Immunology 1976 30 289

Quantitative and thermodynamic measurements on I andi antigens of human red blood cells

C. DOINEL, C. ROPARS & C. SALMON Groupe de Recherche U 76 de l'INSERM et Serviced'Immunologie du Centre National de Transfusion Sanguine, Boulevard Diderot, Paris Cedex 12, France

Received 27 June 1975; acceptedfor publication 23 July 1975

Summary. Different homogeneous IgM cold agglu-tinins (two anti-I, two anti-i and one anti-Ii cross-reacting antibodies) have been used to determinethe antigen site densities of adult I and i erythrocytesand of cord red cells. The equilibrium constants andthe thermodynamic constants of these reactions havebeen determined.The two anti-I antibodies, which did not combine

with i or cord red blood cells, recognized two differ-ent determinants on I red cells. The antigen densityof the I Fla. receptor was 120,000 sites per erythro-cyte and the standard enthalpy change (-AH0) ofthe reaction was 18 to 25 kcal/mole. The antigendensity of the I Loi. determinant varied according tothe red cells tested and the enthalpy change (H-H0)of these reactions was 50-65 kcal/mole.The i and cord erythrocytes reactive structures

were more heterogeneous than those present on Ierythrocytes. The equilibrium constants rapidlydecreased as the temperature rose and the standardenthalpy changes (-AH0) ranged from 50 to 90kcal/mole. Two types of i determinants were ob-served; one of the anti-i antibodies reacted mainlywith an i component present on cord erythrocytes,the other antibody reacted with a different i com-ponent present on i adult red-cells.The determinants, recognized by the cross-reacting

Correspondence: Dr C. Doinel, Groupe de RechercheU 76 de 1'INSERM, 53, Boulevard Diderot, 75571 ParisCedex 12, France.

antibody on I red cells, differed from those on i orcord red cells in equilibrium constant, thermo-dynamic constants, index of heterogeneity of thereaction and in their sensitivity to formalin treat-ment.

INTRODUCTION

Many attempts of classification of the I and i antigensinto subgroups have been proposed (Wiener, Unger,Cohen and Feldman, 1956; Jenkins and Marsh,1961; Marsh, Nichols and Reid, 1971; Marsh andJenkins, 1960; Marsh, 1961). The interpretation ofthe serological reactions is restricted by the semi-quantitative nature of the measurements and by theapparently heterogeneous nature of the I and iantigens (Crookston, Dacie and Rossi, 1956; Maasand Schubothe, 1968). Moreover, the antibodiesseldom have simple specificities in haemagglutina-tion. They are often mixtures of antibodies whichare difficult to separate or cross-reacting antibodies(Jackson, Issitt, Francis, Garris and Sanders, 1968;Boissezon, Marty, Bierme and Ducos, 1970;Dzierzkowa-Borodej, Seyfried and Lisowska, 1975;Doinel, Ropars and Salmon, 1974). It cannot bespecified whether the cross-reacting antibodiesrecognize other different or common antigens on Iand i red blood cells.

Feizi and Kabat (1972) studied various anti-I and

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anti-i, using a quantitative precipitation method, andattempted to group them according to their re-activity with blood group substances. They charac-terized six different anti-I specificities and fourgroups for anti-i. Roelcke, Ebert, Metz and Weickert(1971) found three determinants in the fraction ofglycoproteins with I activity, obtained from theerythrocyte membrane. Feizi, Kabat, Vicari, Ander-son and Marsh (1971) described the biochemicalstructure of an oligosaccharide detected by the anti-I(Ma) but the specificities of the other antibodies weredifferent and are not yet known (Feizi and Kabat,1974). Most antibodies cross-react with the numerousdeterminants of the I antigen complex, each onecombining with a varying number of these antigenicdeterminants.

In this work, we have studied several antigen-antibody combinations in the I, i blood group system.To avoid the problem of mixtures of antibodies weused homogeneous IgM from cold agglutinindisease. The purification and the biochemical char-acterization of these compounds have also enabledus to attempt a quantitative study with radio-chemical methods.The five antibodies we have studied belong to

three distinct groups. Two react with I red bloodcells only, two others with the red blood cells ofadult i subjects, and one with both types.

