Blood, Vol. 56. No. 6 (December), 1980 969

Quantitative Immunoferritin Microscopy of Fya, Fyb, Jka, U, and Dib

Antigen Site Numbers on Human Red Cells

By S. P. Masouredis, E. Sudora, L. Mahan, and E.J. Victoria

The Fy, Fyb. Jk. U. and Dib antigen site numbers and ultrastructural distribution patterns on the human erythrocytemembrane were determined using quantitative immunoferritin microscopy. For homozygous antigen-positive red cells. theaverage number of determinants per red cell was about 1 4,000 for Jk, 1 7,000 for Fy and Fyb, 1 9,000 for Dib, and 23,000

for the U antigen. assuming that the equilibrium binding observed represented 80% saturation of the accessible antigensites. The site numbers for this group of antigens were less than that for the Rh antigens. but considerably more than the

Kell and Cellano antigens. The technique used was capable of demonstrating a twofold difference in antigen densitybetween heterozygous and homozygous Fy (a + ) red cells. More than 85% of the Fy and Fyb antigen sites were lost

following pretreatment of the red cells with papain. consistent with the serologic lability of the Fy antigens followingproteolysis. The ferritin distribution observed following conjugate staining of antibody-sensitized ghost membranes wassimilar for all five antigens studied and showed a random, clustered ferritin pattern. Although the quantitative estimatesare valid, the remarkable similarity in antigen distribution pattern for this diverse group of antigens, as well as otherconsiderations. suggest that the findings with ghost membranes probably do not reflect faithfully the antigen arrangementon the intact red cell membrane.

K NOWLEDGE of the molecular distribution

pattern, as well as the number of blood group

antigenic determinants on the red cell membrane,

provides information useful in analyzing the participa-

tion and role of these receptors in immunologic

phenomena, such as immune hemagglutination,

complement fixation, and antibody-mediated hemoly-

sis. Such information, in addition, may provide clues

for the identification and isolation of antigen-bearing

components from the membrane, and may lead to

insights regarding the molecular organization of the

red cell membrane.

Both radioisotope-labeled antibodies”2 and immu-

noelectron microscopy3’4 have been used to estimate

the average number of blood group receptors on mdi-

vidual red cells. Isotopic techniques have used either

direct labeling of the antibody”2 or an indirect tech-

nique with labeled anti-human IgG,5 whereas immu-

noelectron microscopy has been done with an indirect

technique using ferritin-labeled anti-human IgG #{149}6,7

Antigen densities have been obtained for the ABH,

Rh, Kell, and Cellano antigens using both isotopic and

immunoelectron microscopic techniques.

This report presents receptor site densities obtained

by immunoelectron microscopy for five additional

blood group antigens: Di”, U, Fya, Fyb, and Jk4.

Although the clustered ultrastructural distribution

pattern of these antigens was indistinguishable from

that observed with Rh, Kell, and Cellano antigens, the

number of determinants per red cell was significantly

different.

MATERIALS AND METHODS

Blood Group Antisera

The anti-Dir’, a gift from Dr. Dl. Buchanan, had an antiglobulin(AG) titer of 32-64 against Caucasian DibDib red cells. The anti.Jk’

was obtained from Dr. A.A. Konugres and had an AG titer of

64-128. Three anti-Fy’ sera were used, kindly provided by Drs.

A.A. Konugres, W. Pollack, and W.L. Marsh. They had AG titersof64-l28, 128, and 512, respectively. Two anti�Fyb sera were used,one obtained from Dr. W. Pollack with an AG titer of I 28, and the

other from Dr. P. Sturgeon with a titer of 64. The anti-U was a gift

from Dr. M. Schanfield and had an AG titer of 128. Although the

antisera did not have saline agglutinating activity, no attempt was

made to exclude the presence of minimal amounts of 1gM antibody

except for one anti-Fy’ and one anti�Fyb preparations that were usedas IgG fractions isolated by ammonium sulfate precipitation. The

site density values obtained using whole serum or the IgG fraction

for these antigens did not show a significant difference.

