Yrac. Nutl. Acad. Sci. USA
Vol. 76, No. 10, pp. 4913-4917, October 1979
Biochemistry

Monoclonal antibody to a plasma membrane antigen of neurons

(lymphocyte hybridoma cells/ganglioside/retina/brain)

GEORGE S. EISENBARTH*, FRANK S. WALSH+, AND MARSHALL NIRENBERG

/
I    Laboratory of Biochemical Genetics, National Heart, Lung and Dlood Institute, National Institutes of Health, I$&hes&, Maryland 20205
   Contributed by Marshall Warren Nirenberg, July 2,1979

ABSTRACT  Fusion of spleen cells from a mouse immunized
with chicken embryo retina cells with clonal mouse myeloma
cells yielded a lymphocyte hybrid ceil line that produced anti-
body that bound to neural tissue such as retina, brain, spinal
cord, and dorsal root ganglia but not to other tissues tested. The
antigen was shown by indirect immunofluorescence to be as-
sociated with plasma membranes of most, or all, neuron cell
bodies in chicken retina, hut little or no antigen was detected
on axons or dendrites, Miiller cells, or retina pigment cells. The
activity of antigen AZB5 is relatively stable at lOO"C, is insen-
sitive to trypsin, exhibits the solubility properties of a ganglio-
side, and is destroyed by neuraminidase. Antibody A2B5 cyto-
toxicity against retina cells is inhibited by a CQ ganglioside
fraction from bovine brain (estimated half-maximal inhibition
at 0.2 PM) or by N-acetylneuraminic acid (half-maximal inhi-
bition at 5000 PM) but not by other purified gangliosides tested.
These results suggest that the antigen is a complex ganglioside
in plasma membranes of retina neuron cell bodies but not axons
or dendrites.

Chicken embryo retina cells have been used to study biological
processes that require interactions between neurons, such as cell
aggregation (1, 2), adhesiveness (3), and synapse formation
(4-7). To define surface molecules of retina neurons, we have
used the technique, introduced by Milstein and coworkers
(8-lo), of antibody production by hybrid cells formed by fusion
of mouse myeloma cells with spleen cells from mice immunized
with chicken embryo retina cells. This approach is useful be-
cause lymphocyte hybridomas can synthesize large quantities
of monoclonal antibody with specificity for a single antigen
determinant. Hybridoma cell lines have been reported which
synthesize antibodies specific for cell surface antigens as diverse
as the Forsmann antigen (ll), HLA determinants (12),
tumor-specific antigens (13), and an antigen detected on human
neuroblastoma cells and fetal brain (14) (also see ref. 15).

Other techniques also have been used to produce antisera
against nervous system antigens. These include xenogenic im-
munization followed by extensive absorption with non-neuronal
tissues (16, 17) and immunization with neural cell lines (18-ZO),
partially purified synaptosomes, plasma membranes, and
protein fractions (21. 22).

In this report we describe the characterization of a mono-
specific antibody synthesized by lymphocyte hybrid A2B5 cells
that recognizes a surface antigen restricted to cell bodies of
most, or all, retina neurons.

METHODS AND MATERIALS

Immunization of Mice and Production of Hybrid Cells.
Female BALB/c mice were immunized by intraperitoneal
injections at zero time and 7 days with 1 X lo7 S-day-chicken
embryo retina cells [dissociated with 0.2 mM sodium ethylene
glycol bis(/l-aminoethyl ether)-N,N,N',N'-tetraacetate and

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this fact.

fixed with 0.1% glutaraldehyde in Dulbecco's phosphate-buf-
fered saline without Ca2+ or Mg2+ (Pi/NaCI) for 5 mini, sus-
pended in 0.25 ml of complete Freund's adjuvant, followed at
37 days by intravenous and intraperitoneal injections of 5 X 106:
cells (each) without adjuvant. On day 41, two spleens were re-
moved, the cells were dissociated mechanically, and 2 X 10'
spleen cells were fused with 2 X lo7 P3X63 Ag8 mouse mye-
loma cells (8) [obtained from John Minna] in the presence of
0.8 ml of 50% polyethylene glycol 1000 (Baker) according to
the methods of Galfre et al. (23). After fusion, cells were sus-
pended in medium A [Dulbecco's modification of Eagle's
minimal essential medium containing 100 GM hypoxanthine,
1 /.LM aminopterin, 16 PM thymidine, and 20% fetal bovine
serum (heat inactivated at 56oC for 30 min)] and inoculated
into 92 wells (2.2 X 10' cells per ml of medium per well per
2-cm2 surface area) of Costar 3524 multiwell plates. A2B5 hy-
brid cells (also termed F12A2B5) also were grown as ascites
tumors in BALB/c mice that had been injected intraperitone-
ally with 0.5 ml of pristane (Aldrich) 7 days prior to injection
of l-10 X 10" cells.

