Monoclonal antibodies against adult and fetal red blood cells

Monoclonal antibodies against adult and fetal red blood cells

Clinical Immunology Newsletter Volume 7, No. 12 December 1986 Monoclonal Antibodies against Adult and Fetal Red Blood Cells L. Edelman, M.D. lnstitu...

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Clinical Immunology Newsletter Volume 7, No. 12

December 1986

Monoclonal Antibodies against Adult and Fetal Red Blood Cells L. Edelman, M.D. lnstitut Pasteur Paris, France

P. Edelman, M.D.


Clinique Universitaire Baudelocque Paris, France

J. Reviron, M.D. CTS H6pital Saint-Louis Paris, France


W. Vainchenker, M.D. lnserm U 91 Cr~teil, France

In immunohematology, monoclonal antibodies create many possibilities for research. The use of high-titered, monospecific, identically batched antibodies can be of great value in routine typing in transfusion centers, for the study of weak groups, and for a better understanding of the antigen structure (9). Characteristics Anti-A, Anti-B, and Anti-H blood group monoclonal antibodies obtained to date have characteristics similar to those of classical polyclonal test sera used routinely (10) and present the following characteristics (Tables 1, 2):

1. By agglutination test: Anti-A (D3DI) aggutinates A red blood cells only, including A2, A3" (from a panel of 50 cells), and CisAB erythrocytes (4). Anti-B (B183) agglutinates B cells only, including B3, Bx, 3/4 acquired B (8). Anti-H (B2-10) weakly agglutinates A2, and strongly agglutinates

CIMNDC 7(/2) 177-192, 1986



O erythrocytes. There is no reaction with A I and Bombay red blood cells (3). By inhibition of salivary tests: There is inhibition by the saliva of each corresponding secretor subject (Tables 3, 4). By immunofluorescence tests: The different monoclonal antibodies were able to detect ABH antigens on human kidney tissue sections (glomeruli, distal tube, endothelium vessels) and on pancreatic tissue (acini and endothelium vessels) (Fig. 1). Affinity constants: These were determined by thermodynamic study. Thus, a value of 2.10 7 L/M was found for anti-A, and 3.10 8 L/M was found for anti-B. Antigen identification: By fixation on oligosaccharide immunoadsorbent, the structure recognized by anti-An monoclonal antibody is

Table 1 Main Characteristics of Monoclonai Anti-Blood Group Antibodies Monoclonal Antibodies

Intensity Avidity Titer Association constant Class

GALNAC( 1- 3)GAL( 1- 3)GAL Nac Fuc

The structure recognized by anti-B is GAL( 1- 3)GAL( 1- 3)GINac



In This Issue Monocional Antibodies against Adult and Fetal Red Blood Ceils . . . . . . . . . . . . . . . . . . . . . . . .


Emphasizes their practical applications in transfusion centers Monoclonal Antibodies to the Thyrotropin (TSH) Receptor in the Study of Graves' Disease: Idiotypes and Antiidiotypes . . . . . 182

A discussion of the types of autoantibodies and their respective clinical correlation Detection of Microbial Antigens in Spinal Fluid and Blood . . . . . .




An evaluation of the sensitivity, specificity, and prognostic value of standard immunoassays

+ + + 6" 256

+ + + 4" 512

Meeting Announcements . . . . . . .


2.107 L/M IgM

3.108 L/M IgM

Meeting Notices . . . . . . . . . . . . . .


Index to Volume 7 . . . . . . . . . . . .



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Table 2

Main Characteristics of Human Test Sera ICNTSI Um-B

•D m - q

Intensit}. . . . Avidity Trier Association constant






4 I0.15"

+ + + '" 512 l0 s L/M

+ f + II)"

