J. Cell Sci. 10, 525-533 (1972) 525Printed in Great Britain

THE LOCATION OF BLOOD GROUP

ANTIGEN A ON CULTURED RABBIT KIDNEY

CELLS AS REVEALED BY FERRITIN-LABELLED

ANTIBODY

ELIZABETH DIMMOCK,* D. FRANKS ANDAUDREY M. GLAUERTfStrangeways Research Laboratory, Cambridge and Department of Pathology, Universityof Cambridge, England

SUMMARY

Fluorescein- and ferritin-labelled antibodies were used to locate blood group antigen A onthe surfaces of cultured rabbit kidney cells (RK 13). Fluorescent anti-A revealed the antigen onsome cells, but not on others. Controls indicated that specific staining could be obtained.Comparable observations were made with ferritin-labelled antibody, and the comparativelyhigh resolution of this method allowed individual antigen molecules to be located. Measure-ments were made of the distances between the centres of the ferritin molecules and the cellsurface, and the position of the blood group antigen relative to the visible components of themembrane is discussed in relation to this.

INTRODUCTION

Although much is now known about the chemical composition of biologicalmembranes (e.g. see Cook, 1968), there is little information concerning the locationof these components within membranes. The detection of the sites of individualantigens is possible with the ferritin-labelled antibody technique (Singer & McLean,1963) and the aim of the present study was to use this method to locate a specificcomponent, blood group A substance, of the surface membrane of a tissue cell asaccurately as possible.

An epithelioid line of rabbit kidney cells (RK 13) was chosen for examination sincethe distribution of blood group antigen A on these cells had already been studied withthe mixed agglutination technique (Franks & Dawson, 1966; Dawson & Franks, 1967).

MATERIALS AND METHODS

Cells

The characteristics and method of culruring RK 13 cells have been described in an earlierpublication (Dimmock, 1970).

• Present address: Department of Physiology, College of Medicine, University ofSaskatchewan, Saskatoon, Saskatchewan, Canada,

f Sir Halley Stewart Research Fellow.

526 E. Dimmock, D. Franks and A. M. Glauert

Preparation of conjugate of fluorescein isothiocyanate and anti-A globulin

A globulin fraction was separated from a high titre anti-A serum by sodium sulphateprecipitation. The titre of the serum by the anti-globulin test was i: 1024 and of the redissolvedglobulin fraction 1:51200c The protein concentration was 96 mg/ml; 2-1 ml of the globulinsolution was mixed with 3 mg of fluorescein isothiocyanate and stirred at 4 °C for 17 h (Riggset at. 1958). After conjugation, the mixture was titrated to pH 7-4, the globulin was separatedfrom unconjugated fluorescein isothiocyanate on G 25 Sephadex and subsequently fractionatedby stepwise elution from DEAE-cellulose. After concentration and dialysis against phosphate-buffered saline, the fluorescein content of each fraction was measured with a spectrophotometerby absorption at 495 run, and the molar fluorescein: protein ratio was calculated (Lachmann,1964). The anti-A titres and staining abilities of all the fractions were measured, and 2 fractions(numbers 3 and 6) were found to give staining which was bright enough for use in a series ofexperiments to investigate the specificity of the staining reaction.

Preparation of a conjugate of ferritin and anti-A globulin

One millilitre of the globulin fraction of the anti-A serum was conjugated with 1 ml of 7 %ferritin using bis-diazotized benzidine (Williams & Gregory, 1967). After conjugation for 1 hat o °C the conjugate was dialysed against buffer and the unconjugated globulin was separatedfrom the conjugate by centrifugation (Rifkind, Hsu & Morgan, 1964) 3 times at 100000 g for4 h .

Preparation of formalin-fixed rabbit liver hotnogenates

Rabbit tissue preparations for absorption of fluorescein- and ferritin-conjugated anti-Apreparations were made from the livers of A-positive and A-negative rabbits. The A antigen onrabbit tissue cells cross-reacts with the blood group antigen A found in humans. The rabbitswere grouped by a mixed agglutination test on their buccal cells (Hawes & Coombs, 1959).Homogenates of rabbit liver were washed in saline and fixed with 1 % formalin at 4 °C over-night. They were then washed in saline and stored in 20 % glycerol at — 20 °C. The homo-genates were washed again in saline before use.

Fixation of RK 13 cells

It is desirable to fix cells which are to be treated with ferritin-conjugated antibody since thisprecludes the possibility of pinocytosis of the conjugate (Metzger & Smith, 1962), but fixationmay destroy the reactivity of the antigen and consequently preliminary tests are necessary. Itwas found that glutaraldehyde-fixed RK 13 cells gave the expected reactions in a mixedagglutination test with several anti-A sera and no reaction with a control AB serum. It was alsoobserved that glutaraldehyde-fixed red blood cells of blood group A were agglutinated by anti-A but not by anti-B grouping sera.