Cytotoxicity and Absorption Assays. Synthesis of anti-retina
antibodies by hybridoma A2B5 cells was measured by com-
plement-dependent cytoxicity. Retinas obtained from White
Leghorn chicken &day embryos (Truslow Farms, Chestertown,
MD) were dissociated by incubation with 0.5% trypsin (crys-
tallized 3 times, Worthington) for 5 min and washed by cen-
trifugation. Approximately 1.2-1.6 X l@ cells were suspended
in 4 ml of Eagle's minimal essential medium supplemented with
10% fetal bovine serum containing 400 PCi of NaslCrOd (4.8
PM; 1.5 X lo7 becquerels) incubated for 40 min at 37oC in an
atmosphere of 95% sir/5% Con. Cells were washed twice with
Pi/NaCl, incubated at 4oC for 30 min in Pi/NaCl, washed three
times with Pi/NaCl, and then suspended in P,/NaCI. 51Cr-
Labeled retina cells (1 X 1O'j cells, approximately2000-10,000
cpm) were incubated in a mixture of 95 ~1 of Pi/NaCl, 5 ~1 of
guinea pig serum as a source of complement, and 50 ~1 of cul-
ture medium or diluted antibody in microtiter U plates (catalog
no. 76-205-042, Flow General, Hamden, CT). The cell sus-
pension was incubated for 30 min at 37oC; cells then were
sedimented at 1000 X g; the supematant solution was harvested
by using Titertek harvesting filters (Flow General, catalog no.
78-210-05) and the radioactivity in the supernatant solutions
that was absorbed by the filters was determined. Saturating
concentrations of antibody A2B5 in the presence of complement
released 50-60% of radioactive material from cells compared
to that released by 0.3% Triton X-100. In the absence of com-
plement, <5% of the radioactive material was released.

Abbreviation: Pi/NaCI, Dulbecco's phosphate-buffered saline without
CaZC or Mg"+.
* Present address: Box 3021, Department of Medicine, Duke Univer-
sitv. Durham, NC 27710.
+ Present address: Muscular Dystrophy Laboratories, Institute of
Neurology, National Hospital, Queen Square, London WC1 N 3BC,
England.

4913


Cells used for absorption of antibody were prepared by
mincing the embryonic tissues into I-mm pieces, triturating
the suspension with a lo-ml pipette, and forcing the cells
through a Nitex 130 nylon filter. Various concentrations of cells
suspended in 100 ~1 of Pi/NaCl were incubated with 0.4 pg of
antibody [antibody concentration determined by assay with
1251-labeled protein A (24)] for 30 min at room temperature.
Cells were removed by centrifugation at 8000 X g for 2 min,
and 50 ~1 of the supernatant solution (absorbed antibody) was
added to each well to assay cytotoxicity against 8day-embryo
retina cells.

Antigen Characterization. White Leghorn chicken eggs
fertilized 8 days previously were obtained from Truslow Farms.
Ten 8-day-embryo retinas and three 8day-embryo brains were
homogenized separately in 3.3 ml of methanol/chloroform/
water, 2:1:0.3, (vol/vol), in a Potter-Elvehjem homogenizer.
The homogenate was centrifuged at 1000 X g for 20 min at
4oC the supernatant fraction was removed, and the pellet was
extracted with 3.8 ml of methanol/chloroform/water, 2:l:O.g
(vol/vol). After centrifugation (1000 X g for 20 min at 4oC).
the supernatant fractions were combined (fraction A). Two
milliliters of chloroform and 2 ml of Hz0 were added to the
pooled supernatant fraction, the mixture was centrifuged at
1000 X g for 20 min, and the lower chloroform phase (fraction
B) and upper methanol/Ha0 phase were separated. Methanol
was removed from the upper phase by flash evaporation
(fraction C), and the residue was tested for ability to inhibit
antibody A2B5-dependent cytotoxicity. In other experiments,
the upper phase was dialyzed overnight at 4oC against 50 vol
of Hz0 (changed three times) and lyophilized, and the residue
was dissolved in chloroform/methanol/HzO, 10:5:1 (vol/vol)
(fraction D). Solvents were removed from fractions by evapo-
ration under a stream of nitrogen or air. The residues were
dissolved in Ha0 and assayed for inhibition of cytotoxicity.