+ f + +" 512 I 0 s L/M



2. I (I7 L/M

Table 3 Salivary Test with Anti-A Monoclonal Antibody Saliva Dilutions Sul~jects

ASc BSe OSc Osc



-++ ++ ++ ++ + + ++



++ ++ ++ ++ + + ++

1/16 1/32

+ ++ ++ ++

++ ++ ++ + +

Table 4 Salivary Test with Anti-B Monoclonal Antibody Saliva Dilutions Sul)]ects

B Se ASc OSe Ose




1/18 1/16 1/32

. . . . + + + + + + + + + + + ++ + + + + ++ ++ ++ + + ++ ++ + + ++ + + ++


I(F L,'M

of m a k i n g adaptations to each change of reagent lots in individual laboratories and should offer better assurance against difficulties with grouping. On a national or even internatkmal plane, standardization should allow useful comparisons. Because the use of monoctonal antibodies in isolation or in a pool is well adapted to instruments for automatic blood grouping, it is possible to utilize fully the potentials of two advanced technologies. The first study' undertaken on the Groupmatic M G 50 has shown no disagreement whatever, out of more than 10,000 donors, between the monoclonal antibodies and the classical polyclonal test sera (studied in Paris). In addition to the advantage of disposing reagents of perfectly defined

specificity, thcre is thc po,sihility ut obtaining test sera against rare antigens. Anothcr major advantage ol USill~ tnonochmals i', awfiding voluntarx immunization. In fact, because of strengthened control of blood groupings, the n u m b c r of transfusion accidents is smaller, and the possibility of obtaining high-titcred irumunc antibodies has become rare. Voluntary hlllnunization is thus on the increase and raises ethical questions. A frequent problem is that or typing of thc Rh system and especially the difficulties of obtaining suitable anti-l) test sera. Until expert cvaluation obtained in other fields (notably in antitumt+r antisera and in Jmmunoptcs,,ion) allows us to envisage thc therapeutic injcction of thc products ot" niurinc hybridomas. the use of nll_lrine monoclonals should, be reserved tor p h c n o t y p m g of the Rh system, with the p r e \ c n t i o n of hcruolytic diseasc of the ne~bot'n being perfom~ed, as before, by thc injection of human y-globulins. The present possibilit,, of c u h u r m g h u m a n ruyehmm allows us to foresee the fusion of the sensitized peripheral h u m a n lymphocytcs with such m y e l o m a cells. These human monochmal anti-

The structure recognized by anti-H is GAL(1-3)G1Nac



Practical Applications The practical applications of monoclonal antibodies in transfusion centers are indeed numerous. In particular, there is the important possibility of producing monospecific and invariant reagents in theoretically unlimited quantities. Of special interest is the standardization possible with monoclonal antibodies; this would avoid the necessity,

Figure 1, Human kidney tissue section labeled with anti-A mom~clonal antibody (endothelium vessel is strongly positive).

('lini~al lmmunolog~ (]SSN ()IV7

1~5VI i, i ~ u c d monlh]~ in one illdcxcd ~olulll¢ pCZ ~c;u h~, I l~cxic~ 5,cicn~e Puhli~hm E ( ' ~ . Inv . $2 \ a n d c r h h \ ~ c m l c N c ~ "i~,Ek N~ I ~ 1 7 %ubxcllptl~,n pHcc pci year: $b2 ()) For ~urlace airlilt to Furupc. add $21 OO F~/: air mailclscwhcrc, add $ 2 4 0 0 Sct~lnd clas. poslagc pending at Nc~ York, N~. and at addltiouat maihng oU{cc> Pt~dnla*tc: Svnd addict, ~hangc, to ('lineal Imlnlolol,*w Neuslcth't [il,c~icr Science Puhli*hing ('<~ lnc . 52 Vandcrlqlt \ x c n u c , Ncx~ Y~wk. N~ I!~)t 7



I85WSO/SO.O0 ~


~ 1986 E l s e v i e r S c i e n c e P u b l i s h i n g ( ' o . . Inc.

C l i n i c a l [ m u l u n o l o u ) N c x ~ q c t l c l 7 : 1 2 , ItlS{~

MW x10-3


2 3" 4

5 6


bodies should thus widen the range of test sera useful for immunohematology.

9 1011

Monoclonal Antibodies Against Membrane Glycoproteins Anti-N Like


Figure 2. lmmunostaining ~!1 human red cell glycolwotein.~ on nitrocellulose sheets by iB5 monoclonal antibody. Lane /, NN: lane 2, Ett(a ): lane 3. S - . ~ - U - N + M (Set.): lane 4, S - s - U - N - M + (Ger.): lane 5. "l)t (Ba): lane 6, Immozygous M ,~ (Gen. 1: lane 7. M"M (Cor.); lane 8, MM: lane 9, pj/Stqa + )/: lane I0, heterozygot~s Mi. V (Chr.): lane 11, homoo~gous Mi.lll (C.H.).