Methods of staining RK 13 cells with fluorescein- and ferritin-labelled antibodies

Glutaraldehyde-fixed monolayer cultures of RK 13 cells on cover-slips (Dimmock, 1970)were exposed to fluorescein-conjugated anti-A for 10 min and subsequently washed twice. Thepreparations were mounted in buffered glycerol (Mrenova & Albrecht, 1966) and examined witha Reichert Zetopan microscope fitted with an HBO 200 mercury lamp.

The ferritin-conjugated antibody was applied to glutaraldehyde-fixed RK 13 cells for30 min and then the cells were washed 3 times in cold phosphate buffer, fixed in osmiumtetroxide and dehydrated in ethanol. The layer of cells was removed from the coverslip duringdehydration and embedded in Araldite (Dimmock, 1970). Thin sections were stained withuranyl acetate and lead citrate, or with lead citrate only, and examined in an AEI EM 6Belectron microscope.

Location of antigen A on cells in culture 527

RESULTS

The application of fluorescein isothiocyanate-labelled anti-A to RK 13 cells

The application of fraction 3 or fraction 6 of the fluorescein isothiocyanate-labelledanti-A to RK 13 cells resulted in the fluorescent staining of some but not all cells(Fig. 1). Somewhat less than half the cells were stained and the unstained cells werescattered irregularly throughout the preparations.

Table 1. Summary of the results of experiments with fractions 3 and 6 ofthe fluorescein isothiocyanate-anti-A conjugate

Type of test Fraction

ControlBlocking testFalse blocking testAbsorption testAbsorption testBlocking test on absorbed fractionBlocking test on absorbed fractionBlocking test on absorbed fractionBlocking test on absorbed fractionControlBlocking testBlocking testAbsorption testAbsorption test

33333333366666

Fluorescence: + + + + very bright; + + •faint; ( —) almost certainly none; —

Reagent

Blockingagent

NoneAnti-AAB serumNoneNoneAnti-AAnti-AAB serumAB serumNoneAnti-AAB serumNoneNone

+• bright; +definitely none; N.'

Absorbingagent

NoneNoneNoneA — veA +veA — ve

A +veA — veA +veNoneNoneNoneA — veA +ve

ResultsA

' F ' ^Undiluted diluted

fraction i in 2

+ + + + + + +N.T. ( + )N.T. + ++ + ( —)( + )/— (—)——(+)—+ + / + + + + +( + ) / - -+ +++

+ faint; + very faint; ( + ) very, veryr. not tested.

The application of unconjugated anti-A serum to the cells for 30 min before theapplication of fraction 3 of the conjugate resulted in the almost total inhibition ofstaining (Table 1). By contrast serum from an AB person had only a very slight effecton the brightness of the staining.

Fraction 3 of the conjugated antibody was absorbed with rabbit liver homogenatesby incubation 3 successive times for 15, 30 and 60 min respectively. Absorption withblood group A-negative rabbit liver homogenate slightly reduced the brightness of thestaining, while absorption with the A-positive liver homogenate abolished the stainingcompletely or almost completely.

The application of unconjugated anti-A serum before application of the absorbedconjugate fraction resulted in the total inhibition of staining by the conjugate fractionabsorbed with the A-negative liver preparation; and as expected there was no stainingby the conjugate fraction absorbed with the A-positive liver preparation after appli-cation of the unconjugated serum. The substitution of serum from an AB person hadno significant dimming effect on the staining given by the conjugate fraction absorbedwith the A-negative liver homogenate (Table 1).

528 E. Dimmock, D. Franks and A. M. Glauert

The application of ferritin-labelled anti-A to RK 13 cells

The fine structure of RK 13 cells has been described by Dimmock (1970) and is notaffected by the application of ferritin-conjugated anti-A. Ferritin was never identifiedin preparations of cells to which it had not been applied.

When unabsorbed ferritin-labelled anti-A globulin was applied to RK 13 cells,ferritin was found scattered along the surfaces of some but not all cells (Fig. 2). It wasseen along both upper and lower surfaces and between adjacent cells.