GMr, GDr, and CT1 were obtained from Supelco; GTr and
GQ fractions were kindly provided by Peter Fishman (Labo-
ratory of Neurochemistry, National Institutes of Health). The
GQ fraction purified by DEAE column chromatography and
silicic acid column chromatography (25) was approximately
IOOO-fold enriched in GQ compared to unfractioned bovine
brain gangliosides.

Indirect Immunofluorescence. Chicken neural retina was
fixed with 3% formaldehyde in Pi/NaCl for 30 min, washed
with PJNaCl, immersed in liquid nitrogen, and then sectioned
(16 pm). Sections were incubated with 50-100 ~1 of A2B5 as-
cites fluid diluted 1:190 (2-4 pg of antibody) for 45 min at room
temperature and then washed with PJNaCl for 45 min. For
immunofluorescence of monolayer cultures, cells were disso-
ciated from 8-day-embryo retina (0.5% trypsin for 5 min at
37oC) and cultured for 24 hr in 35-mm petri dishes (1 X 106
cells per dish) in 90% Eagle's minimal essential medium
(GIBCO)/lO% fetal bovine serum. The cells were fixed as de-
scribed above, washed, and incubated with 4 pg of antibody
in 109 ~1 of Pr/NaCl for 45 min. Cultured retina cells or sections
were incubated with fluorescein-labeled rabbit antibody di-
rected against mouse IgG for 30 min at room temperature and
then were washed with PJNaCI.

RESULTS

Spleen cells from a mouse immunized with chicken retina cells
from 8-day embryos and P3X63 Ag8 myeloma cells which lack
hypoxanthine phosphoribosyltransferase activity (EC 2.4.2.8)
(8) were fused in the presence of polyethylene glycol. Two
weeks after fusion, colonies were present in all wells. Part of the
medium was removed and assayed (i) for antibody directed
against retina cells by assaying binding of 12SI-labeled protein

A to 8-day embryo retina cells with bound immunoglobulin (26)
and (ii) for complement-dependent cytotoxicity by using 8-
day-embryo retina cells labeled with 51Cr as targets. Media
harvested from 45 of the 92 wells assayed contained antibodies
that bound to retina cells and to protein A. Cytotoxic antibodies
were detected in three wells and the properties of one, A2B5,
is described in this communication. Antibody was obtained by
harvesting the medium from cultured cells or ascites fluid from
BALB/c mice with A2B5 ascites tumors. The ascites fluid was
cytotoxic, at a dilution of 125,000, to 8day-embryo retina cells
and was used for most of the studies described. A2B5 cells were
cloned six times by dilution (0.8 cell/well) in multiwell plates.
After 2 weeks of incubation for each cloning cycle, approxi-
mately 30% of the wells contained colonies. Initially, both
positive and negative sublines of A2B5 were obtained with re-
spect to the synthesis of antibody directed against chick retina;
however, cycle 5 of cloning resulted in a subline (A2B5 clone
105) that, when cloned again, gave rise only to positive colonies.
Ascites fluid from mice bearing subline A2B5 clone 105 was
cytotoxic, at a dilution of 1:300,000, to 8-day-embryo retina
cells.

Different tissues were assayed for antigen A2B5 by incu-
bating the antibody with the cells and, after absorption, de-
termining the remaining cytotoxic activity against 8-day-
embryo retina cells. The cytotoxic activity of antibody A2B5
was removed by 8- and 16day-embryo retina or brain cells but
not by muscle, heart, kidney, or liver cells or erythrocytes (Fig.
1). The antigen also was detected in human and bovine brain
and chicken dorsal root ganglion (data not shown).

The neural retina of the chicken 16-day embryo consists of
three layers of cell bodies separated by two layers of axons and
dendrites connected by synapses. The distribution of antigen
A2B5 in sections of 16- and 8-day-embryo retina and adult

NUMBER OF CELLS USED FOR ABSORPTION

FIG. 1.  Residual cytotoxicity of antibody A2B5 after absorption
with different concentrations of cells from 8- or 16.day-embryo
tissues: o , &day, brain; 0,16-day, brain: * , 8-day. retina; O , 16.day.
retina; A. &day, liver; A, l&day, liver; A', 16-day, muscle: 8, l&day.
heart; m, 16.day, kidney: e, l&day, erythrocytes. Residual cytotox-
icity = (cpm released due to antibody A2B5 after absorption/cpm
released due to unabsorbed antibody AZB5) X 100. One hundred
percent residual cytotoxicity corresponds to the release of 785 cpm
from &day-embryo cells labeled with 51Cr, in response to 0.4 fig 01
antibody A2B5.