In each case, we have measured the antigen sitedensities on the red cells, the equilibrium andthermodynamic constants of the reactions.

MATERIALS AND METHODS

Red cellsI erythrocytes were freshly drawn from healthy adultblood donors. The erythrocytes from Caucasian iadults and from cord blood were frozen in liquidnitrogen until used.

SeraThese were obtained from subjects with chronic coldagglutinin disease. Two monoclonal macro-globu-lins without any known antibody activity were usedas a negative control. All the homogeneous IgMhad been purified by delipidation with dextransulphate, 2 M ammonium sulphate precipitation andpreparative ultracentrifugation in a Beckman L 3-50ultracentrifuge at 110,000 g maximum, for 18 h at40 in a 5-20 per cent linear gradient of sucrose in

phosphate-buffered saline. On average, 5 per centof a2-macroglobulin contaminated these IgMpreparations.

Quantitative agglutinationThe agglutinating activity of the various IgM anti-bodies was determined by a quantitative methodusing a Technicon Autoanalyzer (Doinel et al., 1975).The titres were expressed as the reciprocal of thedilution able to agglutinate 50 per cent of a suspen-sion of 800,000 red cells per mm3.

Treatment of the red cells with formalinOne volume of packed cells was treated with twentyvolumes of 20 per cent formalin in PBS, pH 7.3, for20 h at 200, then washed, filtered through glass-wooland stored at 40 until used.

Labelling of the purified antibodies (Bocci, 1974)To 10 mg of purified IgM in PBS (pH 7 2) were

successively added 10 p1 of 3 x 10-3M KI, 110 pCi of125I-labelled Na, 300 pg of chloramine T, and 30min later, 300 pg of Na metabisulphite. The freeiodine was eliminated by dialysis. On an average,two atoms of iodine were fixed per IgM molecule.

Non-specific fixationAnalytical ultracentrifugation of the purified coldagglutinins has shown the presence of componentswith sedimentation coefficients over 20S; the pro-portion of aggregates was increased after the label-ling procedure. Consequently, just before use, thelabelled IgM preparations were ultracentrifugedunder the conditions described above. The peak ofthe aggregates, representing about 20 per cent of theproteins, was eliminated.

In all experiments, the use of a monoclonal IgMwithout any known antibody activity, labelled with1251I, made it possible to determine the amount ofnon-specific fixation included in each measurement.The amount of non-specific fixation was the samefor both IgM preparations.The specificity of the cold agglutinins was deter-

mined by agglutination at 30 of red cells with variousphenotypes in the I, i system. It was confirmed bycomparing the percentage of purified antibody thatcould be absorbed onto these cells.

Quantitative and thermodynamic measurements(a) Determination of the number of bound IgMmolecules per erythrocyte: the experiments were

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carried out in PBS (pH 7 2) containing 1 per centbovine albumin (Miles-Pentex) and 0-15 M glycine.The concentrations of specific antibodies rangedfrom 0 5 to 50 ,ug in 0-5 ml. The red cell concentra-tion determined with a Coultronics Counter, modelB, varied between 1 x 107 and 2 5 x 107 cells/ml. Allexperiments were carried out in duplicate, in plastictubes stored at 30 under continuous agitation for 4 h.The packed cells, centrifuged and washed three timesat 30, were transferred into new tubes; counting was

performed in a CGR-SAIP gamma counter. Themaximum number of IgM molecules, n, which couldbe bound per erythrocyte was evaluated from theexpressions of the mass action law derived byScatchard (1949) or by Ekins, Newman and O'Rior-dan (1967).(b) Determination of the equilibrium constants: to5 pg of specific antibodies in 1 ml of PBS containing1 per cent albumin, various cell concentrations rang-

ing from 5 x 106 to 250 x 106 cells/ml were added.The samples were incubated in duplicate, at 30 and at100, under continuous mild shaking, for 18 h. Theequilibrium constants were calculated from theKarush derivation (1962). The calculation was per-

formed assuming that antibodies behave like univa-lent coumpounds.(c) The standard free energy change AF0, the stand-ard enthalpy change AHO and the standard entropychange AS0 were calculated from the equilibriumconstants at 30 and 100.