RK 13 cells were treated with a mixture of ferritin and unconjugated antibody toinvestigate the possibility that ferritin may stick to the cells even when antibodycannot be responsible for the binding (Cohen, Zuelzer & Evans, i960). Very littleferritin was seen in such preparations; an occasional ferritin molecule was seen on theupper surface of a cell and sometimes on the lower surface. More ferritin was seenin the regions between cells, probably because of non-specific trapping. Afterabsorption of ferritin-labelled anti-A with an A-negative liver preparation theconjugate stained some but not all cells (Fig. 3), and the ferritin was more sparselydistributed than in preparations treated with the unabsorbed conjugate (compareFig. 2). In contrast, there was almost no ferritin present on cells stained with theconjugate absorbed with the A-positive liver homogenate, where the result resembledthat obtained with a mixture of ferritin and antibody.

When anti-A globulin was applied to the RK 13 cells before the application of theferritin-conjugated antibody a total inhibition of ferritin staining was not obtained,but an almost total abolition of staining was obtained by applying the unconjugatedantibody before the conjugate absorbed with the A-negative liver homogenate. Theseresults are consistent with the results obtained with the third fraction of the fluorescentantibody conjugate of the same serum.

It appears, therefore, that the staining with the ferritin-antibody conjugate afterabsorption with an A-negative liver preparation is specific for blood group Asubstance, and that ferritin present on the upper surfaces of the cells in such prepara-tions is conjugated to antibody which is specifically attached to A substance. Thisferritin is irregularly distributed along the upper surfaces of the cells; it is found onthe level parts of the cell surface and on the microvilli but not within the coatedvesicles. The ferritin molecules appear to be distributed singly or in pairs; occasionallythey are in chains (Fig. 3, arrow) or clumps.

Measurements of the perpendicular distance of the centres of ferritin molecules tothe nearest point on a clearly defined membrane surface gave values ranging from 6 to30 nm, with a majority between 8 and 15 nm.

DISCUSSION

The staining of some but not all RK 13 cells with fluorescein-labelled anti-Aindicates the immunological nature of the staining (Franks & Dawson, 1966) and thecontrol experiments show that the reaction between fraction 3 of the fluorescentconjugate, absorbed with A-negative liver homogenate, and RK 13 cells is a specific

Location of antigen A on cells in culture 529

reaction between anti-A and blood group antigen A. The results of the experimentswith ferritin-labelled anti-A are in agreement with those with fluorescein-labelledanti-A, suggesting that ferritin staining with the absorbed conjugate is also specificfor blood group antigen A, and that the ferritin molecules indicate the sites of theantigen on the membranes of RK 13 cells.

The density of staining with ferritin-conjugated anti-A is not very great, indicatingthat blood group A substance is sparsely distributed on the surfaces of RK 13 cells.Some other authors have reported similar densities; for example, in the distribution ofRh0 (D) antigen on human red blood cells (Lee & Feldman, 1964). Various factorssuch as the type of antibody (Haberman, Blanton & Martin, 1967; Lee & Feldman,1964) and the thickness of the sections, which may vary considerably (Williams &Meek, 1966), will affect the apparent density of the ferritin staining. There are reports,however, of variations in the quantity of ferritin present which it seems entirelyreasonable to accept as a genuine indication of antigen distribution. Lee & Feldman(1964) found a considerable difference between the amount of ferritin-anti-A andferritin-anti-Rh0 (D) bound to human red blood cells. Their findings are in agreementwith evidence provided by other methods. Aoki, Hammerling, De Harven, Boyse &Old (1969) found differing densities of ferritin present both in different areas of thesame cell surface and on different cell types in an extensive survey of the distributionof H-2, 6, and thymus leukemia antigens on various mouse cells.

It is possible that the distribution of A antigen and therefore of ferritin along thesurfaces of RK 13 cells might show some variation and association with certain surfacefeatures. Evidence of this was looked for but it appears that the antigen is randomlydistributed and shows no particular association with any identifiable feature such asthe microvilli of the RK 13 cell surface. This is in accordance with the observationthat blood group antigen A is uniformly distributed on the human erythrocytesurface (Lee & Feldman, 1964).

Ferritin-labelled antibodies enable the sites of individual antigen molecules to belocated, the resolution depending on the size of the ferritin-antibody conjugatewhich can be estimated approximately. Ferritin is a spherical molecule with a diameterof 11 nm and it has a central, iron-containing core with a diameter of about 5-5 nm;this core is visible in electron micrographs of thin sections and thus the centre of themolecule can be located. Feinstein & Rowe (1965) estimated that IgG antibody is ina folded configuration with an overall length of 10-5 nm when combined with 1 antigenmolecule, and is extended, with a length of 20 nm, when combined with 2 antigenmolecules, while Green (1969) suggested that the length of the IgG molecule from theantigen-combining site to the tips of the Fc fragment is 12 nm. The sizes of IgMmolecules have been measured by Hoglund & Levin (1965), who give values of20 x 30 nm. More recently Feinstein & Munn (1969) concluded that IgM moleculesare approximately 30 nm long in the extended configuration. Thus the maximumdistance of the antigen from the centre of the ferritin molecule is 10-5 + 5-5 = 16 nm,12 + 5-5 = 17-5 nm or 20 + 5-5 = 25-5 nm with an IgG molecule, and 30 + 5-5 =35-5 nm for an IgM molecule. These are maximum figures, since it is possible thatthe ferritin molecule is attached at some point along the antibody molecule, rather