All antisera were monospecific when tested against a commercial

red cell panel. Red cells used for electron microscopy were group 0

and selected from a commercial red cell panel (provided by Ortho

Diagnostics, Raritan, N.J.). A commercial antiglobulin serum was

used for all titrations (provided by Ortho Diagnostics).Because of the time-consuming demands of the technique, it was

difficult to test a large number of either antisera or red cells for each

specificity. Two different antisera against each Fy’ and Fyb, and

only one antiserum each of Dib, Jk’, and U specificity were used.

The number of antigen-positive and -negative pairs of red cellstested for each antigen were three for Fy’, two each for Jk’ and Fy”,

and one each for Dib and U.

125j JgG Anti-D

Five different labeled IgG anti-D eluates derived from two

antiserum donors were prepared as described previously.8 The

anti-D antiglobulin titers of the source sera ranged from 8000 to

16,000, and the eluate titers ranged from 64-128. The lgG anti-D

content of the eluates was estimated using the specific radioactivity

of the 251 lgG fraction used to prepare the eluate. The IgG content

From the Department of Pathology, University of California.

San Diego.

Supported in part by NIH Grants HL-/2994, HL-071 I 9 (SPM)

and HL-23108 (EJV).

Submitted May 5. /980; accepted July 21. 1980.

Address reprint requests to S.P. Masouredis, Department of

Pathology. T-003, University of California, San Diego, La Jolla,

�‘alif 92093.

(c) I 980 by Grune & Stratton, Inc.

0006-497//80/5606--0004$0/.00/0

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970 MASOUREDIS ET AL.

of the IgG fraction was obtained by the Lowry method9 using astandard curve based on crystalline bovine serum albumin (BSA).

Blood Group Antibody Binding to

Red Cells (Sensitization)

Washed red cells of the appropriate blood group phenotype were

adjusted to either a 5% or 10% suspension and incubated for 1 hr at

37#{176}Cwith antibody containing: serum, IgG fraction, or 251 lgG

anti-D eluates. The sensitized red cells were washed four times with

an excess of cold, pH 6.5 BNS (1 volume of 0. 15 M phosphate

buffer plus 9 volume 0.1 5 M NaCI) containing 0.3% BSA.

Red cells sensitized with 251 anti-D were assayed for their 251

content by gamma spectrometry using a well-type thallium acti-vated NaI crystal. The average number of IgG anti-D moleculesbound per cell was estimated from the cell-bound radioactivity, the

specific radioactivity of the 251 IgG anti-D fraction used to preparethe eluate, and the number of red cells in the suspension asdetermined with a Coulter Counter (Model F, Coulter Electronics,Inc., Hialeah, Fla.).

Enzyme modification using crude papain (Mathson, Colemanand Bell, Norwood, Ohio), crystalline proteinase K (E. Merck,Darmstadt, Germany), or Vibrio cholerae neuraminidase (Calbio-

chem-Behring Corp., La Jolla, Calif.) was as described previously.’0

Cells sensitized by either 251 IgG anti-D or by blood groupantisera were stained with the immunoferritin conjugate as

described below.

Preparation ofFerritin-Conjugated Anti-Human IgG

Ferritin conjugated rabbit anti-human IgG was prepared as

described previously” using toluene 2,4 diisocyanate as a coupling

reagent. Rabbit anti-human IgG serum was adsorbed with washedhuman group 0, A, and B red cells and red cell stromata, after

which the IgG was isolated by ammonium sulfate precipitation and

DEAE chromatography. The ferritin conjugate was separated from

unconjugated ferritin and free lgG by chromatography on Bio-Rad

agarose A-Sm, 6% gel (200-400 mesh) using a 0.05 M phosphate

buffer, pH 7.5. The first peak containing highly aggregated materialwas discarded. Four fractions designated [I], [2], [3), and [4] were

selected from the ascending portion of the second peak. These

fractions were free of unconjugated IgG by immunoelectrophoresis,

but contained some free ferritin, predominantly in fraction [4]. Asindicated below, the conjugate contained variable amounts of fern-tin oligomers in all four fractions. Conjugate antiglobulin titers,determined using a D-positive red cell sensitized with a I :5 dilutionofa high-titered anti-D serum, ranged from 128 to 1024.