RESULTS

(1) Cold agglutinins specificities

The specificities of the cold agglutinin auto-anti-bodies which have been studied, are shown inTable 1. Two antibodies (Fla., Loi.) have anti-Ispecificities. The Fou. and Min. antibodies are

anti-i. The Abg. antibody agglutinated the I ery-

throcytes from adults and, but more weakly, theerythrocytes from cord and i adults. The agglutina-tion titres were the same with the cord and i adultred cells. The two reactivities disappeared simul-taneously and in the same proportion on partialabsorption with I or i erythrocytes. After absorptionand elution from I erythrocytes, the same ratio ofagglutination titres on I and i red cells was obtainedas for the initial antibody. The same result was ob-tained when the antibody was absorbed on and

Table 1. Specificities of the cold agglutinins

Percentage puri-Sera fied antibody

agglutination absorbed attitres at 30 3 'C by:

SpecificityI RBC i RBC* I RBC i RBC*

Fla. 475,000 10,000 73 1 Anti-ILoi. 180,000 10,000 37 0 7 Anti-IFou. 250 8000 <0 5 5 Anti-iMin. 250 32,000 < 0-5 75 Anti-iAbg. 232,500 58,500 83 82 Anti-Ii

* RBC from i adults.

eluted from i red cells. Therefore, the Abg. serum didnot contain a mixture of anti-I and anti-i antibodiesbut cross-reacting antibody.Marsh et al. (1971) have defined two components

for the I erythrocyte antigen: one, called 'IF' couldbe found on all human red blood cells including thei and cord red cells; the other one, called 'ID' wasspecific for I red cells from adults. The Abg. anti-body, having a strong agglutinating activity with I,i and cord red cells, could correspond to an anti_1Fantibody, and the Fla. and Loi. antibodies to theanti-ID antibody. Dzierzkowa-Borodej, Hudrewicz-Hubicka and Mejbaum-Katzenellenbogen (1971)have shown that desialization of the erythrocytesincreased their agglutinability by the anti-ID, but notby the anti-IF. In other experiments, the details ofwhich are not given here, we found that the agglutina-tion scores of the Abg. antibody were multiplied bya factor of 12 when the cells were treated withVibrio cholera neuraminidase. This increase was notvery significant as the agglutination scores of theFla. and Loi. antibodies were increased in thesame proportion.

(2) Conditions for determining the number of antigensites

The number of sites that combine with the variousantibodies is represented by the maximum number ofspecific IgM molecules fixed per red blood cell.Evans, Turner and Bingham (1965) observed that thetemperature of mixing of antibodies and erythro-cytes influenced the number of IgM molecules whichcould be fixed later at 40 by these erythrocytes. Wehave carried out this experiment again; the anti-body suspensions and red cells were shaken for 10

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C. Doinel, C. Ropars & C. Salmon

( a )

014 1

0

0

\0I I1 X

02 0-6 10 14

0*1 F

006 -

002

(b)

0

0

0-00

,\0.1 0-3 0.5

r (kLg per RBC X 107)

Figure 1. Plots of r/(A) against r according to the Scatchard equation:r/(A) = K (n-r)

where r is the equilibrium concentration of antigen-antibody complex, (A) is the free antibody concentration, K is the equilib-rium constant and n the concentration of antigen-antibody complex when all the antigen is bound to antibody. The extrapolationto the abcissa gives n, expressed here by the maximum number of IgM (Abg) bound per I adult red cell (a) and i adult red cell (b).

20-

O R=0R63 +0 79 AT

0

0

00

2

0-4 8 12 16 20 24

ATiFigure 2. Results plotted according to the Ekins equation:

100

50 [

10

( b )

R=13 03 +2-17 AT /

0~~

II

8 16 24 32 40 48

(/Lg per RBC X 107)

2+ AT+R ( AgT K.AgT) K.AgT

R is the ratio of the free-to-bound antibody concentrations, AgT is the total antigen site and AT the total antibody con-

centration. This equation represent a hyperbola. The slope of the experimental curve approches that of the asymptote repre-sented by the equation:

R= T

AgT K.AgT

The maximum quantity of bound IgM (Abg) per I red cell (a) and i adult red cell (b) is given by the reciprocal of the slope ofthe asymptote.

