Influence of pH Environment on the Agglutination Ability of Anti-A-Monoclonal Antibodies and Their Inhibition by A-Glycoconjugates of Lipid and Protein Origin with Different Isoelectric Properties

Research Article

J Blood Disord. 2023; 10(2): 1077.

Influence of pH Environment on the Agglutination Ability of Anti-A-Monoclonal Antibodies and Their Inhibition by A-Glycoconjugates of Lipid and Protein Origin with Different Isoelectric Properties

Yu P Delevsky*

Institute of Pathology of the Spine and Joints, Prof. MI Sitenko AMS of Ukraine, Kharkiv, Ukraine

*Corresponding author: Yu P Delevsky Institute of Pathology of the Spine and Joints, Prof. MI Sitenko AMS of Ukraine, Kharkiv, Ukraine. Email: valentinka_1987@ukr.net

Received: June 07, 2023 Accepted: July 06, 2023 Published: July 13, 2023

Abstract

The effect caused by medium acidification to pH 6.5 on the agglutinating ability of seven anti-A MAbs (2-8, 2-17, 2-19, 2-22, 2-23, 2-28 from Workshop IV and BRIC-145) and their inhibition by glycoconjugates obtained from the membranes of A erythrocytes by enzymatic treatment and chloroform-methanol method, followed by ion-exchange gel chromatography, was evaluated.

Medium acidification most significantly reduced the agglutination of a erythrocytes by IgM MAbs 2-17, 2-19, 2-22 and 2-28 and had a weaker manifestation in more alkaline IgM MAbs 2-8, 2-23 and BRIC-145.

The inhibition of the lipid isotypes a lp-00, A lp-0, and Alp-3 (with the isoionic pH points of 8.1, 8.0 and 6.55) and the protein ones A pr-1 and A pr-3 (with the isoionic pH points of 7.15 and 6.45) was assessed in scores. Acidification to pH value 6.5 for MAbs 2-28 and 2-17 caused a considerable reduction in inhibition with acid Alp-3 and Apr-3 with slightly increased inhibition with alkaline A lp-00; MAbs 2-22 and 2-19 insignificantly and selectively altered the inhibitory capacity by more alkaline types of glycoconjugates. MAb 2-8 hardly changed inhibition.

All the above illustrates a significant role played by both the charge of glycotopes and antibodies and the specificity - the selective avidity of MAbs to certain isotypes of a glycotopes.

Keywords: Transferase; Erythrocyte; Agglutination; Blood; Detection.

Introduction

As shown by previous studies, acidification of the medium to pH 6.8-7.0 affects the severity and rate of agglutination of erythrocytes by polyclonal group-specific antibodies. It turned out, that agglutination was weakened and slowed down only in the absence of one of the two types of antibodies in the donor's serum (there are agglutinins a, but no anti-A antibodies or agglutinins b, but no anti-B antibodies). In turn, the absence of anti-A or anti-B antibodies was observed in donors with an atypical group characteristic of the type of the ANAP phenomenon in the ABO system - agglutination is negative, absorption with complement fixation is positive, for example Ax, (or, conditionally, O(I) Ac'+) [1].

It would seem that the phenomenon is due only to the type of reacting antibodies. However, the weakening of agglutination in a slightly acidic medium was also observed with a complete set of antibodies in the serum donor (for example, a and anti-A), but provided that the donor did not have a second, non-agglutinogenic isotype A group antigen tested by absorption of antibodies and in cell electrophoresis at complement fixation, such as AB(Ac'-Bc'+). It turned out to be difficult to explain this phenomenon in terms of the nature of the antigenic determinants glycolipid or glycoprotein. In particular, when the medium was acidified, it was possible to "remove" protein-bound antigenic determinants of ABH from the surface of erythrocytes with trypsin. However, even after such treatment, erythrocytes did not lose the ability to absorb both a and anti-A antibodies, and the decrease in titer in the agglutination test when using them was small.

New opportunities in the study of the phenomenon opened up with the use of a wide range of monoclonal antibodies presented at Workshop IV, and especially in connection with the discovery of isoelectric differences in A, B and H glycotopes (glycoconjugates) associated with both lipids and erythrocyte membrane protein [2]. Significant differences in the isoionic point of glycoconjugates (from pH 8.1 to 6.5) indicated differences in the charge of glycotopes acquired at blood pH [3,8]. There are grounds to suggest that the change in the charge of glycotopes upon acidification of the medium (contrary to the previously expressed opinion about the absence of charged groups in antigens representing group-specific substances [4]) is one of the significant factors in changing the agglutinability of erythrocytes. The foregoing determined the task of this study - to evaluate the effect of medium pH on the agglutinating properties of various anti-A MAbs and their inhibition by A glycoconjugates of various nature and with different isoelectric properties.

Materials and Methods

Monoclonal Antibodies (MAbs) were obtained for testing under the program of the 2nd section of the IV International Workshop on monoclonal antibodies to red blood cell antigens (Paris, 2001). Among the used MAbs anti-A 2-17, anti-A 2-19, anti-A 2-22, anti-A 2-23, anti-A 2-8, anti-A 2-28, anti-A BRIC - 145.

Antigens: Isolation of glycosphingolipids in the composition of polar lipids from erythrocyte membranes was carried out by the chloroform-methanol method according to J. Folch et al. (1957) in the modification of M. Brockhaus [5], and glycoprotein fragments, using the treatment of erythrocytes with 1% trypsin solution (Spofa, Slovakia) under the previously described conditions at pH 6.6 [7]. Further isolation and purification of glycoconjugates was carried out on DEAE-cellulose (Reanal, Hungary) with elution in a NaCl gradient and decreasing pH and on DEAE-sephadex A-25 (Farmacia, Fine Chemicals, Sweden). Chromatography of glycolipid antigens on DEAE cellulose was performed using 33% ethanol solutions.

The yield of fractions was evaluated photometrically (at 205, 280 nm and full UV spectrum) on an SF-26 spectrophotometer, Specord and serologically.

Serological studies: The hemagglutination reaction was performed on 96-well panels according to the standard method using O, A, and A2 erythrocytes [6] and taking into account visually (+) and under a microscope (+m) both the titer and the severity of the reaction and dilutions (Score). A2-erythrocytes were tested by the nature of the reaction with polyclonal antibodies, with MAB anti-H and the absence of agglutination (visually) with MAB anti-A 2-24.

Score was calculated according to W. Marsh [9] with an indication of the degree of agglutination at dilutions (points - pt), using the scale: and a 60 minute exposure with a suspension of test erythrocytes.

Results and Discussion

Analyzing the change in the agglutinating ability of seven MAbs anti-A with a change in pH from 7.4 to 6.5, one has to state significant differences in the nature of the effect of pH on Mabs. Nevertheless, in all cases, there was a decrease in titer and especially Score in the agglutination test. The only difference, although significant, was the degree of this decrease. By arranging Mabs according to the degree of decrease (Table 1), we were able to distinguish 2 groups of Mabs - with a significant weakening of the ability to agglutinate (inhibition index > 5 –MAbs 2 – –28, 2 – – 22,2–19, and 2––17) and with less significant weakening (inhibition index < 5 - MABs 2-8, BRIC-145).

Citation: Delevsky YP. Influence of Ph Environment on the Agglutination Ability of Anti-A-Monoclonal Antibodies and Their Inhibition by A-Glycoconjugates of Lipid and Protein Origin with Different Isoelectric Properties. J Blood Disord. 2023; 10(2): 1077.