Blood group antibody IgG coated red cells were converted toghosts by hypotonic lysis at an air-water inferface, and picked up

from above on carbon-strengthened collodion-coated electron

microscopy grids by touching the grids to the ghost-bearing water

surface.’ The grids were conditioned with a solution of 5% BSA andstained by adding a drop of the conjugate to the grid. After 4-S mm

of incubation at room temperature, the grids were washed withbuffer, air-dried, and examined in a Zeiss Model 9S electronmicroscope (Carl Zeiss, Inc., N. Y.).

Scoring ofElectron Micrographs and Estimation of

Cell Bound IgG

Electron micrographs (EM) of the red cell membranes wereobtained at 20,000 x magnification and the negative printed at

about 3-4 times more magnification. The EM prints were scored by

visually counting the number of fernitin grains per �tm2 surface area

as determined by using a calibration grating replica photographed

at the same magnification.

The number of fernitin grains per cell bound IgG was determinedusing 251 anti-D IgG sensitized red cells stained with each conju-

gate. The quantity ofcell bound IgG anti-D was estimated from thecell bound radioactivity and correlated with the fennitin grain

counts. Values obtained ranged from 2 to 6.8 grains pen IgG for the

six different conjugates used in these studies. The higher values

probably represent conjugates containing fernitin oligomers.

The number of fernitin grains per membrane-bound, antiserum-derived IgG was assumed to be the same as that found with the

membrane-bound 251 anti-D IgG. A value of 145 �.tm2 was used for

the total surface area’ in calculating the average number of cell-

bouf,d IgG molecules per red cell.All studies were controlled by using an antigen negative red cell

treated with the antiserum and stained with the conjugate.

RESULTS

Conditions Affecting Conjugate Staining of I25� Anti-

D JgG Sensitized Red Cells

Table 1 shows the effect of conjugate staining time

(study A) and conjugate dilution (study B) on the

yield of ferritin grains per cell bound anti-D IgG.

There was no significant difference in the quantity of

ferritin grains on the anti-D IgG sensitized

membranes as the staining time was progressively

increased from 4 to 10 mm.7 Saturation of cell bound

IgG was achieved at 4 mm with this conjugate prepa-

ration that had an antiglobulin titer of 80. The quan-

tity of ferritin grains per cell bound IgG progressively

decreased from 6.7 to I .5 as the conjugate was diluted

(study B). As a result of these findings, red cells

sensitized with IgG blood group antisera were

routinely stained 4-5 mm using conjugate prepara-

tions with antiglobulin titers of 160 or greater.

Table 2 shows that similar grain counts were

obtained when a D-positive red cell was sensitized with

an 1251 anti-D IgG containing eluate, an unlabeled IgG

Table 1 . Relationship of Rabbit Anti-Human lgG Ferritin

Conjugate Dilution and Staining Time on the Number of Ferritin

Grains per Cell-Bound � Anti-D lgG

No.

Study

Stainin9

Time(mm)

ConjugateDilution

Anti-D

lgGper sm2

Ferritin

Grains

per �zm2

Ferritin

Grains

per lgG

A 4

68

10

Undiluted

Undiluted

Undiluted

Undiluted

50

50

50

50

367 ± 60401 ± 50397 ± 101

384 ± 69

7.38.07.9

8.4B 4

44

44

Undiluted

1:21:4

1:81:16

52t

52t52t

52t52f

346 ± 37

250±43158±40

114±2980±29

6.7

4.83.0

2.21.5

Conjugate preparation J-2[1] with an antiglobulin titer of 80 used in

both studies.#{149}0.1 ml of a 10% R ,r red cell suspension bound 0.2 13 �g IgG

following incubation with 1 .47 zg of 1251 anti-D IgG.

tO.1 ml of a 10% R,r red cell suspension bound 0.236 �g IgGfollowing incubation with 0.74 zg of 1251 anti-D IgG.

