Quantitation of the Number of Molecules of Glycophorins C and D on Normal Red Blood Cells Using RadioiodinatedFab Fragments of Monoclonal Antibodies By Jon Smythe, Brigitte Gardner, andDavid J. Anstee Two rat monoclonal antibodies (BRAC 1 and BRAC 1 1 ) cytes. Fabfragments of BRAC 1 1 and ERIC 10 gave values have been produced. BRAC 1 recognizes an epitope com- of 143,000 molecules GPC per red blood cell (RBC). Fab mon to the human erythrocyte membrane glycoproteins fragments of BRAC1 gave 225,000 molecules of GPC and glycophorin C (GPC) and glycophorin D (GPD). BRAC 11 GPD per RBC. These results indicate that GPC and GPD is specific for GPC. Fabfragments of these antibodies and together are sufficiently abundantto provide membrane at- BRlC 10, a murine monoclonal anti-GPC,were radioiodin- tachment sites for all ofthe protein 4.1 in normal RBCs. ated and used in quantitative binding assays to measure 0 1994 by The American Societyof Hematology. the number of GPC and GPD molecules on normal erythro- HE SHAPE AND deformability of the mature human (200,000)" and those reported for GPC (50,000).7 This nu- Downloaded from http://ashpublications.org/blood/article-pdf/83/6/1668/612763/1668.pdf by guest on 24 September 2021 T erythrocyte is controlled by a flexible two-dimensional merical differencehas led to the suggestion that a significant lattice of proteins, which together comprise the membrane proportion of protein 4.1 in normal erythrocyte membranes skeleton.' The major components of the skeleton are spec- must be bound to sites other than GPC and GPD.3 The trin, actin, ankyrin, and protein 4.1. The skeleton is at- value of 50,000 molecules of GPC per RBC was obtained tached to the cytoplasmic face of the lipid bilayer through using radioiodinated murine monoclonal IgG anti-GPC.7 interactions involving ankyrin and protein 4. l. It is known This value is a minimum estimate, since accessibilityto the that ankyrin interacts with the N-terminal cytoplasmic do- relevant epitope on all GPC molecules may beimpeded by main of the integral membrane protein, band 3. The way in steric hindrance resulting from the size of whole IgG mole- which protein 4.1 binds to the plasma membrane is less cules. In this report, we have measured the abundance of clearly understood. Evidence has been presentedto support GPC using Fab fragments of rat and murine monoclonal interaction of protein 4.1 with band 3,2-4glycophorin A,' anti-GPC antibodies. We also describe the use of Fab frag- glycophorin C (GPC) and glycophorin D (GPD),6s7 and ments from a rat monoclonal antibody that recognizes an phospholipid.' The relative importance ofthese interactions epitope common to GPC and GPD. remains unclear. However, there is strong evidence in sup- MATERIALSAND METHODS port of a role for GPC and GPD in vivo. GPC andGPD are Human RBCs were obtained from Blood Services South West, present in skeletons preparedfrom normal erythrocytes,but Bristol, UK. Rat monoclonal antibodies (BRACs 1 and 11) were are absent from skeletons prepared from the erythrocytes produced in LOU rats in response to two intraperitoneal injections of individuals with hereditary elliptocytosis resulting from of human RBCs (0.2 ml of 50% suspension in phosphate-buffered homozygous protein 4. l defi~iency.~,~Erythrocyte mem- saline [PBS], pH 7) 24 days apart. Three days after the second im- branes of the Leach phenotype, which have a total defi- munization, splenocytes were fused with Y3.Ag. 1.2.3. myeloma ciency of GPC and GPD,7lack a membrane binding site for cells," in the ratio 4: I, using PEG 3500. Hybrid cells were selected protein 4.1, since incontrast to normal membranes, protein by culture in Dulbecco's modified Eagle's medium (DMEM; con- 4.1 is readily extracted from Leach phenotype membranes taininghypoxanthine, thymidine, aminopterin [HAT]) supple- mented with 20% fetal bovine serum (F'BS) and 10% Y3 culture under the low ionic strength conditions used to isolate spec- supernatant in the presence of irradiated 3T3 fibroblasts. BRICs 4 trin." and 10, murine monoclonal antibodies to GPC, were as previously One problem concerning interpretation of the in vivo sig- described.' Pronase, trypsin, and chymotrypsin treatment of RBCs nificance of GPC and GPD as membrane binding sites for were as described el~ewhere,'~ was as the treatment of RBCs with protein 4.1 has been the apparent difference between the sialidasei4and with 2 aminoethylisothiouronium bromide (AET).'' number of protein 4.1 molecules per red blood cell (RBC) Immunoblotting was performed as described el~ewhere,'~except that 5% wt/vol bovine milk powder was used as the blocking agent, Immobilon-P membranes (Millipore Ltd,Watford, UK) were used, From