Volume 68 December 1975


Section of Clinical Immunology & Allergy President K Citron MD

Meeting 10 February 1975

Blood Group Antigens Dr Ten Feizi (Clinical Research Centre, Watford Road, Harrow, Middlesex, HA1 3UJ)

et al. 1961, Feizi & Hardisty 1966), and infectious mononucleosis (Jenkins et al. 1965). The majority of the persistent cold agglutinins are monoclonal proteins which are indistinguishable from Waldenstrom macroglobulins (Fudenberg & Kunkel 1957). Many of the transiently occurring cold agglutinins also show restricted heterogeneity (Feizi & Schumacher 1968, Feizi 1967). Because of their homogeneity individual cold agglutinins would be expected to have uniform binding sites with restricted specificities. The reactions of these antibodies with the complex I-i active glycoproteins described in this report are compatible with such restricted specificities. Each antibody presumably reacts with one component of a complex antigen system.

Ii Antigens

The I and i antigens are the most common targets of the red cell autoantibodies which are known as cold agglutinins (Dacie 1962, Marsh & Jenkins 1960). The I antigen is well represented on the red cells of the majority of adults (Wiener et al. 1956). The i antigen is strongly expressed on cord blood cells; in the course of the first year of life the I antigen increases to adult levels and the i antigen diminishes (Marsh 1961). Rare healthy adults (approximately one in ten thousand) lack the red cell I antigen and have i antigen well expressed. In certain hmmatological disorders Relationship ofIi Antigens to associated with bone marrow stress, e.g. thalas- Blood Group Precursors ssemia major, there is increased expression of In the last several years evidence has been erythrocyte i antigen (Giblet & Crookston 1964). accumulating to suggest that the I-i antigens are Both of these antigens are present on leukocytes closely related to the precursors of the ABH and (Franks 1966, Shumak et al. 1971, Thomas 1973). Lewis blood group substances. The first indications of such a relationship arose in the course of Autoantibodies with Ilnd i Specificities studies on an I-active antigen extracted from The autoantibodies (cold agglutinins) which human milk. The sugar composition of this recognize these antigens occur in high titres material resembled that of blood group subpersistently in the chronic cold agglutinin syn- stances; however, its content of fucose was drome (Dacie 1962) and transiently in association unusually low (Feizi et al. 1971a). In this respect with Mycoplasma pneumonike infection (Chanock it resembled two blood group precursor-like /JDGaI


/iDGlcNAc J/DGal 1-3) ;DGICNAC(1-3)


/iDbal a-3)/;DGlcNAca-3)I3DGal (1-3),DGaINAC-serine ;DGal (I-4sDGICNAca---6)




ioDGIcNAc 3



Fig 1 Proposed structure of the oligosaccharide moiety of the precursor-like substance OG (Vicari & Kabat 1970)


800 Proc. roy. Soc. Med. Volume 68 December 1975



0 m














Micrograms antigen









Micrograms antigen

Fig 2 Quantitative precipitin reactions of two different types ofanti-I sera, Ma and Step, with the 1 antigen of human milk ('milkfraction C', * ) and with the two precursor-like substances OG (0) and Fl (+). A, reactions of 15 ,ul of anti-I Ma in a total volume of 400 pl. B, reactions ofanother anti-I serum, Step (5 pAI serum in a total volume of 125 1,u), which recognizes a different kind of I determinant. (Modifiedfrom Feiziet al. 1971b)

substances, OG and Fl (named after the patients from whom they were isolated). These substances 4. are glycoproteins which have been isolated from human ovarian cyst fluids by Vicari & Kabat ._M1, 44 (1969, 1970) and by Watkins & Morgan (1959) respectively. The studies of Vicari & Kabat have 01 3 indicated that 'precursor' OG contains a branched oligosaccharide moiety made with /3DGal (1 -->3) E flDGlcNAc (1 -÷3) -1 (Type 1 chains) and cw 2 I3DGal (1 ->4) 3DGIcNAc (1 -+6) (Type 2 chains) (Fig 1). Precursor Fl presumably contains Type 2 Original A, B & H chains since it reacts with antisera against substances pneumococcus Type XIV polysaccharide. Anti-I antibodies which reacted strongly with the milk antigen also reacted with these two precursor substances. For example, the anti-I serum Ma (Fig 2A) reacted well with all three antigens. Inhibition of precipitation assays have shown that the Type 2 chain is involved in the specificity .2_ of the anti-I Ma (Feizi et al. 1971b). However, anti-I Ma is representative of a cC, minority of anti-I specificities. The majority of anti-I antibodies differ from this antibody in the I determinants that they recognize. For example, !aE another anti-I serum, Step, reacts well with determinants on OG but it reacts poorly with il A, B & H substances ly degraded A & B those on Fl and the milk antigen (Fig 2B). The I O differences between these two antibodies are 40 50 20 30 with further evidenced by their reactions partially CB Micrograms antigen degraded blood group A, B and H substances. Fig 3 Quantitative precipitin assays of anti-I sera Ma Anti-I Ma reacts well with determinants revealed (A) and Step (B) with human A, B and Hsubstances after partial acid hydrolysis (Allen & Kabat before and afterpartial degradation. Symbols: 1959) or the first stage of periodate oxidation and 7, A, O = original A, B and Hsubstances Smith degradation (Lloyd & Kabat 1968) of A respectively; *. U--first stage ofperiodate -