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ANTIGEN SITE NUMBERS 971

Table 2. Fernitin Grain Densities on Red Cells Sensitized With

Serum, lgG and Eluate �l Anti-D lgG

Rabbit Anti-Human Rabbit Anti-Human

IgG Ferritin IgG Ferritin

No.Conjugate [lit Conjugate [3it

Anti-D Ferritin Ferritin Ferritin Ferritin

Red Cell IgG Grains Grains Grains Grains

Sensitization’ per �tm2 per �tm2 per IgG per �tm2 per IgG

R,r + 1251

anti-Deluate 61� 417 ± 29 6.8 324 ± 23 5.3

R,r + lgG

anti-D - 394 ± 137 - 356 ± 42 -

R,r + serum

anti-D - 398±77 - 418±107 -

rr + serum

anti-D - 19 ± 12 - 47 ± 12 -

The anti-D antiglobulin titers using an R 1r red cell suspension were

8000 for the serum anti-D, 4000 for the IgG anti-D and 64 for the 1251

anti-D lgG eluate.

tThe chromatographic fractions [ 1 ] and [3] of conjugate preparationK-2 were used. Both had an anti-globulin titer of 80.

�O.1 ml of a 10% R,r red cell suspension bound 0.322 �sg lgG

following incubation with 1 .47 zg of 1251 anti-D IgG.

anti-D fraction, or the original anti-D serum. Similar

degrees of conjugate staining with anti-D preparations

that differed in titer from 64 to 8000 indicate satura-

tion of D sites was achieved with titers in excess of 64.

These results provide justification for using the ratio of

ferritin grains per cell bound IgG obtained with 251

anti-D IgG sensitized red cells to calculate the cell

bound IgG with the various unknown blood group

antisera.

Table 3 shows the ferritin grain density following

sensitization of antigen-positive and -negative red cells

with five different blood group antisera (Dib, j�a u,Fya and Fyb). The ferritin grain densities have been

converted to molecules of cell bound IgG for each

conjugate preparation using the experimentally deter-

mined ratio of ferritin grains to cell bound IgG

obtained with 1251 anti-D IgG coated red cells. This

value varied from 2 to 4.8, probably reflecting differ-

ences in the content of ferritin oligomers in the

different conjugates.

The antigen-negative control cells contain less than

15% of the ferritin grains on antigen-positive cells

except for one experiment in which the Fy(a -b + ) cell

had 29% of the grains found on the heterozygous

Fy(a+b+) cell, conjugate 1-3.

The variability of the ferritin grain counts per ,um2

on antigen-positive cells as reflected in the standard

deviation ranged from a low of 12% to a high of 29%.

This variation was due in large part to the presence of

irregularly distributed ferritin clusters as shown in the

electron micrographs in Figs. I , 2, and 3.

The number of antigen receptors per red cell at

equilibrium was obtained on the assumption that the

antibody IgG binds univalently to the receptor and

that the red cell surface area is 145 .tm2. The equilib-

rium site values for homozygous red cells for the five

blood group antigens ranged from I 3,000-i 8,000. The

heterozygous Fy(a + b + ) red cell had an equilibrium

value of 6900 Fy5 sites, as compared to 1 3,300 for a

homozygous Fy(a + b - ) red cell.

Figures 1 and 2 show representative areas of the

electron micrographs scored for ferritin grains

presented in Table 3. The antigen density patterns

found with all five blood group antibodies was similar,

showing a random distribution of ferritin with areas of

ferritin clustering.

Table 4 presents the effects on conjugate staining

produced by pretreatment of red cells with enzymes

before antibody binding. Antibody sensitization of

protease-treated Fy(a+) and Fy(b+) red cells

resulted in a marked reduction in conjugate staining,

Tabl e 3. Ferritin Grain Dens ities and Red Cell Rece ptor Sites at Equili bnium for Five Blood Gr oup Antigens

Rabbit

Anti-Human

Probable IgG Ferritin Ferritin Antigen Sites

Red Cell Ferritin Grains’ Grainst per Red Cell

Antiserum Genotypes Conjugate per IgG per .tm2 at Equilibrium

Di” DibDib

Dj’Di’

J-3[2]

J-3[2]

4.8

4.8

510 ± 80

7 ± 6

15,400 ± 2,400

220 ± 190

it. Jc’Jk’ L-3[3]