the International Blood Group Reference Laboratory, Bris- and rabbit anti-rat peroxidase conjugate (DAKO, High Wycombe, tol, UK; and the Department of Haematology, St Mary's Hospital UK) was used with the BRAC antibodies. The rat monoclonal anti- Medical School, London, UK. body isotypes were determined using a dot-blot method essentially Submitted May 25, 1993; accepted November 5, 1993. as described by McDougal et IgG was purified from cell-culture Presented in part at the Annual Meeting of the British Blood supernatant using Protein A Sepharose (Pharmacia, Milton Transfusion Society, York, UK, September 8-11, 1992 and pub- Keynes, UK) andIgM was purified using ion exchange on Q Seph- lished in Transf Med 2.69, 1992 (suppl l). arose fast-flow followed by gel filtration through Sephacryl S300 Address reprint requests to David J. Anstee, PhD, Director, ln- (Pharmacia) as recommended by the manufacturer. Methods for ternational Blood Group Reference Laboratory, Southmead Rd, the purification of Fab fragments, radiolabeling of whole immuno- Bristol BSI0 5ND,UK. globulin and Fab fragments, and their use in quantitative binding, The publication costs of this article were defrayed in part by page functional affinity, and competitive inhibition assays were as pre- charge payment. This article must therefore be hereby marked viously described." "advertisement" in accordance with 18 U.S.C.section I734 solely to RESULTS indicate this fact. 0 1994 by TheAmerican Society of Hematology. Characterization of rat monoclonal antibodies BRAC 1 0004-4971/94/8306-0002~3.00/0 and BRAC 11. BRAC 1 and BRAC l1 agglutinated nor- 1668 Blood, Vol83, No 6 (March 15). 1994: pp 1668-1 672 WANTITATION OF GLYCOPHORINS C AND D ONRBCs Origin - MW 40K - MW 32K - Downloaded from http://ashpublications.org/blood/article-pdf/83/6/1668/612763/1668.pdf by guest on 24 September 2021 MW 30K Fig 1. lmmunoblotting with BRAC 1 and BRAC - 11. Erythrocyte membrane components were sep- arated under nonreducing conditions by sodium do- decyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) using 10% (wt/vol) acrylamide gels with a 3% (wt/vol) overlay." Lanes a and 1, normal erythrocyte membranes.Lanes b and g, mem- branes from Leach phenotype erythrocytes. Lanes ! c and h, membranes from trypsin-treated normal erythrocytes. Lane d. membranes from Gerbich- negative erythrocytes of the Yus type. Lane e, a bcde fgh membranes from Gerbich-negative erythrocytes of the Ge phenotype. BRAC 1 BRAC 11 mal RBCs, but failed to react with RBCs ofthe Leach phe- ration (Scatchard analysis; Fig 2 and Table l). Values ob notype, which lack GPC and GPD. The reactivity of both tained with BRAC 1 gave site numbers for GPC plus GPD antibodies was unaffected by prior treatment of normal averaging 7,000 per RBC using IgMand 238,000 (corrected RBCs with chymotrypsin, sialidase, or AET. BRAC 1 re- to 225,000 assuming binding to Leach phenotype RBCs acted with pronase-treated RBCs and gave markedly re- [Table l] represents nonspecific binding) using Fab frag- duced reactions with trypsin-treated normal RBCs. BRAC 11 failed to react with either pronase-treated or trypsin- treated normal RBCs. Table 1. Number of Available Binding Sites on RBCs for Anti-GPC (BRAC 11, BRlC 4, BRlC 10) and Anti-GPC/D (BRAC 1) Analysis of BRAC 1 and BRAC I l by immunoblotting and Values of Affinity Constants showed that BRAC 1 bound to both GPC and GPD in nor- Sites per RBC mal RBC membranes, but failed to react with membranes Affinity of the Leach phenotype and gave very weak reactivity with Antibody Normal Leach K (x 10' mol/L") trypsin-treatednormal membranes (Fig la throughc). BRlC 4 IgG 62,525 ? 3,9613.000 (1) l .4 f 0.7 BRAC 1 also reacted with the abnormal GPC found in (16) (4) Gerbich-negative RBCs of the Yus type, but not with the BRlC 10 IgG 47,683 ? 4,4964.000 (2)4.9 f 1.8 abnormal GPC found in Gerbich-negativeRBCs of the Ge (4) (16) type (Fig Idand e). BRAC 1 l bound onlyto GPC in normal BRlC 10 Fab 142,463 f 13,660 ND 0.67 t 0.02 RBCs and failed to react with membranes ofthe Leach phe- (8) (7) notype or trypsin-treatednormal membranes (Fig If BRAC 1 1 IgG 29,456 f 1,065 ND 2.3 (2) through h). (5) Quantitative binding experiments using rat and murine BRAC11Fab 143,410t10.448 ND 0.12 (2) (4) IO monoclonalantibodies GPCand GPD. Rat monoclonal BRAC 1 IgM 7,374 ? 1,225 ND ND antibodies BRAC 1 and BRAC 1 1 and murine monoclonal (5) antibody BRIC 10 were purified and Fab fragments pre- BRAC 1 Fab 238,721 ? 22,38013,380 t 2.464 0.22 (2) pared from eachantibody. The purified antibodies and Fab (3) (4) fragments were radioiodinated and usedin quantitative Mean t SD values are givenfor three or more measurements (the num- binding assays.