Biochemistry: Eisenbarth et al.

Proc. Natl. Acad. Sci. USA 76 (1979)   4915

rrtina, determined by indirect immunofluorescent staining,
is shown in Fig. 2. All regions of the 18day-embryo retina oc-
cupied by cell bodies bound antibody A2B5, but the antigen
was not detected on axons or dendrites of neurons in the inner
or outer synaptic layers of the retina or on axons of ganglion
neurons (Fig. 2A). Layers of cell bodies separated from pro-
cesses are not present in 8-day-embryo retina, and antibody
A2B5 bound to cells in all regions of 8-day-embryo retina (Fig.
2B). In adult chicken retina (Fig. 2C), antibody A2B5 bound
to cell bodies of photoreceptor cells, amacrine neurons, hori-
rental neurons, bipolar neurons, and ganglion neurons but not
to cell processes in the inner and outer synaptic layers of the
retina. In this and other retina sections, the inner segments of
photoreceptor cells were intensely fluorescent and fluorescence

A

F ;.?        : . .
         `.

FK;. 2. Distribution of'antigen
A2B.i in sections of chicken retina
I'rom l&day embryo (A), X-day
embryo (H), and adult CC'). Letters
rel'er to retina layers: R, photore-
ceptor cell layer; 0, outer synaptic
laver; IN, inner nuclear layer con-
taining cell bodies of' horizontal,
bipc&r, and amacrine neurons and
Miiller cells; IS, inner synaptic
layer comprised ot' processes and
synapses ol'amacrine, bipolar, and
ganglion neurons; G, ganglion
neuron cell bodies; A, ganglion
neuron axons. (Bar, 4 Fm.)

was detected both within the cells and on the surface mem-
brane. Thus, the antibody may be reacting with both plasma
membrane and cytoplasmic antigens. Less antibody A2B5
bound to the central part of the inner nuclear layer which
contains Miller cell bodies (retina glial cells) than to other areas
of the retina with cell bodies. Antibody A2B5 also did not bind
to processes of Miiller cells which traverse the inner and outer
synaptic layers. In contrast to the pattern of fluorescence with
antibody A2B5, antibodies produced by other hybrid cell lines
and rabbit anti-retina antibodies bound to all layers of the retina
(data not shown).

The distribution of antigen A2B5 on retina cells dissociated
from 8day-embryo and cultured for 24 hr also was determined.
These retina cells adhered to the substratum, extended pro-

FIG. 3.  Distribution of antigen
A2B5 on chicken retina cells dis-
sociated from &day-embryo and
cultured for 1 day, detected by in-
direct immuntrtluorescellce. (A)
Bright-field view of the cultured
cells showing cell bodies and pro-
cesses (denoted hy arrows). (HI
Same field showing tluorescent cell
bodies and cell processes without
t'luorescence. (Bar, 20 pm.1


                        Residual
                   cytotoxicity.
     Antibody A%5 plus:           %

     No addition               100f 5
     Pellet                     4f5
     Pellet. :< min at 100oC         2:j o 5
    Pellet. 30 min at 100oC         40 zt 4
    Pellet. 5 min with trypsin        6 f 3
    Pellet. 30 min with trypsin        a f 6
     Pellet extracted with
      chloroform/methan(,1, ?:I     87 f 1:\
     Material extracted from
     100.000 X :: pellet into
      chloroform/methanol, 2:l      38 f 9

I'rllet fraction corresponded to 44 p:: of unfractionated retina
protein; ant ibody A%5 was 0.3 p,u per well. Release of "`Cr from 8-
day-embryo retina cells was determined. Where specified, the pellet
t'raction was incubated with 0.8 m,u of trypsin in 400 ~1 at 37oC for 3
or :((I min: then 0.8 mg of soybean trypsin inhibitor was added and the
eflect on cytotoxicity was determined. Residual rytotoxicity is defined
in the legend to Fig. 1. One hundred percent residual cytotoxirity
corresponded to 1515 cpm released from X-day-embryo retina cells
labeled with :"Cr. Each value is the mean f SEM of three determi-
nations.

cesses, and formed aggregates (Fig. 3). Antibody A2B5 bound
to >60% of the cell bodies but little or no antigen was detected
on cell processes. The antibody did not bind to retina pigment
cells or to cells with the morphology of fibroblasts or epithelial
cells (these comprise <2O% of the cell population). The re-
maining cells that lack antigen A2B5 may be Mtiller cells. An-
tigen A2B5 was detected on the soma and on the processes of
cultured chicken embryo dorsal root ganglion cells; thus, lo-
calization to the cell soma is not an invariant property of the
antigen.