L-3[3]

4.7

4.7

366 ± 101

37 ± 12

11,300 ± 3,100

1,100 ± 370

U U pos

U neg

Fy’Fy’

C-3[3]

C-3[3]

1-3(3]

2.2

2.2

3.8

274 ± 79

16 ± 9

180 ± 16

18,100 ± 5,200

1,100 ± 610

6.900 ± 610

Fy’ FybFyb

Fy’Fy’

Fy’Fy’

I-3[3]

L-3[3]

L-3[3]

3.8

4.7

4.7

52 ± 6

432 ± 114

25 ± 6

2,000 ± 24013,300 ± 3,500

770 ± 190

Fy” FybFyb

Fy’Fy’

X-2[3]

X-2[3]2.0

2.0189 ± 55

19 ± 13

13,700 ± 4,000

1,400 ± 940

Estimated for each conjugate preparation using 1251 anti-D lgG sensitized red cells.

tMean ± SD.

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Fig. 1 . Electron micrographs of antibody-sensitized red cells stained with ferritin rabbit anti-human lgG. (A) Fy(a + b - ) red cellsensitized with lgG anti-Fy. (B) Fy(a - b + ) red cell incubated with lgG anti-Fy’ (antigen-negative control). (C) Fy(a - b + ) red cell sensitizedwith lgG anti-Fy’. (D) Fy(a + b - ) red cell incubated with lgG anti�Fyb (antigen-negative control). Conjugate preparation X-2[3J with anaverage of two ferritin grains per cell-bound lgG. Marker bar 0.2 �m.

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Fig. 2. Electron micrographs of antibody-sensitized red cells stained with ferritin rabbit anti-human lgG. (A) Jk(a + b - ) red cell

sensitized with anti-Jk’, conjugate preparation E-3[3]. (B) U-positive red cell sensitized with anti-U, conjugate preparation B-3[3J. (C)Di(a - b + ) red cell sensitized with anti�Dib, conjugate preparation 1-3(21; (D) R1r red cell sensitized with 1251 anti-D, conjugate preparationJ-3[21. The number of ferritin grains per cell-bound lgG were 4.7 for E-3[3], 4.4 for B-3[3]. and 4.8 for J-3[2]. Marker bar 0.2 �m.

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974 MASOUREDIS ET AL.

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Table 4. Enzyme Red Cell Modification and Ferritin Grain Densities

Rabbit

Anti-Human

IgGProbable Ferritin Ferritin Ferritin Antigen Sites

Red Cell Conjugate Gra,nst Grans* re Red CellAntiserum Genotypes Treatment Preparation per IgG per �m2 at Equilibrium

Fy FV’FV’ Untreated

Proteinase K

Papain

1-3(3]

I-3[3]

I-3[3]

3.8

3.8

3.8

1 80 ± 1 6

26 ± 10

12 ± 5

6,900 ± 6 10

990 ± 380

460 ± 180

Fyb FV”FV’ Untreated

Papain

B-3[3]

B-3[3]

4.4

4.4

387 ± 47

32 ± 27

12.800 ± 1.500

1,050 ± 900

U U pos Untreated

Papain

C-3[3]

C-3[3]

2.2

2.2

274 ± 79

497 ± 1 14

18.100 ± 5,200

32,000 ± 5,700

Dib OpOib Untreated

ProteinaseK

Neuraminidase

J-3[2J

J-3[2]

J-3[2]

4.8

4.84.8

510 ± 80

245 ± 37

438 ± 62

15,400 ± 2,400

7,400 ± 1,100

1 3,200 ± 1.400

A 1 : 1 0 dilution of a 1 % crude papain, a 0.2% solution of proteinase K and 1 00 U of Vibrio cholerae neuraminidase solution were used to modify the

red cells as described previously,hle

tEstimated for each conjugate preparation using 1251 anti-D IgG sensitized red cells.

*Mean ± SD.

