Note: Descriptions are shown in the official language in which they were submitted.
~ 22557
. 1L)893~i~
.
Many medical procedures require a determination of
pre-transfusion or pre grafting blood cell compatibility be-
tw~een donor and'patient. Blood ceil compatibility is deter-
mined by the non-occurrence of an immunological-reaction be-
tween antibodies contained in the blood serum of a patient andantigens present on blood cells from a donor. For example, if
the red cells of a patient are type A (i.e., have "A" antigens
on the red cells), the serum of such a patient's blood will
have anti-B antibodies, i.e., antibodies which will react
with "B-'cells. If such a patient receives a donation of "B"
blood, an immunological reaction will occur between the anti-B
antibodies of the patient-'s serum and the B-antigens of the
red blood cells of the donor. Such an incompatibiiity can
result in serious consequences because of intravascular hemolysis.
Tests for blood cell typing and compatibility are
generally of two types: ti) a test to determine whether a
spec~ific antibody addea to the cells'will cause their agglutina-
tion, and ~ a test to determine whether a specific antibody
added to the tested cells together with serum complement, will
~ause cell lysis.
The first of these two basic tests, agglutination,
refers to a clumping of blood cells containing, for example,
.
type A antigens, to which anti-A antibodies are added in the
-- absence of complement. The A-antigen and the anti-A antibody
react specifically with each other by immunological reaction
with the antibody forming bridges between adjacent cells. This
leads to an interlocked'mass of the blood cells joined to each
other by the added antibodies.
,
` I ~ 22557
. lV893~3
The second of the two tests referred to above, cell
lysis, relates to the disruption of cell membranes leading to
death of the cells and release of their intra-cellular contents.
nCell lysis" is the result of a reaction which occurs between
S c~ll membrane bound antibody and a group of potentially destruc-
ti~e proteins in normal serum (called "complement~).
Both methods described above are used for the typing
and compatibility testing of the cellular blood elements,
erythrocytes, granulocytes, lymphocytes and platelets (thrombocytes).
Although often used only qualitatively, both methods are in-
~rihsically quantifiable and have been used separately, for
assay of antigen, antibody, and serum complement.
Background of the Invention
In blood celi typing and compatibility test procedures
commonly use2 in clinics today, hoth agglutinat-on tests and
cell lysis tests are carried out in a liquid phase, that is,
sera containing antibodies with or without complement to be
tested are mixed with suspensions of the blood cells with respect
to which blood typing or compatibility testing is to be evaluated.
Normally, fixed volumes are employed.
Evaluation of agglutination test results requires
the technician to distinguish agglutination of cells due to
specific antigen-antibody molecular bridging from non-specific
cell aggregation in which unrelated forces also cause some
degree of clumping. The technician must also be able to dis-
tinguish free unagglutinated ce-ls which may be present from
clumped or agglutinated cells. This requires highly experienced
personnel or precise particle sizing and coun~ing with costly
instruments. In addition, measurement o~ the degree of
.
` [ 1 22~57
1(~8~359
specific agglutLnation is either poorly semi-quan~itative or
is costly and complicated to perform.
. .
While some instrumented tests for typing of rea
blood cells by agglutination have been developed, the equip-
ment for these procedures is bo~h costly and complicated touse. For example, one device which has been proposed for typing
red blood cells by instrument is known as the "AutoAnalyzer"
of Berkman et al. descri-bed--in Transfusion, Vol. 11, No. 6,
, . .
pp. 317, et seq. (1971). In the AutoAnalyzer, blood samples
and antibody sera are combined under special circumstances in
complex tubular coils designed to bring about agglutination.
The reaction coils then pass a "T~ connection with the leg in
a downward position so that agglutinates which are formed tend
to be removed. Agglutination can be detected measuring the
decrease in optical density of the effluent from the "T" carry-
ing the non--~gglutinated fraction (Rer~m~r. et al.), or by
trapping the agglutinates from the ~T" on filter paper ~Shield
et al., Transfusion, Vol. 9, pp. 348, 1969). The apparatus,
however, is complex and costly, and does not lend itself to
a simple automation technique suitable for routine use in
blood banking clinics.