530 E. Dimmock, D. Franks and A. M. Glauert

than at the farthest point from the combining site with antigen. Furthermore theferritin-antibody conjugate may be inclined at an angle to the beam withinthe section, with a consequent reduction in the apparent distance of the ferritin fromthe antigen. It can therefore be concluded that the antigen lies at a distance of 35-5 nm orless from the centre of the ferritin molecule. If it is known that only IgG is present,then this distance is reduced to 25-5, 17-5 or 16 nm, depending upon the configurationof the antibody.

In the studies of the location of blood group antigen A on RK 13 cells the serumused contained IgG antibody in high titre, but no attempt was made to remove theIgM antibody, so that the antigen may be 35-5 nm from the centre of the ferritin.Measurements from the micrographs indicate that there is a peak in the distributionof ferritin molecules at a distance of 8-15 nm from the membrane. It seems likely thatIgG molecules predominate in this serum, and assuming that the antibody is in theunextended configuration, it can be concluded that the blood group antigen A islocated either within the visible structure of the membrane, which in these micro-graphs appears as a single dark line about 10 nm thick, or in the unstained surfacelayer of the membrane (Dimmock, 1970). Studies with fixation methods that revealmore of the membrane structure and on micrographs at higher magnification will benecessary before it will be possible to localize the antigen more precisely.

We are grateful to Dame Honor Fell, F.R.S., for her continued interest in this work and toMr R. A. Parker for skilled technical assistance. One of us (E.D.) is grateful to the MedicalResearch Council for a Scholarship for Training in Research Methods, and also to Dr L. B.Jaques and his secretarial staff for help with the typescript.

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COHEN, F., ZUELZER, W. W. & EVANS, M. M. (i960). Identification of blood group antigens andminor cell populations by the fluorescent antibody method. Blood 15, 884-900.

COOK, G. M. W. (1968). Chemistry of membranes. Br. med. Bull. 24, 118-123.DAWSON, A. & FRANKS, D. (1967). Factors affecting the expression of blood group antigen A in

cultured cells. Expl Cell Res. 47, 377-385.DIMMOCK, E. (1970). The surface structure of cultured rabbit kidney cells as revealed by

electron microscopy. J. Cell Sci. 7, 719—737.FEINSTEIN, A. & MUNN, E. A. (1969). Conformation of the free and antigen-bound IgM

antibody molecules. Nature, Lond. 224, 1307-1309.FEINSTEIN, A. & ROWE, A. J. (1965). Molecular mechanism of formation of an antigen-antibody

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clonal cultures of rabbit cells. Expl Cell Res. 42, 543-561.GREEN, N. M. (1969). Electron microscopy of the immunoglobulins. Adv. Immxin. n , 1-30.HABERMAN, S., BLANTON, P. & MARTIN, J. (1967). Some observations on the ABO antigen

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(Received 16 April 1971 - Revised 3 September 1971)

532 E. Dimmock, D. Franks and A. M. Glauert

Fig. i. Black and white print of a fluorescence colour photograph of RK 13 cellsstained with fluorescein isothiocyanate-conjugated anti-A globulin at a dilution of 1 in2. Stained cells appear green (light with prominent nuclei, arrow, in black and white).Unstained cells appear purplish (grey in black and white prints). Fixation withphosphate-buffered glutaraldehyde. x 420.

Fig. 2. Electron micrograph of a thin section through the upper surface of an RK 13cell stained with ferritin-conjugated anti-A globulin. Ferritin (small black dots) ispresent along the surface. Some ferritin is present in clumps (arrow). Fixation withphosphate-buffered glutaraldehyde and phosphate-buffered osmium tetroxide.Section stained with uranyl acetate and lead citrate, x 60000.

Fig. 3. The upper surface of an RK 13 cell treated with ferritin-conjugated anti-Aantibody which had been absorbed with A-negative formalinized rabbit liver homo-genate. Ferritin is sparsely scattered along the cell surface. A linear array of ferritinmolecules (arrow) is present. Fixation and staining as for Fig. 2. x 60000.

Location of antigen A on celb in culture 533