ANTIGEN SITE NUMBERS 975

with ferritin grain densities that were less than 10% of

those found on untreated cells. In contrast, protease

treatment increased by 26% the ferritin grain density

on U(+) cells. Neuraminidase treatment of Di(b+)

cells did not affect the ferritin grain density, but

proteinase K treatment reduced antibody binding by

about 50%. Electron micrographs of anti�Fyb sensi-

tized Fy(a - b + ) red cells before and after protease

treatment are shown in Fig. 3. Similar results were

obtained with anti�Fya following enzyme modification

(Table 4).

DISCUSSION

Estimates were obtained, for the first time, of the

red cell antigen densities for the Duffy, Kidd, Diego,

and U blood group systems. The equilibrium values for

homozygous red cells ranged from I 3,000-I 8,000 sites

per cell as determined by immunoelectron microscopy.

These estimates were based on the assumption that the

number of ferritin grains per cell bound IgG obtained

using 1251 IgG eluate anti-D can be used to convert

ferritin grains to cell bound IgG when cells are sensi-

tized with whole serum containing IgG antibody.

Support for this assumption was obtained by showing

that under saturating conditions, the quantity of fern-

tin grains on the membrane was similar regardless of

whether the sensitizing reagent was labeled anti-D in

an eluate, unlabeled anti-D in an IgG fraction, or

anti-D containing serum (Table 2). The overall neli-

ability of the technique was supported by both the

demonstration of a dosage effect and by the appropni-

ate changes in fernitin density following enzymatic

modification of red cells. Specifically, a homozygous

Fy(a+) red cell at equilibrium had 13,300 sites as

compared to 6900 sites on a heterozygous cell, and

protease pretreatment of Duffy red cells, as expected,

resulted in a loss of more than 85% of the antigen sites

(Table 4).

The reliability of the values obtained in this study

should be assessed with an awareness of the technical

difficulties of quantitating immunoelectron micros-

copy data. Many of the problems associated with this

method have been reviewed.7 Overestimation of the

number of antigenic determinants can result from

unconjugated fernitin in the conjugate preparation,

from nonimmunologic binding of the conjugate, and

from the presence of conjugates containing aggregated

fernitin. The more than threefold variation in number

of fernitin grains per cell bound 1251 anti-D IgG (2-6.8)

indicates that the conjugates used contained fernitin

oligomens. The effect of fernitin aggregates was mini-

mized by using the experimentally determined ratio of

fernitin grains to cell bound 251 anti-D IgG in

analyzing the fernitin grain densities on red cells

sensitized with unlabeled blood group antisera. The

contribution of nonimmunologic binding of either free

fernitin or the fernitin conjugate was controlled by the

use of antigen-negative cells which, in most cases, had

less than 10% of the fenritin grains found on antigen-

positive cells.

Underestimation of site densities could result from

the use of low-titered sera that fail to saturate the red

cell determinants, the presence of IgM antibodies in

the sera that would block determinants available to the

stainable IgG, and the presence of conjugates contain-

ing apofernitin that would not be visualized. In addi-

tion to these systematic sources of error, there are

other problems associated with the method. These

include the presence of variable numbers of poor

quality membranes due to the relatively uncontrolla-

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976 MASOUREDIS ET AL.

ble hypotonic lysis step, observer bias in photograph-

ing only the better quality membranes, and observer

fatigue in visually counting fernitin grains. In view of

these limitations, the values reported could vary by as

much as 20%-30%.

Another reservation is that the data are based on

only I or 2 antisera and only 1-3 different red cell

donors. Unfortunately, this was necessitated by the

overwhelming time and resource demands of the tech-

nique used. It is conceivable that the site numbers for

some of these antigens may not be representative of

the values found when larger numbers of red cells and

antisera are tested.