292

0 8 F

0*6

0 4 _

0 2 _

O

I and i antigens

min at temperatures ranging from 60 to 370, then for1 h at 40, centrifuged and washed at 4°. The quan-tities of antibodies fixed did not depend on theinitial mixing temperature.For high concentrations, the fixation of antibody

was followed by erythrocyte agglutination whichcould cause non-specific trapping in the agglutinatesor prevent the antibodies from reaching the antigensites within the agglutinates. Preliminary formoliza-tion of the red cells prevented these phenomena butlead to some disadvantages which will be discussedlater. Consequently, our experiments were carriedout on non-treated red cells, and the number ofantibodies trapped in the agglutinates was deter-mined by using, as a control, IgM labelled with 1251and unlabelled IgM cold agglutinin simultaneously.Under the above-mentioned conditions for antibodyand erythrocyte concentration, the amount of trap-ped IgM was always less than 0 5 per cent.

(3) Antigen site density measurement (Table 2)

The results achieved by extrapolation from theScatchard curves were nearly the same as thoseobtained by the method of Ekins (Figs 1 and 2).The Fla. and Loi. antibodies did not react with the ired blood-cells. The Fou. and Min. antibodies didnot combine with the I adult red cells.The experiments carried out on normal 0 red

cells from many adult subjects with Fla., Loi. andAbg. IgM made it possible to estimate that theaverage number of I sites was 1-1 ±0 3 x 105 pererythrocyte. The results of Abg. antibody on A andB red cells were about the same.The IgM antibody,Loi., gave a value of 1-1 25 x 105 sites per redcell on some healthy 01 adults, but other examples ofred cells only gave values of 4-5-5 x 104 per red cell.This antibody may react with a determinant of the Iantigen complex which can be found in varyingquantities on the adult red cells.The results achieved with the Min. and Fou.

antibodies showed the heterogeneity of the structuresdetected on the i and cord erythrocytes. The Fou.antibody recognized a structure, present in largerquantity on the cord erythrocytes than on the iadult erythrocytes. In the presence of i and corderythrocytes the cross-reacting Abg. antibody be-haved like the Fou. and Min. antibodies. Solheim(1972) found that the rabbit red cells bound 1-8 x 105molecules of IgM anti-I; our results with the IgMAbg. and anti-i Min. with rabbit cells are shown in

Table 2. Contrary to human cells, in the presence ofrabbit red cells, the Abg. antibody gave a linearScatchard plot.

(4) Equilibrium constants and indices of heterogeneity(Fig. 3)

The equilibrium constant values are given in Table 3.Each antibody had a characteristic equilibriumconstant towards the red cells bearing one antigendeterminant. The equilibrium constant of Loi.towards the I red cells was independent of the num-ber of sites detected, as described above.The determinants of the i adult red cells and those

of the cord red cells could be distinguished by differ-ent association constants with the same antibody.Fou. might be a better fit with the structure or theantigen conformation present on i erythrocytes fromadults, and Min. might fit better the antigen struc-ture present on the cord erythrocytes.The values of the indices of heterogeneity of the

Fla., Loi. and Abg. antibodies showed the highdegree of homogeneity of these IgM and of theantigens with which they reacted. The reactions ofthe i red-cells from adults and cords are character-

+1 F-

0

-1

-10

Figure 3. Re.Karush (1962)

a (30) = 1-0

a (10°)=0.87

-8 24 -7 85

il I «l-8-9 -7

log (Ag)

sults plotted according to the derivation of

log-= alog K+a log (Ag),(A)

where (Ag) is the free antigen concentration, r the equilibriumconcentration of antigen-antibody complex and (A) the freeantibody concentration.The slope of the line gives the heterogeneity index, a. When

log = 0, then log K = -log (Ag). (0) 3 °. (o) 10.(X4)

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C. Doinel, C. Ropars & C. Salmon

Table 2. Density of I and i antigen sites density on red blood cells

Fla. Loi. Abg Fou. Min.