Table 5 Reactivity of FA6 152 with Several Hemopoietic Cell Lines Percent

Lymphoblastoid Burkitt's lymphoma T-ALL U-937 HL 60

(five cell lines (five cell lines) (three cell lines)


HEL K 562

75 - + 10 "~_+9

Putko (hybrid between K 562--P3 HR-1) Dutko (hybrid between K 562 and Daudi)

>90 >90


M. BFU-E CFU-E Proery. M~rQw e l y .







A monoclonal antibody (iB5, lgGlk) reacting with human red blood cells was produced alter immunization of BALB/c mice with cord red cells, followed by fusion of the spleen cells with the murine myeloma cell line Ag 86-53 (2). The monoclonal antibody agglutinated blood group N + much more effectively than M + N - red cells but did not recognize erythrocytes from rare individuals typed as M + N - S s - U - and those from an E n ( a - ) individual (M.E.P.). However, S-s-Udonors typed as M + N + or M - N + and E n ( a - ) red cells from donor G.W. were agglutinated. The erythrocyte receptors for iB5 are completely destroyed by papain treatment and significantly decreased by neuraminidase. In addition, the iB5 antibody failed to agglutinate trypsintreated N + M - S - s - U erythrocytes. Other investigations have shown that the monoclonal antibody precipitated glycophorin A and B from N + red blood cells and only glycophorin B from M + N - erythrocytes. The reactivity of iB5 was further explored by immunostaining after the electrophoretic transfer to nitrocellulose sheets of membrane proteins from common (M and N) and rare erythrocytes [ E n ( a - ) , S - s - U - MgMg, McM, St(a + ), MiV, MiIlI, Tn] separated by SDSpolyacrylamide gel electrophoresis (Fig. 2). These studies have clearly demonstrated that the monoclonal iB5 antibody is directed against the homologous N-terminal domain of glycophorin A and B, a specificity that explains the serologic reactivity of iB5 against common and rare erythrocytes (5).

Anti-Glycophorin A CFU-GdVl


Marrow pr~ursors



Figure 3. Schematic representation of the expression of FA6-152 on adult and fetal hemopoietic cells. A, adult; F, fetus.

Clinical hnmunology Newsletter 7:12. 1986

:~: 1986 Elsevier Science Publishing Co., Inc.

This monoclonal was characterized serologically as an anti-Ena; by Western blot analysis, it seemed directed against glycophorin A only. These reagents are of great value in characterizing membrane glycoproteins (1, 7).

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Figure 4. Proervthrc~b/ast

Monocional Antibody Against a Fetal Erythroid Antigen and Erythroid Progenitors A monoclonal antibody FA6 152 against human fetal red blood cells has been obtained after BALB/c immunization with 20-week fetal erythrocytes.

the cell membraJle ~hows monerozcs gold lmrlic/(,~,

This antibody recognized fetal red blood cells by agglutination test until 32 weeks. There was no reaction with cord, adult common red blood cells, or rare adult red blood cells. By the indirect immunofluorescence test, B lymphocytes, T lymphocytes, and granular

Figure 5. Polychromatic er>'throblast--gold particles are concentrated in several parts o/ the membrane.


0197-1859/86/S000 ~, 02.20

~ 1986 EIsevier Science Publishing Co.,Inc,

cells were not recognized. However, rnonocytes and platelets from peripheral blood are fluorescent. Identical results were obtained from fetal cells: only platelets and monocytes tested positive. Results of the fluorescence test, using different cell lines, are shown in Table 5 (6). The study on medullary progenitors was performed on frozen bone marrow obtained from normal donors for bone marrow transplantation. After indirect fluorescent labeling with FA6 monoclonal antibody, marrow cells were analyzed and sorted on the basis of fluorescent intensity. The positive fraction used for clonogenic assays showed the following results: mature BFU-E, CFU-E, and erythroblasts were positive. The antigen identified by FA6 monoclonal antibody was synthesized at an early stage of the erythroid differentiation, earlier than glycophorin A, as the colonies derived from blood BFU-E were labeled as early as day 5 whereas a monoclonal antibody against glycophorin A did not stain the colonies before day 8 (11). In contrast, the same antigen was detected on a latter

(linical Immun~Ho~5 Nc~sletter 7:12. ItiN6




Figure 6. Reticulo(3'te--there is no &beling of the membrane.

stage of differentiation on the megakaryocytic and monocytic lineage; the granular lineage remained negative. The same results were obtained from fetal liver. Results are summarized in Figure 3. A study using the electron microscope was carried out after staining with FA6 monoclonal antibody and an antiglobulin coupled to colloidal gold: • On proerythroblast numerous gold particles were found on the membrane indicating antigenic sites recognized by FA6 antibody (Fig. 4). • On polychromatic erythroblast gold particles are less numerous and were concentrated on a part of the membrane (Fig. 5). • On early reticulocyte, a few particles remained.