~ n alternative device is known as the '~Gro~pàmati
and can cost several hundred thousand dollars (see Garretta
et al., Vox Sang., Vol. 27, pp. 141, 1974). In the Groupamatic
device, sera and blood cell suspensiohs are combined to pro-
duce agglutination. The presence of agglutination is detected
by passing the suspension across two light beams, one of which
passes through the center of the reaction cuvette while the
other passes through the periphery. A difference in the
-4-
~ ~ 22557
893S9
transmission of the beams is taken as the measure of the
strength of agglutination. Sophisticated circuitry is re-
quired, however, placing the instrument beyond the means of
ail but the largest blood ~ank operations.
Tests based upon immune lysis present no problem when
the cell surface antigen-antibody reaction interacts efficiently
with complement, as for tissue (e.g., HL-A) typing. Unfor-
tunately, this is rarely true with human red cells where either
the cell membrane antigen-antibody complex interacts ineffi-
ciently with complement or antibody concentration must be
limited to prevent intense agglutination that will mechanically
interfere with immune lysis.
These problems gravely affect the operation of blood
banking serology laboratories where even routine red cell
typing remains a time-consuming, manual operat~on tha* demands
morç skillful and experienced pe~sonnel than are available.
Furthermore~ relatively few blood banks can undertake lympho-
cytotoxic tests required for tissue typing. There are no
direct immunologic tests a~ailable to determine the pre-transfusion
compatibility of granulocytes, and only an indirect test for
platelets, such as 14C-serotonin release. ~While granulocytes
and platelets may be assigned HL-A types by selected and
appropriate tests, such typing will not guarantee their com-
patibility).
Statement of the Invention
.
Broadly within the scope of the present invention,
we have discovered that both agglutination and celi lysis test
procedures on human blood cells are significantly improved
when a monolayer of reactive cells is irreversibly ~ound to
~ 22557
10~39359
a solid matrix. Thereafter that layer is contacted with a
solution (or serum) containing antibodies potentially reactive
by immunoadsorbence on the cell laye~, and the extent to which
immunoaclsorbence occurs is determined. In an alternative pro-
cess employing the basic principles we have developed, thepresence of immunological factors (i.e., antibodies or antigens)
present on the cell surfaces Qf a cell population can be
assayed by~
~a) applying a first suspehsion of cells of the
cell population to be tested to a solid matrix u~der condition$
effe~tive to bind irreversibly a layer of such cells to the
matrix,
(b) contacting the cell layer thereby formed with
a solution (or serum) containing antibodies or antigens of
~nown specificity; and
(c) thereafter measuring tne extent of immuno-
adsorbence of the antigens or antibodies onto the cell layer.
By irreversible bonding of cells to a solid matrix,
we are referring to bonding of the reactive cells by molecular
forces, such as the formation of covalent chemical bonds be-
tween sites on the cell surface and the reactive groups on
the substrate, or to the formation of bonds by weaker molecular
forces such as van der Waals forces, columbic forces or hydrogen
bonding that will withstand the effects of further procedural
conditions. Not only is the sensitivity of the test procedure
increased, but the result of the immune reaction is readily
measurable by simple instrument~tion.
.
-6-
)893Si9
By one aspect of the present teachings, a method is
provided for blood cell typing on a solid matrix which comprises
the steps of
(a) forming a first layer of blood cells
irreversibly bound to a solid matrix;
(b) contacting the layer (a) with a first solution
containing antibodies, the blood type of the blood cells of the
first layer or the antibodies in the solution being known; and
(c) thereafter measurina the extent of immunoadsorp-
tion of the antibodies to the blood cells.