Abbreviations: DGal = D-galactopyranose; LFuc = L-fucopyra-

nose; DGlcNac =N-acetyl-D-glucosamine;



oxidation of A, B and Hsubstances; *, B substance after partial acid hydrolysis. Volumes ofsera and total reaction volumes as in Fig 2. (Modifiedfrom Feizi et al. 1971b)

Section of Clinical Immunology & Allergy



:;DGal 4



L Fuc 1

2 6 or DoGal (1-31 ,DGIcNAc(1-3) B gene DWl (1-3 eJoGal (1-3)-;DGlcNAc(i-3),,DG-II / 4 Agene DGaINAc(1-3)

A gene



1a (1-3A3) 2 (1-4MDGIcNAc-6o)sGC C 4oren s;GICNAC'

1 6 trine -3),DG5INAc-se



H gene


and B substances (Fig 3A), while anti-I Step fails to react with these but it reacts well with the first periodate stage of H substance (Fig 3B). The above method of periodate oxidation destroys only the terminal non-reducing sugar residues; thus the determinants revealed from A and B substances are different from those revealed from H substances. On A and B substances (Fig 4) the terminal fucoses and galactoses and the 3DGalNAc residues would be destroyed leaving a precursor type structure with Type 1 and Type 2 chains. However, on H substance the terminal a-D-GalNAc and aDGal residues would be absent and there would occur destruction of the terminal fucoses and fDgalactoses, as well as the subterminal galactoses to which the H determining fucoses are attached, leaving a structure on which /3DGlcNAc residues would have been terminal non-reducing ends. Inhibition of precipitation assays with anti-I Step have so far been entirely negative (Feizi & Kabat 1972); however, the limited amounts of oligosaccharides, especially those with terminal fDGlcNAc residues, precluded assays at high concentrations. Thus we cannot be sure whether the I determinant recognized by anti-I Step involves entirely different residues or whether it involves a determinant of larger size and the smaller oligosaccharides were not used in sufficient concentrations to inhibit. Table 1 Classification of anti-I and anti-i sera on the basis of their reactions with blood group related antigens (modified from Feizi & Kabat 1972)

PrecursorFl Partially degraded 'Precursor' Cow 21@ Hydatid A & B S. OG Milk fraction C cystfluid Group

Anti-I 1 2 3 4 5

6 Anti-i


+-+ +

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

+++ +++ +++ ++

-or +++



+++ -or


++ -


+±++ ++ -or _ or:



(+ one


Fig 4 Proposed composite megalosaccharide structure showing the relation of the A, B and H determinants on secreted blood group glycoproteins. The oligosaccharide structure shown in Fig I would be a precursor ofH substance which in turn would be a precursor of A or B substances. (Modifiedfrom Lloyd et al. 1968, Lloyd & Kabat 1968, Vicari & Kabat 1970, Kabat 1970)

In the meantime it has been possible to classify anti-I antibodies on the basis of their quantitative precipitin reactions with an antigen panel of I-active glycoproteins (Table 1). There is evidence for 6 types of I specificity (Anti-I Ma belongs to Group 1 while anti-I Step to Group 3). In addition, there is evidence suggestive of more than one kind of i specificity (Feizi & Kabat 1972). Precursor OG must have a more complex structure' and more varied determinants than Fl and the other I active glycoproteins, for it contains all of the I and i determinants, while only some of these determinants are present on the other glycoproteins. Use of Anti-IAntibodies for Isolation of I Antigens We are currently using insolubilized monoclonal anti-I antibodies as immunoadsorbents for isolating I-active antigens from crude extracts of biological fluids, e.g. sheep hydatid cyst fluid and human amniotic fluid. Sheep hydatid cyst fluid, which contains blood group PI and I activity, can thus be fractionated into an effluent (not retained) P, active fraction and a specifically retained (PI +1) active fraction (Feizi & Kabat 1974). Pooled human amniotic fluid containing A, B, H, I and i activities can be fractionated into an effluent fraction with ABH activity and an eluted fraction, which is a minor component of the starting material, but is highly enriched in I, i as well as A, B and H activities (Feizi et al. 1975). Thus anti-I immunoadsorbents are not only excellent reagents for the specific isolation of antigens they recognize from biological fluids; they also provide interesting information about the association of I antigens with other blood group antigens. They may well prove useful for isolating cell membrane antigens as well.