• On late reticulocyte there was no staining (Fig. 6). This antibody will permit a better understanding of both normal and leukemic erythroid differentiation and can be a reagent for fetal red blood cell identification. References 1. Bigbee, N. L., M. Vanderlaan, S. S. N. Fong, and R. M. Jensen (1983). Monoclonal antibodies specific for the M and N forms of human glycophorin. A. Molec. lmmunol. 20:1353. 2. Dahr, N., G. Uhlenbruek, and H. Knott (1975). Immunochemical aspects of the MNSs blood group system. J. Immunogenet. 2:87. 3. Doinel, C., L. Edelman, P. Rouger, J. Leblanc, J. Reviron, and C. Salmon (1983). A murine monoclonal





antibody against blood group H type 1 and 2 structures. Immunology 50:215. Edelman, L., P. Rouger, C. Doinel, J. F. Bach, J. Reviron, and C. Salmon ( 1981). Thermodynamic and immunological properties of a monoclonal antibody to human blood group A. Immunology 44:549. Edelman, L., D. Blanchard, P. Rouger, C. Doinel, J. P. Cartron, C. Salmon, and J. Reviron (1984). A monoclonal antibody directed against the homologous N-terminal domain of glycophorin A and B. Exp. Clin. lmmunogenet..1:129. Edelman, P., C. Vinci, W. Vainchenker, R. Miglierina, P. Rouger, J. Reviron, J. Breton-Gorius, and L. Edelman (1986). A monoclonal antibody against an erythrocyte ontogenic antigen identifies fetal and adult erythroid progenitors. Blood 67:1. 56. Furthmayr, H. (1978). Structural comparison of glycophorins and immunochemical analysis of genetic variants. Nature (London) 271:519. Rouger, P., L. Edelman, C. Doinel, J. Reviron, C. Salmon, and J. F. Bach (1983). Study of blood group B antigen with a specific monoclonal antibody. Immunology 49:77. Salmon, C., J. P. Cartron, and P. Rouger (1983). The human blood groups. Masson. New-York. Voak, D., S. Sack, T. Alderson, F. Takei, E. Lennox, J. Jarvis, C. Milstein, and J. Darnboroug (1980). Monoclonal anti-A from a hybrid myeloma: evaluation on a blood grouping reagent. Vox Sang. 39:134. Vokochi, T., M. Brice, P. S. Rabinovitch, T. Papayannopoulov, and C. Stamatoyannopoulos (1984). Monoclonal antibodies detecting antigenic determinants with restricted expression on erythroid cells: from the erythroid committed progenitor level to the mature erythroblast. Blood 63:6. 1376.

Meeting Notices

April 4 - 7 , 1987: "Second International Symposium on Immunobiology in Clinical Oncology and Immune Dysfunctions." Nice, France. Contact International Society for Preventive Oncology--ISPO, 217 East 8th Street, Suite 303, New York, NY 10028, USA. Phone (212) 496-1900.

Clinical Immunology Newsletter 7:12, 1986

June 1-5, 1987: "III International Conference on AIDS." Washington, DC. Contact International Conference on AIDS, 655 15th Street, NW, Suite 300, Washington, DC 20005, USA. Phone (202) 347-5900. Telex 440487 COURTESY. June 3 - 5 , 1987: "Covalently Modified

© 1986 Elsevier Science Publishing Co., Inc.

Antigens and Antibiotics in Diagnosis and Therapy." Lyon, France. Contact (from North and South America and Australia) John D. Rodwell, Cytogen Corporation, 201 College Road East, Princeton, NJ 08540, USA, or (from Europe, Asia, and Africa) Geard A. Quash, Unite de Virologie 1, place J. Renaut, 69371 Lyon Cedex 08, France.


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