Abstracts section 2

Transfus Clin Biol 2002 ; 9 : 61-2 © 2002 E´ditions scientifiques et médicales Elsevier SAS Tous droits réservés S12467820010218X/MIS

ABSTRACTS

Section 2: Immunochemical, immunohistological and serological analysis of monoclonal antibodies with carbohydrates

TESTING OF THE MONOCLONAL ANTIBODIES AGAINST A, B ANTIGENS

SPECIFICITY MAPPING OF MONOCLONAL ANTIBODIES USING GLYCOLIPIDS AND IMMUNOSTAINED THIN LAYER CHROMATOGRAMS

The specificity and activity of monoclonal antibodies against A-, B-, AB-antigens was investigated. All antibodies were tested by the microplate method with untreated O, B, A1, A2, A2B, AB RBCs. Three concentrations : normal (without dilution), diluted 1 :10 and 1 :100 in phosphate buffer with 2 % BSA of all MAbs were examined. It was showed that MAbs belong to four categories : 1) anti-A1 #24 : reacted only with A1 RBCs ; 2) anti-A1,A2 - ## 1-11, 16-20, 22, 23, 28 : reacted as with A1 as A2 RBCs ; 3) anti-B - ## 44-48, 51, 53-55, 57-63 : reacted with B RBCs ; and 4) anti- AB - ## 30-33, 37-41 : reacted with both A and B RBCs. © 2002 Éditions scientifiques et médicales Elsevier SAS

J. CAMPBELL, S. HENRY

GLYCOSCIENCE RESEARCH CENTRE, AUCKLAND TECHNOLOGY, AUCKLAND, NEW ZEALAND Correspondence and reprints : [email protected]

UNIVERSITY

OF

The polymorphic ABH, Lewis and P glycotopes occur naturally on a variety of glycoconjugates – especially glycolipids. It has been previously shown that specific glycoconjugates (either natural or synthetic) better reveal the true specificity of monoclonal antibodies (MAbs) than does the agglutination of red cells. We therefore prepared a panel of glycolipids (from red cells, plasma and small intestine) which are representative of the glycolipids expressed in the major and many of the minor blood group phenotypes. These glycolipids were separated by high-performance thin layer chromatography and then reacted with the MAbs. Specific MAb binding was visualised with alkaline phosphatase labelled anti-antibody reactivity with substrate (EIA-TLC). Eighty-six MAbs and three polyclonal reagents were evaluated by EIA-TLC and clustered according to their reactivity and specificity patterns. Eight MAbs were non-reactive by EIA-TLC. Twenty-seven monoclonal anti-A reagents were grouped into four clusters ; 17 anti-B into three clusters ; 11 anti-A/B into nineclusters ; six anti-H into one cluster ; eight anti-Prelated into three clusters ; and eight anti-Lewis into six clusters. The EIA-TLC patterns of reactivity and cross-reactivity unique to each cluster revealed specific information on the glycotopes recognised by each MAb. It is anticipated that these reactivity patterns will be useful in predicting the value and serological specificity of individuals MAbs. © 2002 Éditions scientifiques et médicales Elsevier SAS

ANALYSIS OF ANTI-H, LEWIS AND P ANTIBODIES BY IMMUNOHISTOCHEMISTRY AND ELISA ON SYNTHETIC OLIGOSACCHARIDES J. LE PENDU*, J. ROCHER

INSERM U419, INSTITUT DE BIOLOGIE, 9 QUAI 44011 NANTES, FRANCE *Correspondence and reprints : [email protected]

MONCOUSU,

Anti-H, Lewis and P antibodies were tested by immunohistochemistry on human gastrointestinal mucosa of known ABO, Lewis and Secretor phenotypes. In addition, the carbohydrate specificity of the anti-H antibodies was tested by ELISA using conjugates of synthetic oligosaccharides. The results allowed the definition of suitable and unsuitable reagents for immunohistological analysis. Among suitable reagents, their limitations in terms of potential cross-reactivities with related carbohydrate epitopes have been underlined. © 2002 Éditions scientifiques et médicales Elsevier SAS

N. MINEEVA, N. BODROVA, O. ZAVARZINA

RUSSIAN INSTITUTE OF HEMATOLOGY AND TRANSFUSIOLOGY, ST. PETERSBURG, RUSSIA Correspondence and reprints : [email protected]

PHOTONIC TEST OF MONOCLONAL ANTIBODIES – SECTION 2 R.J. RASIA1*, J.R. VALVERDE2, N. DE ISLA3, J.F. STOLTZ3, A. RAPAILLE4, D. SONDAG4