The total number of receptors for the blood group

antigens range from 14,100-22,600 if the equilibrium

values obtained in this study with homozygous cells

represent about 80% saturation of the available sites.

ika appears to be at the lower range, and U and Dib at

the upper range. This range for the Duffy, Kidd, U,

and Diego antigens differs significantly from the anti-

gen site densities reported for the ABH, Rh, and Kell

and Cellano antigens. The Kell antigen, by both

isotopic’2 and electron microscopy’3 and the Cellano

by electron microscopy’3 have relatively low site densi-

ties of about 6000 sites per cell. At the other extreme,

there are about I 06 A, sites per cell as measured by

either isotopic techniques’4 or by electron microscopy

with fernitin conjugated Dolichos biflorus lectin.’5 A

sites on A2 red cells consistent with their serologic

behavior were significantly reduced in number, I .5-

2.5 x 105,15.16 whereas H sites varied from 3 x IO� on

group 0 cells to I x lO� on group A,B cells.’7 Rh

antigen site values for c, C, D, e, and E on homozygous

cells by immunoelectron microscopy ranged from 2.5-

3.9 x lO�� and were in the same range by direct

isotopic techniques,’8 except for c, which had a site

value of 7.0-8.5 x 104,18 and C with a value of 4.2-

5.6 x lO�.’� Two conclusions can be drawn from this

brief review of blood group antigen site values. One is

that the density of antigenic determinants for different

blood group systems segregate into distinct classes on

the basis of their site numbers. Secondly, there is

remarkably good agreement between the estimates

obtained by either isotopic or immunoelectron micro-

scopic techniques. It should be evident, however, that

both techniques measure only antibody-accessible

determinants on the intact red cell and may not reflect

the total antigen content of isolated membrane compo-

nents.

The wide disparity in antigen site values between

ABH (106), Rh (4 x IO�), Fy, Jk, U, and Diego (2 x

lOg) and Kell-Cellano (5 x lOs) suggest that these

antigens are associated with different membrane

components. About 80% of the membrane protein

mass is resolved into about 9 or 1 0 principal polypep-

tides by sodium dodecyl sulfate polyacrylamide gel

electrophoresis (SDS-PAGE).2#{176} Only one major poly-

peptide, component 3, and three or more glycoproteins

are found on the outer surface of the red cellmembranes. The number of these polypeptides range

from 2.5 x I O� to I 06 copies per cell. Except for the

ABH system, which may be fortuitous since these

antigens are believed by some to reside predominantly

on glycolipids,2’ the estimated number of accessible

blood group antigens do not correlate directly with the

number of membrane polypeptides defined by SDS-

PAGE. It appears that these antigens may reside on

polypeptides present in tOO low a concentration to be

identified by protein stains or that the bands resolved

by SDS-PAGE contain more than one polypeptide,

each having in common the same molecular weight.

Only one of the antigens studied has been associated

with a membrane component. The U antigen has been

assigned to the carbohydrate-free portion of the

membrane sialoglycoprotein PAS-3.22 The increase in

U antigen sites following protease treatment is consis-

tent with this assignment. The association of U anti-

gen activity with a protein determinant is reinforced,

although not proved, by the similarity in the response

of U and Rh antigens to protease treatment.

Uncommon blood group antigens were intentionally

selected for this study with the thought that they

might reveal an ultrastructural distribution pattern

that would differ from the random, clustered ferritin

pattern found with Rh antigens.7 The pattern observed

with all antigens studied, in spite of the wide range in

antigen site densities (5 x lO� to 4 x lOg) is indistin-

guishable from that observed with the Rh antigens. As

discussed previously,7 the use of ghost membranes for

immunoconjugate staining probably does not reflect

the antigen distribution pattern on the intact

membrane. Disruption of the membrane cytoskeleton

during hypotonic lysis leads to altered antigen mobili-

ty, which facilitates ligand-induced antigen clustering.

The antigen clustering found with Rh, Kell, Cellano,

and the four antigens in this study suggest that they all

have in common a membrane arrangement that is

disrupted by hypotonic lysis of the intact red cell that

results in increased mobility of the antigen-bearing

membrane component.

ACKNOWLEDGMENT

The authors are grateful to the San Diego Blood Bank forgenerous supplies of normal donor blood, to Dr. J . M . Bowman of the

Canadian Red Cross at Winnepeg for high-titered anti-D sera, and

to Joan DeChernie for confirmation of the specificity of the antiseraused.

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ANTIGEN SITE NUMBERS 977

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1980 56: 969-977  

SP Masouredis, E Sudora, L Mahan and EJ Victoria antigen site numbers on human red cellsQuantitative immunoferritin microscopy of Fya, Fyb, Jka, U, and Dib 

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