By a further teaching there is provided a method of
measuring blood cell compatibility on a solid matrix which com-
prises the steps of
~ a) forming a layer of blood cells irreversibly
bound to a solid matrix, the blood cells being from a first person; ~ -
(b) contacting the layer (a) with serum from theblood of a second person; and
(c) thereafter determining the extent to which
antibodies from the serum of the second person are immunoadsorbed
by the blood cells from the first person.
By yet a further teaching there is provided a method
of assaying for antigens on the surface of a cell population com-
prising the steps of
(a) applying a first suspension of cells of a popula-
tion to be tested to a solid matrix under conditions effective to
bind irreversibly a layer of the cells to the matrix thereby form-
ing a first layer of cells to be tested on the matrix;
(b) contacting the layer (a) with a solution contain-
ing antibodies of known specificity reactive by immunoadsorption
with the antigens to be assayed on said cell surface; and
(c) measuring the extent of immunoadsorption which
occurs.
-6a-
-- 108~3S9
I~ accordance with a further teaching, a method is
provicled for assaying cell type or compatibility on a solid
matri~c comprising the steps of
(a) forming a first monolayer of cells irreversibly
bound to a solid matrix, the cells having intrinsic or acquired
antigens thereon;
(b) contacting the layer (.a) with a solution con-
taining antibodies, whereby a layer of an'ibodies is bound by
immunoadsorption, to the extent that the antibodies of the
solution are reactive with the antigens of the cells of the
first layer;
(c) thereafter applying a suspension of second
cells, the second cells having antigens bound thereon; and
(d) subsequently measuring the extent of immuno-
adherence of the second cells by antibody bound to the first
layer of cells.
By a further teaching there is provided a method
for assaying cell type or compatibility on a solid matrix
comprising the steps of
(a) forming a first monolayer of erythrocytes
irreversibly bound to a solid matrix, the erythrocytes having
. intrinsic or acquired antigens thereon;
(b) contacting the layer (a) with a solution
containing antibodies, whereby a layer of antibodies is
bound b~ immunoadsorption to the extent that the antibodies
of the solution are reactiYe with the antigens of the erythro-
cytes of the first layer;
(c) thereafter applying a suspension of second
erythrocytes, the second erythrocytes having antigens thereon;
and -~
-6b-
'~
':
~ V8~3S9
td) subsequently measuring the extent of immuno-
adherence of the second erthrocytes by antibody bound to the ~ :
first layer of erythrocytes.
In a further aspect, there is provided a method
for assaying agglutinins in a serum comprising the steps of
(a) forming a first monolayer of cells irrever-
sibly bound to a solid matrix, the cells being known to have
intrinsic or ac~uired antigens corresponding to the agglutinin
to be assayed;
(b) contacting the layer (a~ with a solution to be
assayed for agglutinins;
(c) thereafter applying a second suspension of
cells, the second cells known to have or to carry antigens
to the agglutinins to be assayed; and
(d) subsequent measuring the extent of immuno-
adherence of the second cells to the first cell layer.
By yet a further aspect a method is provided for
assaying cell type or compatibility on a solid matrix comprising
the steps of
(a) forming a first monolayer of washed erythro-
cytes irreversibly bound to a solid matrix, the erthrocytes
having intrinsic or acquired antigens thereon;
(b) contacting the layer (a) with a solution
containing antibodies under conditions of reduced ionic con-
centration, whereby a layer of antibodies is bound by immuno-
adsorption to the first monolayer of erythrocytes to the extent
that the antibodies of the solution are reactive with the
; antigens of the erythrocytes of the first lay~r;
(c) thereafter aPplying a suspension of second
erythrocytes, the second erythrocytes having antigens thereon,
-6c-
` 10~S~3~9
and allowing the second erythrocytes to settle on the solid
matrix as a second monolayer over the first layer of erythro-
cytes;
(d) after the said second monolayer has formed,
adding protamine sulphate ~o the second suspension in an amount
sufficient to promote immunoadsorption of the second erythro-
cytes to antibodies bound to the first monolayer; and
(e) thereafter washing to remove second erythro-
cytes which are not immunobound, and measuring the extent of
immunoadherence of the second erythrocytes to antibodies bound
to the first layer of erythrocytes.