1 IFIR (CONICET-U.N.ROSARIO), BVRD. 27 DE FEBRERO 210 BIS, 2000 ROSARIO, ARGENTINA ; 2FAC. DE CS. BIOQ. Y FARMAC., U.N.ROSARIO, SUIPACHA 531, 2000 ROSARIO, ARGENTINA ; 3 ANGIOHEMATOLOGIE-HEMORHEOLOGIE, FAC. MED., U. H. P, 54505 VANDŒUVRE, FRANCE ; 4CENTRE DE TRANSFUSION SANGUINE, 41, RUE DOS FANCHON, 4020 LIÈGE, BELGIUM *Correspondence and reprints : [email protected] ; [email protected]

Seventy monoclonal antibodies (mAbs) of A, B, AB, H, Lea, Leb, P1, P and Pk against human red blood cells (RBC) carrying the specific antigen were tested applying the Photonic method. This method was developed to obtain a normalized parameter reflecting the energy exchanged during the agglutination reaction related to the potency of the mAb. It uses laser retrodiffusion to determine the work done in dissociating mechanically agglutinates by flow shear in a Couette shear field. © 2002 Éditions scientifiques et médicales Elsevier SAS

REACTIVITY OF MONOCLONAL ANTIBODIES AGAINST ABH, LEWIS AND P-RELATED ANTIGENS J. TAKAHASHI1, T. SENO2, N. YAMASHITA1, M. TANAKA1, F. HIRAYAMA1, H. YAMANO1, H. SHIBATA1, Y. TANI1*

1 OSAKA RED CROSS BLOOD CENTER, OSAKA, JAPAN ; 2REFERENCE MURAKAMI LABORATORY, JAPAN *Correspondence and reprints : [email protected]

Seventy-seven ABH, Lewis and P-related monoclonal antibodies (Mabs) were provided by the Workshop and 72 Mabs were evaluated for suitability as blood grouping reagents. Mabs (2-9, 10) were suitable as anti-A, Mabs (2-26, 27) as anti-A1, Mabs except two Mabs (2-35, 39) as anti-AB, and Mabs (2-57, 63) as anti-B. Mabs (2-72, 73) were suitable as anti-H, since they reacted with AB and cord cells. Anti-Lea and Leb reacted weakly in saline but strongly in LISS. P-related Mabs tested here were suitable for blood grouping. © 2002 Éditions scientifiques et médicales Elsevier SAS

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Analysis of mAbs with carbohydrates

HAVANA REPORT ON SECTION 2 : SEROLOGY OF ANTIBODIES TO ABO AND OTHER CARBOHYDRATE ANTIGENS

Y. ALFONSO VALDES, A.A. BENCOMO HERNANDEZ, M. DIAZ SALAZAR, R.A. RIVERO JIMENEZ INSTITUTE OF HEMATOLOGY AND IMMUNOLOGY, POB 8070, HAVANA-8, CP 10800, CUBA Correspondence and reprints : [email protected] A total of 64 monoclonal antibodies (MAbs) submitted to Section 2 of the Fourth International Workshop on MAbs against Human Red Blood Cells (RBC) and Related Antigens were evaluated in Havana. Potency of MAbs with ABH specificity was performed by titration using a microplate technique and selected ABO RBCs. MAbs were also evaluated in a spin-tube test using ABO cord RBCs. Weak A2BHw variant RBCs were assessed with

anti-A MAbs. Anti-Lea, -Leb and -P1 were evaluated by tube test with RBCs of different phenotypes ; when a negative result was obtained an enzyme method was performed. Anti-A MAbs 2-5, 2-8, 2-9, 2-18, 2-20, 2-22, and 2-23 fulfilled all requirements. However, five anti-A MAbs (2-7, 2-12, 2-13, 2-14, and 2-19) did not recognize A2BHw variants and five others (2-1, 2-2, 2-3, 2-4, and 2-17) did not react with either A2BHw or A2B adults’ RBCs. Expected results were obtained with the anti-A1 MAb 2-24, but 2-28 also reacted with A2 and A2B adults’ RBCs. Anti-B and Anti-AB MAbs reacted strongly with adults’ and cord RBCs. Anti-H 2-66, 2-67, 2-71 and 2-73 gave classic ABO RBC patterns, while MAbs 2-65, 2-68 and 2-74 showed a predominant anti-HI reactivity. Anti-Lea (2-77, 2-79) and anti-Leb (2-80) were specific against the RBCs used ; however, anti-Lea 2-78 and 2-81 gave negative results, even by the Papain technique. All anti-P1 MAbs except 2-98 and 2-99 recognized the appropriate RBCs. Forty-nine of the 64 MAbs in general reacted in accordance with their designated specificity and potency. © 2002 Éditions scientifiques et médicales Elsevier SAS

Transfus Clin Biol 2002 ; 9 : 61–2