycosylated plasma proteins is suitable for routine analysis in the clinical laboratory. There have been several recent reports on the use of simple Reprint requests and correspondence to Dr BJ Gould. 16affinity column methods for the measurement of glycosylated haemoglobins. \11-14 A similar chromatographic procedure should senarate glycosylated plasma proteins from their non-glyccsylated counterparts. The get used is a III-aminophenylboronate agarose, which binds selectively to cis-diols.

Glycated albumin by affinity chromatography and radioimmunoassay in the management of diabetes mellitus

“…The dramatic fall of fasting glucose levels from 606 mg/ dL on day 1 to 105 mg/dL on day 13 was accompanied by an equally impressive decrease of GALB from 7.6% on day 1 to 0.9% on day 16. GALB levels increased in response to glucose elevation from day 40, reaching a maximal value of 4.9% on day 68 before returning to within RI of 0.6% on day 103.…”

Glycation of monoclonal antibodies impairs their ability to bind antigen

“…The CM5 sensor chips, P20 surfactant, amine coupling kit containing N-hydroxysuccinimide (NHS), [15] and using a periodate-based colorimetric method developed by us [16].…”

“…The unglycated MoAb was treated identically but with no glucose added during the 21-day incubation and no glucose added to the MoAb when it was diluted 1:17000 and mixed with antigen. However, 15 mm glucose was added to the antigen-antibody mixture immediately before adding the mixture to an antigencoated plate, to rule out the possibility that the presence of 15mM glucose in the wells alters Kd rather than the extent of MoAb glycation. The mean Kd (n = 5) of glycated MoAb continually exposed to 15mm glucose was significantly higher at 4 0 x 10-9/M (+0 12) compared with 3 4 x 10-9/M (+ 0-11) for incubated control MoAb (P < 0 02, n = 5).…”

Fingerstick Glycosylated Hemoglobin, Plasma Protein, and Albumin

“…One of the best techniques for determination of glycohemoglobin is aminophenylboronic acid affinity chromatography (1). This technique is also applicable to the measurement of glycosylated plasma protein (2) and albumin (3,4). The ability to reliably measure glycoalbumin is an important supplement to glycohemoglobin measurement in the management of diabetes.…”