By a further embodiment there is provided a substrate
for assaying red blood cell type or compatibility consisting
essentially of
(a) a light transmitting solid support membrane
having a face to permit measurement of a surface layer applied
thereon; and
(b) a monolayer of red blood cells irreversibly
adhered to the face, the red blood cells having been lysed to
~ render the cells transparent, the cells carrying the antigenic
determinants to be assayed.
In another embodiment there is provided a substrate
for assaying red blood cell type or compatibility consisting
essentially of
(a) a light transmitting solid support member having
a face adapted to permit measurement of a surface layer applied
thereon;
(b) a layer applied to the face having reactive
chemical groups thereon, effective to bind cells to the substrate;
(c) a monlayer of red blood cells adhered to the face
by the layer (b), the red blood cells having been lysed to render
the cells transparent, the cells carrying the antigenic deter-
-6d-
10893S9
minants to be assayed.
By a further aspect there is provided a substra~e
for assaying cell type or compatibility consisting essentially
of
(a) a light transmitting solid support member of
polystyrene with a face adapted to permit measurement of a
surface layer thereon;
(p) a layer of fibrinogen bound to the face of the
polystyrene substrate; and
(c) a layer of polylysine bound to the fibrinogen,
the polylysine layer being capable of irreversibly binding a
monolayer of cells to be assayed thereto.
By yet a further teaching a method is provided for
assaying cell type or compatibility on a solid matrix compri~ing
the steps of
(a) forming a first monolayer of cells irreversibly
bound to a solid matrix, the cells having or carrying antigens
thexeon;
(b) forming a first monolayer (a) with a solution
containing antibodies capable of forming a layer of antibodies
bound by immunoadsorption to the first layer of cells to the
extent that the antibodies of the solution are reactive with the
antigens of the cells of the layex; and
(c) thereaftex measuring the extent of immunoad-
sorption of the antibodies by applying a second solution
containing lytic complement effective to lyse the cells of the
layer (a) in the presence of immunoadsorbed al.tibody and
observing the extent of immune lysis which oc_urs.
By way of a simple illustration, the blood typing
test referred to above may be carried out in three steps:
(1) a monolayer of erythrocytes (by way of illustr~tion,
-6e-
-r
~ 22557
10~93~i9i
. .
reference will be made to type A) are bound irreversibly to
a solid matrix; (2) using the cell monolayer as an immuno-
ads;orbant, antibodies such as anti-A antibodies can be applied
and adsor~ed specifically; and (3) the antibody-coated cells
S may now be tested either for their susceptibility to immune
lysis by complement ~solid~phase lysis) or for their capacity
to bind a second monolayer of cells that carry appropriate
antigens (solid-phase agglutination).
.The test result may be evaluated in any convenient
fashion, such as by examination under a microscope; however,
of particular importance to the present invention is that the
test results are especially suited to be evaluated by densito-
metric techniques using standard, and.readily available, in-
struments. For solid-phase hemagglutination, the test plate
prepared in the foregoing manner can be subjected to static
or scanning densitometry at wave lengths where the hemoglobin
conte~t of the tested erythrocytes is absorbant to light (for
instance, blue light having a wavelength of 415 nm is suitable).
Scanning densitometry involves only the use of well-established
laboratory equipment which is readily available and provides
quantitative answers to questions such as (1~ Is a second
layer formed?; ~2) How many cells does a second layer repre-
sent?; and (3~ What is the distribution of the second layer?
(8y "distribution", reference is made to the uniformity of
the second layer. A uniform second layer implies that 100%
of cells in th~ applied suspension carry the necessary antigen
to bind to the first layer, whereas a non-uniform distribution
implies the occurrence of some "negative" cells in the applied
suspension).
