Generation and characterization of monoclonal antibody against Advanced Glycation End Products in chronic kidney disease

Abstract

Advanced Glycation End Products (AGEs) are toxins that are involved in structural and functional alterations of several organs and tissues, resulting in various pathologies. Several types of AGEs have been described but carboxymethyllysine (CML) is the major antigenic AGE compound. In this study, three different immunogenic carrier proteins (KLH, keyhole limpet hemocyanin; BSA, bovine serum albumin; and HSA, human serum albumin) were modified by glycation. The glycated molecules were used to produce epitope-specific monoclonal antibodies able to recognize the CML domain and to detect uremic toxins in the serum of patients with chronic kidney disease (CKD). A competitive ELISA was standardized in order to quantify CML in the sera of CKD patients. An increase in uremic toxins can compromise the clinical condition of these patients, thus, the detection and quantification of these toxins should contribute to a better management and understanding of this disease.

Keywords: Age; CML; Chronic kidney disease; Monoclonal antibody.

Fig. 1

Molecular characterization of the modified proteins. (A) Schematic drawing of CML-protein formation. (B) The synthesized samples were separated by SDS-PAGE after Coomassie staining. MW, molecular weight marker. (C) Mass spectrometry HSA and CML-HSA by MALDI TOF MS.

Fig. 2

Reactivity of polyclonal and monoclonal antibodies by ELISA direct. (A) Reactivity of polyclonal antibodies produced against glycated proteins and cross-reactivity against native protein. (B) Reactivity of cell supernatants against glycated and non-glycated proteins. (C) Specificity of monoclonal antibodies against CML-HSA and cross-reactivity to HSA. (D) Dose dependence of mAb 2D6G2 against CML-proteins.

Fig. 3

Cloning of mAb2D6G2 cDNA encoding antibody variable domains. (A) Agarose gel electrophoresis of cDNA encoding IGkV (lane Vk) and IGHV (lane VH) domains. M: 100-bp DNA ladder. (B) “Collier de perles” of mAb 2D6G2 antibody variable domains.

Fig. 4

Detection of synthesized CML-HSA by competitive ELISA using mAb 2D6G2. (A) A standard curve of the known concentrations of CML-HSA. The results are expressed as decreasing absorbance correlated with increasing competitor. (B) Principle of the assay. CML-HSA (10 μg/mL) was immobilized on plate. The sample to be tested (CML-HSA 0.4-200 ng) was mixed with 0.1 μg/mL of mAb 2D6G2 anti-CML antibody. A portion of the mixture (100 μL) was added to the plate prepared above. The reactions were confirmed by adding anti-immunoglobulin.

Fig. 5

Detection of CKD serum CML levels. (A) Analysis of the competition between the CML-HSA levels in human serum and the immobilized synthesized CML-HSA for binding to mAb 2D6G2. The results are expressed as increasing inhibition correlated with increasing competitor. (B) A curve correlating inhibition and CML-HSA levels. (C) Correlation between CKD stages and CML-HSA concentration. (D) Relationship between serum levels of CML-HSA and GFR. CKD stages were performed following KDOQI Clinical Practice Guidelines for Chronic Kidney Disease.