Antigen A2B5 was assayed by inhibition of antibody A2B5-
dependent cytotoxicity (Table 1). The antigen was inactivated
only 36% on incubation at 100oC for 30 min, was insensitive
to trypsin, was sedimented by centrifugation at 100,000 X g for
30 min, and was extracted from the pellet into chloroform/

1500 MOO0 1:2000 No Ab 1:500 1:lOOO NoAb
    ANTIBODY A2B5TITER

FIG. 4. Inhibition of antibody A2B5-dependent cytotoxicity
by molecules extracted from &day-embryo chicken brain or retina
into chloroform/methanol/H~O, 3:1:0:3. Antigenic activities (corre-
sponding to antigen extracted from 3.9 mg of brain or 1.0 mg of retina,
wet weight) in the chloroform phase (fraction B) and in the metha-
nol/H;?O phase (fraction C) are shown in A and B, respectively.

Table 2.  Eflecl 01 neuraminidasr 101 antigen ~\?tKi

                      Residual
                   cytotoxicity.
  Additions                     c,

None
Antibody A2BS
Antibody A2B5 + antigen A'S5
Antibody A2B5 + neuraminidase
Antibody A2B5 + antigen A2B5
+ neuraminidase
Antibody A2B5 + antigen A2R5
+ boiled neuraminidase

0
100 f 2
53 + 4
92 f 6


86 * 3


43 f 2

Antigen A2B5 extracted from 7 mg (wet weight) of chicken 8-day-
embryo brain (fraction 1)) was incubated at 37'C for 60 min in a r~
action mixture (final volume 3"OpI) containing 30 mM sodium acetatt
(pH 5.5), 0.48 pg of neuraminidase protein from Clostridium pf'r-
/ringpn.s purified h., ff'
       \ a unity chromatography (47 units/mg protein
Sigma), or heat-inactivated neuraminidase (1OO'C. 10 min). Then, %I
~1 of the reaction mixture containin, u antigen A2H5 extracted from
440 pg (wet weight) of brain and 0.025 pg of neuraminidase or hrat-
inactivated neuraminidase protein was added to each well assayed
for cvtotoxicity (0.4 peg of antibodv AS5 per well). Residual cyto~
toxicity is defined in the legend of' Fig. 1. One hundred percent re-
sidual cytotoxicity corresponded to the release of 196 cpm. Each valur
is the mean f SE!M of three determinations.

methanol, 2:l (vol/vol). Antigen A2B5 was solubilized by e\-
traction of S-day-embryo chicken brain or retina with metha-
nol/chloroform/H20,2:1:0.3 (vol/vol). Two additional volumes
of chloroform and 2 vol of H20 were added. The lower chlo-
roform phase and the upper methanol/H20 phase were sepa-
rated, taken to dryness, and assayed for inhibition of antibody
AQBSdependent cytotoxicity (Fig. 4). All of the antigen ea-
tracted from brain or retina partitioned into the methanol/H20
phase and thus exhibited the solubility of a ganglioside. A2Bj
antibody-dependent cytotoxicity was inhibited by compounds
in the methanol/H20 phase (Fig. 4B) but not by material in the
chloroform phase (Fig. 4A). When material in the methanol:
Hz0 phase was fractionated by thin-layer chromatograph!
[silica gel plates; propanol/HgO, 70:30 (vol/vol)], antigenic
activity remained close to the origin (RF = 0.02), with GQ
gangliosides.

Because the antigenic material exhibited the properties of
a ganglioside, the effect of neuraminidase (purified by affinit!

UNFRACTIONATED
GANGLIOSIDES

     CONCENTRATION -Log (MOLARITY)
FIG. 5. Inhibition of antibody A2B5-mediated cytotoxicit? "'
purified ganglioside preparations and saccharides. Each point ret'
resents the mean of three determinations. The effects of ganghoP ,di.
GQ (a), GM* (m), GD, (A). and GT, (o), bovine gangliosides cpl
N-acetylneuraminic acid (A), and glucosaminic acid (0) were stud"
at the concentrations shown.