Summary There is evidence for the existence of several types of I and i determinants. These antigens are all found on a blood group precursor-like sub-

serum) * Blood group substance isolated from the stomach of a cow

(Beiser & Kabat 1952) * A and B substances subjected to partiat acid hydrolysis or one stage of periodate oxidation and Smith degradation

'The structure of the sugar moiety of OG substance as proposed in Fig 1 represents a complete chain. In common with other mammalian glycoproteins this substance must contain chains of varying degrees of completeness, due either to incomplete synthesis or to degradation within the cyst cavity

802 Proc. roy. Soc. Med. Volume 68 December 1975 stance. Certain I determinants are revealed on partially degraded A, B and H substances. One of the I determinants involves the type 2 chain of precursor substance. Anti-I antibodies are useful reagents for specifically isolating the antigens they recognize from biological fluids; at the same time they are providing information on the association of the Ii antigens with other blood group antigens. REFERENCES Allen P Z & Kabat E A (1959) Journal of Immunology 82, 340 Beiser S M & Kabat E A (1952) Journal ofImmunology 68, 19 Chanock R M, Rifkind D, Kravetz H M, Knight V & Johnson K M (1961) Proceedings ofthe National Academy of Sciences of the United States ofAmerica 47, 887 Dacie J V (1962) The Hamolytic Anemias. 2nd edn. Grune & Stratton, New York; Pt II Feizi T (1967) Nature (London) 215, 540 Feizi T, Cederqvist L L & Childs R (1975) British Journal of Haematology 30, 485 Feizi T & Hardisty R M (1966) Nature (London) 210, 1066 Feizi T & Kabat E A (1972) Journal ofExperimentalMedicine 135, 1247 (1974) Journal ofImmunology 112, 145 Feizi T, Kabat E A, Vicari G, Anderson B & Marsh W L (1971a) Journal ofExperimental Medicine 133, 39 (1971b) Journal ofImmunology 106, 1578 Feizi T & Schumacher M (1968) Clinical and Experimental Immunology 3, 923 Franks D (1966) Vox sanguinis (Basel) 11, 674 Fudenberg H H & Kunkel H G (1957) Journal of Experimental Medicine 106, 689 Giblet E R & Crookston M C (1964) Nature (London) 201, 1138 Jenkins W J, Koster H G, Marsh W L & Carter R L (1965) British Journal of Haematology 11, 480 Kabat E A (1970) In: Blood and Tissue Antigens. Ed. D Aminoff. Academic Press, New York; p 187 Lloyd K 0 & Kabat E A (1968) Proceedings of the National Academy of Sciences ofthe United States of America 61, 1470 Lloyd K 0, Kabat E A & Licerio E (1968) Biochemistry 7, 2976 Marsh W L (1961) British Journal of Haematology 7, 200 Marsh W L & Jenkins W J (1960) Nature (London) 188, 753 Shumak K H, Rachkewich R A, Crookston M C & Crookston J H (1971) Nature New Biology 231, 148 Thomas D B (1973) European Journal ofImmunology 3, 824 Vicari G & Kabat E A (1969) Journal ofImmunology 102, 821 (1970) Biochemistry 9, 3414 Watkins W M & Morgan W T J (1959) Vox sanguinis (Basel) 4, 97 Wiener A S, Unger L J, Cohen L & Feldman J (1956) Annals ofInternal Medicine 44, 221

The following papers were also read: ABH and P Antigens Professor Winifred Watkins (Lister Institute ofPreventive Medicine, Chelsea Bridge Road, London SWI W8RH) Rh Antigens Dr N Hughes-Jones (Department of Hematology, St Mary's Hospital Medical School, London W2)


Meeting 14 April 1975

HL-A and its Association with Clinical Disease Dr J C Woodrow (Department ofMedicine, University of Liverpool, Ashton Street, Liverpool, L69 38X) HL-A Associations in Cliniical Research

Discussing the multigenic control of susceptibility to Friend virus disease in the mouse, Lilly (1972) commented: 'The difficulty is that in trying to understand the basic mechanisms involved in polygenic control there is no substitute for reaching into the black box and picking out the genes one at a time, identifying them, mapping them and studying their individual mechanisms of action.'

Although this may prove to be a counsel of perfection when it comes to the study of disease in man, it is evident that the discovery of associations between disease and the HL-A system represents at least a start in this direction. Genes underlying susceptibility to a surprisingly wide variety of diseases, thought to be under multigenic control, occur at loci in the HL-A chromosomal region. One immediate problem relates to the indirect way in which the presence of these genes is revealed (McDevitt & Bodmer 1974). If indeed most of the genes of importance in this respect do not determine the serologically defined HL-A antigens but are closely linked to the HL-A loci there is an important corollary. This is that the relative importance of such genes in the pathogenesis of a particular disease is not necessarily related to the strength of the association with any particular HL-A antigen. The presence of a gene causing susceptibility to a disease may be suspected because of a rather weak association with a particular HL-A antigen in a population study, but the contribution of the gene to disease susceptibility may be very important. This, together with the statistical problems inherent in studying associations between the HL-A system and disease (Svejgaard et al. 1974), leads to difficulties in assessing the potential importance of data obtained in these studies. Family studies can contribute to providing further useful evidence and this can be illustrated by studies that Dr Andrew Cudworth and I have carried out in relation to diabetes mellitus (Cudworth & Woodrow 1975). We first looked at

Blood group antigens. Ii antigens.

15 Volume 68 December 1975 799 Section of Clinical Immunology & Allergy President K Citron MD Meeting 10 February 1975 Blood Group Antigens Dr Te...
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