01 adult 120,000-140,000 40,000-55,000* 80,000-120,000 < 1000 0100,000-125,000

Oi adultGui. < 1000 < 1000 35,000 35,000 50,000Tou. n.d.t n.d. 60,000 30,000 70,000

Bi Ro. n.d. n.d. 55,000 60,000O Cord n.d. n.d. 35,000-45,000 45,000-65,000 20,000-30,000Rabbit 150,000 n.d. 0

* Two antigen sites density groups were detected with the Loi. antibody.t n.d. = The number of sites was not determined owing to a very low uptake of antibody onto the cells.

Table 3. Values of equilibrium constants (x 10-8 l.mole- 1) and heterogeneity indices 'a' determined at 30 and 100 for variousantibodies in the I,i system

Fla. Loi. Abg. Fou. Min.

K3 Kjo K3 K10 K3 K10 K3 K10 K3 Kjo

01 adultK 1-5-2 0-7-0-8 0-70-0-85 003-007 4 1-5-2 - - -a (1-08) (0-96) (0-94) (0 68) (1-05) (1-05)

i adultK - - 3-4 0-5-1 3-4 0-1-0-2 0-1-0-4 0-005-0 02a (068) (0-61) (0-61) (0-61) (0-91) (065)

CordK - - 1-2-2-1 0-2-0-5 1-5 0o05 2 0 25)a (0 66) (0 61) (0 64) (0 56) (0-82) (0 82)

Table 4. Standard enthalpy change AH' (kcal.mole-1) and standard entropy change AS' (cal.degree- mole-1) in the I,i blood group system

Fla. Loi. Abg. Fou. Min.

01 adult-AHO 18-25 50-65 10-15-AS0 (30-50) (150-200) (3-15)

i adult-AHO 30-35 75-85 60-65-AS0 (80) (230-260) (185-250)

Cord-AHO 30-35 90 45-50-ASo (75-90) (285) (125-150)

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I and i antigens

ized by lower indices of heterogeneity, which sug-gests several different cross-reacting antigenicstructures.

(5) Thermodynamic constants

The various values of the standard enthalpy changes,AHO, and of the standard entropy changes AS', areplotted in Table 4. The Loi. antibody was clearlydifferent from Abg. and Fla. by its much strongerAH', between -50 and -65 kcal/mole.The temperature effect was greater with adult i and

cord red cells than with I red cells. The cross-reactingantibody Abg. showed this particularity clearly.The standard free energy changes, AF', were cal-

culated from the equation: AF0 = -4 575 T. log K.,the values achieved, ranging from -9 to -11 kcal/mole, may be compared with those obtained byother authors.

(6) Effect of formalin treatment on the reactivity of theIi antigens

Whereas the I sites of the adult red cells were littleaffected by the treatment with formalin (Table 5), theantigens of the i red cells were greatly altered. The

Table 5. Effect of formalin treatment on the reactivity of Iand i antigens with Abg. antibody

Untreated Formalin-treatedRBC RBC

Antigen site density01 adult 105,000-130,000 90,000-115,000Oi adult Gui. 35,000 10,000

Equilibrium constants(x 10-8l.mole-1)at 3 ° and 10 K3 K10 K3 K10

OI adult 3-4 1-1-5 2-5-3 0-5-1Oi adult Gui. 2 5 0-1 0 4 0-015

treatment produced a decrease of the i antigen sitedensity and reduced the affinity constant, even at 40.This observation, with the Abg. antibody, was con-

firmed with the Min. antibody on i adult and cordred cells.

DISCUSSION

The different IgM antibodies which we have purifiedwere monoclonal cold agglutinins according to theusual physicochemical and immunological criteria.In the Karush plot, the two anti-I had heterogeneityindices near to 1 but the Scatchard plot did not give astraight line. With i and cord erythrocytes the re-actions were more heterogeneous since the hetero-geneity indices were much below 1 and the curvaturein the Scatchard plot was more strongly marked. Theheterogeneity did not result from the labelling pro-cedure and was not an effect of the antibodies, since,in the presence of rabbit red cells, the Abg. antibodygave a linear Scatchard plot. The curve observedwith human red cells could be the result either ofsteric factors or of there being several types of I,iantigens on the erythrocyte membrane. In additiontreatment of the red cells with a proteolytic enzyme,such as papain, made the Scatchard curves linearand increased the affinity of the anti-i and Abg.reactions with i and I red cells (Doinel, unpublisheddata).Our results corroborated the heterogeneity of the