Biochemistry: Eisenbarth et al.

column chromatography) on the antigenic activity was deter-
mined (Table 2). Incubation with neuraminidase almost com-
pletely blocked the inhibitory effect of antigen A2B5 on anti-
body-dependent cytotoxicity.

The effects of partially purified ganglioside fractions and
several monosaccharides on re!ease of 5'Cr from &day-embryo
retina cells due to antibody A2B5 cytotoxicity are shown in Fig.
5. A GQ ganglioside fraction (ganglioside molecuIes with four
N-acetylneuraminic acid residues) purified approximately
10OO-fold inhibited cytotoxicity 50% at an estimated concen-
tration of 0.2 PM. A crude bovine ganglioside fraction and
N-acetylneuraminic acid also inhibited the cytotoxic effect of
antibody A2B5 with half-maximal inhibitions estimated to be
600 and 5000 PM, respectively. Gangliosides GTl, GDl, and
CM~ and glucosaminic acid had little or no effect on cytotox-
icity. These results suggest that antibody A2B5 recognizes an
antigen with the properties of a complex ganglioside such as a
GQ ganglioside. Further work is needed to define the structure
of the antigen.

In a preliminary study, antibody A2B5 was used to explore
the possible role of the antigen in synapse formation. Saturating
concentrations of antibody A2B5 did not inhibit the formation
of synapses between dissociated 8-day-embryo chicken retina
cells and rat myotubes or the transmission across synapses.

DISCUSSION

A hybrid cell line that synthesizes a cytotoxic antibody directed
against chicken embryo retina neurons was obtained by fusion
of mouse myeloma cells with spleen cells of mice immunized
with retina cells. The antigen also was found in chicken embryo
brain, spinal cord, and dorsal root ganglion neurons and in
bovine and human brain but was not detected in heart, muscle,
liver, kidney, or erythrocytes of chicken embryos. The antigen
of chicken retina was shown by indirect immunofluorescence
to be associated with plasma membranes of most, or all, neuron
cell bodies including photoreceptor cells, horizontal neurons,
amacrine neurons, bipolar neurons, ganglion neurons, and
neuroblasts. The antigen was not detected on axons or dendrites
in the synaptic layers of the retina, even though the synaptic
layers contain more plasma membrane than equivalent areas
of the retina composed of cell bodies. Bipolar neurons may
contain less antigen A2B5 because they fluoresced less intensely
than did other retina neurons. In addition, inner segments of
photoreceptor cells fluoresced more intensely than outer seg-
ments. Antigen A2B5 was not detected on retina Miiller cells,
pigment cells, or cells with the morphology of fibroblasts or
epithelial cells. Molecules in chicken retina with a distribution
complementary to that of antigen A2B5 (i.e., preferentially
localized to axons or dendrite plasma membranes) have been
reported, such as cell adhesion molecules (27). acetylcholines-
terase (EC 3.1.1.7) (28). nicotinic acetylcholine receptors (29),
and muscarinic acetylcholine receptors (30).

A2B5 antigenicity was not destroyed bv trypsin or by incu-
bation at 100oC but was inactivated during incubation with
neuraminidase. The antigen sedimented at 100,000 X g and
was soluble in chloroform/methanol (2:l). A GQ ganglioside
fraction from bovine brain purified 1000-fold inhibited the
cytotoxic effect of antibody A2B5 on retina cells. The concen-
tration for half-maximal inhibition was estimated to be 0.2 PM.
In contrast gangliosides with three or less N-acetylneuraminic
acid residues that were tested had no effect on cytotoxicity at
concentrations of 1 mM. Antibody A2B5 may not interact with
aII molecules in the GQ ganglioside fraction; thus, the observed
half-maximal inhibition of cytotoxicity may be a minimal es-
timate of the affinity of the antigen for the antibody.

hoc. Nat!. Acad. Sci. USA 76 (1979)   4917

The localization of antigen A2B5 on the exterior surface of
cell bodies of neurons raises questions concerning the function
of the antigen. Whether the antigen is involved in the sorting
out of neurites from cell bodies or in determining the relative
positions of neuronal soma in the retina is unknown. However,
the availability of large quantities of monoclonal antibody
should be useful for further purification of the antigen, for
separating populations of neurons that possess the antigen from
other cell types, and for studies on the synthesis, localization,
and function of the antigen.

We thank Dr. Peter Fishman for generously supplying us with a
purified GQ ganglioside fraction and Dr. Roscoe Brady for helpful
suggestions concerning fractionation of chicken brain gangliosides.

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30


31

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