I ~ ~ 22557
893~9
- In the alternative method described above to assay
imm~nological factors on cell surfaces, the presence of anti-
bodi,es or antigens immunoadsorbed from said solution can be
conveniently detected by applying a second suspension of t~e
cell population to be tested after the bound monolayer has
been reacted with the antigen (or antibody) solution and
measuring the formation of a second cell layer in which the
antigen ~or antibody) of known specificity acts as a cross-
linking agent.
The procedure of this invention can also be used to
test for competitive reactions between solutions or suspensions
o~ materials suspected of sharing antigenic properties. Thus,
an irreversibly bound monolayer of cells is prepared as des-
cribed above. This layer is then contacted with a mixture of
two solutions, one containing antigens or antibodies of known
specificity capable of immunoadsorbence onto the bound cell
monolayer, and a second solution or suspension which is to
- be tested. Competitive binding of the known antibodies or
antigens by factors present in the second solution or suspen-
sion will be reflccted by a reductlon in the formation of
immunoadsorbence reaction with the irreversibly b~und cell
monolayer.
Paralleling this procedure is the preparation of a
cell monolayer with a second layer of antigens bound thereto
.
as described above. Competitive reactions can then be measured
between a suspension of cells capable of forming a second cell
monola~er and a second solution or suspension containing un-
known factors which are to be assayed. The occurrence of
competitive in~unological reactions between these two
.
--8--
10~93S9
suspensions (or suspension and solution? will be reflected
by a reduction in the formation of a second cell monolayer.
Within these general guidelines, several specific
assay procedures are contemplated:
l. Assaying an*igens by competitive immunoadsorp-
tion. This assay comprises the steps of ta) applying to a
solid matrix a first suspension of cells known to carry anti-
gens to be tested under conditions effective to bind irreversibly
a layer of said cells to said matrix; (b) contacting said layer
10 (a) with a mixture of ti) a solution of an antibody reactive -
by immunoadsorption with antigen carried by the cells of said
layer (a) and (ii) a second solution or suspension to be
assayed for antigen by competitive inhibition of the antibody
in said solution (i); and (c) thereafter measuring the extent
15 to which said antibody in solution (i) is bound by immunoadsorp- -
tion to said layer (a).
2. Assaying antigens by competitive inhibition. This
assay comprises the steps of (a) applying to a solid matrix a
first suspension of cells known to carry antigens to be tested
under conditions effective to bind irreversibly a layer of said
cells to said matrix; (b) applying to said layer (a) a solution
containing an antibody reactive by immunoadsorption with antigens
carried by the cells of said layer (a); and (c) thereafter
applying a mixture of (i) a second suspension of cells which are
known to carry the antigens to be tested, and (ii) a suspension
or sQlution containing an antigen competitively reactive with
the immunoadsorbed antibody of said solution (b) and measuring
the extent to which cells of said second suspension form a
second layer of cells on said matrix~
-8a-
l r . '~5~l
. '
1089359
3. ~ssavina antibodies on a cell population. This
assay comprises the steps of (a) applying a first suspension
. of cells of'a population to be tested.to.a solid matrix under
conditions effective to bind irreversib~y a layer of said
cells to said matrix thereby forming a first layer of cells
to be tested on said matrix; (b) contacting said layer ~a)
with a solution containing antigens of known specificity re-
acti~e by immunoadsorption with the antibodies to be assayed,
. . on said cell surfa,ce; and tc) measuring the extent of immuno-
adsorption which occurs.
4. Assayin for antigens in the presence of anti-
g _ _ _
bodies on a cell population. This assay comprises the steps
' of ~a) applying to a solid matrix a first suspension of cells
known t~ carry antibodies under conditions effective to bind
irreversibly a layer of said cells to said matrix, (b) con-
tacting said layer ~a) with a mixture of (i) a solution to ~
assayed for antigen, and (ii) a second solution or suspension
containing antibodies reactive by immunoadsorption with the
antigen to be assayed by competitive inhibition;-and ~c) there- ~
' 20 af~er measuring the extent to whi.c~ said antigen in solution
~i) is bound by immunoadsorption to said layer (a).