I antigen on the red blood cells from adults andshowed at least two I determinants. The Fla. anti-body reacted with a receptor, the density of whichwas about 120,000 sites per red cell. The Loi. anti-body did not react with the same receptors as Fla.and Abg. antibodies: e.g. the 0 erythrocytes (JPC)fixed 90,000 to 100,000 molecules of Abg. or Fla.IgM, but only 55,000 molecules of Loi. IgM. Thedensities of the I Loi. antigen site can thus be dividedinto two groups, according to the adult subjectstested; this determinant has its own associationconstant and is characterized by a high standardenthalpy change (Tables 2, 3 and 4).The results of the antigen density experiments

showed that there were at least two distinct i deter-minants, one present mainly on i red cells, the otheron cord erythrocytes. The Min. and Fou. antibodiesreacted both with these two determinants. The Min.antibody had a stronger affinity for the i componentof cord erythrocytes, whereas the Fou. antibodyreacted better with the i component of adult erythro-cytes (Table 3).The cross-reacting Abg. antibody revealed the

same number of antigen determinants as Fla. on Ired cells; on cord and i red cells, it revealed anantigen structure with a density similar to that of thedeterminants detected by Fou. and Min. The values

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296 C. Doinel, C. Ropars & C. Salmon

of AS' and AH' corresponding to the reaction with iand cord erythrocytes fell between those obtainedwith Fla. antibody on I erythrocytes and thoseobtained with the two anti-i on i adult or corderythrocytes. The Abg. antibody reacted with anantigen determinant common to I, i and cord redblood cells. The reaction of Abg. with I red cells wasdifferent from the reaction with i or cord erythrocytesin affinity constants, thermodynamic constants, andheterogeneity indices (Tables 3 and 4). These resultscould be explained by differences in size of theantigenic site on each cell. Moreover, after formoliza-tion, Abg. reactivity was slighly altered with I redcells and greatly diminished with i red cells. Con-sequently, the Abg. antibody seems to have a widespecificity, reacting strongly with an I site similar tothe I Fla. and more weakly with an antigenstructure on i or cord erythrocytes. This structuremust be a part of the I adult antigen.

Olesen (1966) found 30,000 antigen sites on I redcells with an anti-I antibody (A.J.) and 30,000 sites onI red cells and 70,000-110,000 on adult i or corderythrocyte with an anti-i (M.P.). These results aresomewhat different from those obtained in this work.The anti-i (M.P.) might have been a cross-reactingantibody recognizing an antigen character commonto I, i and cord red blood cells. The anti-I (A.J.)might have reacted with a site similar to our I Loi.Evans et al. (1965) were able to fix 500,000 coldagglutinin IgM per red cell. This particularly highvalue might have been due to the presence ofaggregates among the labelled antibodies. With somepreparations which were purified, labelled andstored at 4° for a few days, we observed an increasein the value of the antigen densities and obtainedsimilar high values. The elimination of the aggregatedIgM, by ultracentrifugation prevented this pheno-menon. From this point of view, the Ekins plot madesit possible to recognize the presence of aggregates(Doinel, unpublished data).

Marcus, Kabat and Rosenfield (1963) have ob-served that the I receptors of A red cells were moresensitive to the action of fi-galactosidase and /3-glucosaminidase from Clostridium tertium than thereceptors of 0 red cells. These authors suggestedthat this phenomenon might be related to a variationin number of I sites depending on the ABO pheno-type. The antigen site densities determined with theAbg. antibody on several Al, A2 and B red cellswere not different from the values obtained with 0erythrocytes. The equilibrium constants and the

thermodynamic constants of these reactions werealso not very different.

ACKNOWLEDGMENTS

We wish to thank Dr J. Brocteur (Liege) for thesample Loi., and Dr De Carbonieres (Paris) forthe sample Min.

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