In each of the foregaing procedures, the extent of
,. .'' ~. immunoadsorption may be determined by one or more of the tech-
.. . niques generally described above.
, . "
.
. ' -8b- , ,
a a
;10~359
Description of the Prior Art
Despite the long-standing clinical problems asso-
ciated with blood banking serology, and the need to upgrade
the! procedures and make those procedures amenable to instru-
mentation and automatic techniques, there has been littleassistance provided by the prior art to those in the field.
~or a number of years, there has been known the so-called
nEldon" cards fox blood typing tests. These have been des- -
cribed, for example, in U.S. patent 2,770,572. The '572 patent
i0 describes a test card for use in typing human blood in which
a support sheet bears on differing portions of its surface
dried specimens of test sera containing antibody factors in
a mixture with conglutinin or conglutinin substitutes. In
effectin~ blood typing tests using the Eldon card, blood
; 15 samples from the patient whose blood is to be typed are placed
in droplets on the various serum spots contained on the card
and examinea, after aIlowing for appropriate reaction time,
for the presence or absence of agglutination. The tests,
however, are limited to anti-A, anti-B, and anti-Rh ~Rho or
D), and even these are associated with significant errors in
interpretation.
- More recently, R. T. Price, in patent 3,666,421,
has described another diagnostic test slide in which serological
- reagents are placed in drops upon a test slide and dried to a
spot that may be subsequently reconstituted and reacted in an
agglutination reaction for the identification of blood type
~or other antigen-antibody reaction systems identifiable by
.
_9.
~ ~ 22557
' ` . . 1~89359
agglutination). The test slide as described ~y Price, how-
ever, remains subject to the defects characteristic of ordinary
li~quid-phase agglutination: the test shows essentially only
the presence or absence of agglu,tination and depends upon
evaluation by s~illed technicians to determine whether the
agglutination is the result of specific antigen-antibody re-
action or is simply the result of non-specific cell aggrega-
tion; it is difficult or impossible to evaluate whether free
unagglu~inated cells are present along with clumped cells of
specific agglutinates.
James E. Smith, in U.S. patent 3,770,380, describes
a device suggested for evaluating immune adherence reactions.
It should be noted in this respect that the immune adherence
reactions to which the Smith patent relates differ from the
immunoadsorption phenomena on which the present invention is
based. Immune adherence is the non-specific clumping of
particles or cells due to the presence of complement; in
immune adherence, it is the complement which causes binding.
Immune adherence is characterized by non-specif~c react~ons
.
between the complement and the particles and cells to which
they bind. Immunoadsorption, by contrast, is a specific bind-
ing between antigenic sites on a cel~ membrane and antibodies
present in a serum. Thus, immunoadsorption, in contrast to
immune adherence, refers to specific binding between antigens
and anti~odiés.
~ he device described by Smith is a flat cell-like
structure supplied on its floor with successive coatings of
; a bacteria or viral material as an overcoating, which is bound
to the base of the cel~ by means of a transparent, dried
. . .
--10--
.
- I ~ 22557
. i0~9359
protein underlayer. Immune adherence reactions between the
bacteria or virus of the thus-prepared cell and red cells
car.rying antibody and complement in a test fluid applied there-
to are then evaluated by deter~ining the extent to which the
specially-preparea red cells in the applied fluid adhere to
the bacteria or virus containing overcoating. The procedure
described by Smith has not found practical clinical value be-
caùse immune adherence itself has not been widely adopted, and
because immune adXerence is non-specific, it is not suitable to
evaluating specific antigen-antibody reactions.
A layer of blood cells imbedded in a solid support
has been used for scientific purposes unrelated to blood typing
a~d compatibility testing. Such layers are not bound by co-
valent or other molecular forces. Thus, Goodman ~Nature, 193:350,
1962) prepared columns of formalinized human red cells imbedded
in polyurethane which he used to fractionate human anti-red
cell antibodies on the basis of the stren~th of their binding
to the trapped red cells~ Edelman, et al., (Proc. Nat. Acad. Sci.,
68:2153, 1971) fractionated blood cells on the basis of their
-
2~ capacity to specifically bind to lectins, antibodies, or antigens
that had been previously bound to partially hydrolyzed nylon
.
fibers.
The principle of binding a prosthetic group (of an
antigen, antibody, enzyme, etc ) to a solid matrix is a well
established biochemical method (see Cuatrecases and Anfinsen,
Annu. Rev. Biochem., 40:259, 1971), and is widely used in
solid-phase radioimmunoassay procedures (see, Brit. Med. Bull.,
30:1~103, 1974). However, blood or other cells have not been
bound irreversibly to a soIid matrix to ~acilitate blood or
tissue typing, compatibility testing, etc.
--11-- .
-. L .
t ~ [
10893~
Detailed D scription of the Invention
The following is a description of the invention with
respect to presently used tests of erythrocytes. Polystyrene
test tubes are coated with fibrinogen (applying 0.5 mg/mll
which binds irreversibly. After washing, polylysine (0.1 mg/ml)
is applied to the bound fibrinogen. After further washing,
there is introduced a suspension of either normal or protease-
treated erythrocytes (RBC). These RBC are bound irreversibly
(~or purposes of testing) as a monolayer to the polylysine-
fibrinogen-coated polystyrene surface. The binding density
of 2 x 106 RBC per square centimeter is in good agreement with
that expected for RBC having a diameter of 7 micrometers.
The monolayer of bound RBC is stable and will, in
turn, serve as i~munoadsorbant to bind antibodies from any
applied solution (or serum) which are specific to antigenic
,sites on the membranes of the bound RBC.
The ~ethod of binding blood cells as a monolayer
on a~ solid màtrix need not be restricted to the above. The
literature relating to coupling proteins to polymers (see
Cuatrecases and Anfinsen, Annu. Rev. Biochem., 40:259, 1971j
indicates that as an alternative, one may use preparations
- of polymers (sheets of polyurethane, polystyrene, etc.) tha~
contain on their surface reactive groups such as glutar-
aldehyde, cyanogen bromide, amino or carboxyl groups, and
others that will allow direct covalent coupling of blood cells
to the matrix. It is obvious that the binding substance must
be immunologically inactive with respect to any antigens or
antibo~ies which may be present in test solutions contem-
plated for use.
-12-
1(~893X9
- Other substrates or matrices for use in this inven-
tion may be any convenient material which is (i) of such a
character that a cell monolayer may be irreversibly bound to
-it as described a~ove; and (ii) suitable for use in view of
the detection method t~ be used. Since the most convenient
detection methods are based on light transmission -- such as
microscopic counting and densitometric scanning -- it is
preferred that a light-transparent substrate be used. How- -
ever, if counting of radioactivity is used, use of material
transparent to light is obviously unnecessary.
For purposes of the present invention, the substrate
or matrix may simply be the interior surface of a test tube.
If a densitometric scanning procedure is to be used to evaluate
the test results, a flat surface is appropriate. For purposes
of large scale testing, strips of matrix having cell binding
properties car. be us~d to prepare the first layer or cells.
Subsequently, antibodies can be spotted and tested for their
- capacity either to support immune lysis in the presence of
complement or to bind a second layer of cells of unknown type;
The quantitative result of either test can then be determined
by scanning densitometry with loss of color indicating lysis
and gain of color indicating the binding of a second layer
of red cells. For the latter purpose, it may be app~Dpriate
as a part of the spot preparation to hypotonically lyse the
first layer of cells so that background due to the first
layer need not be subtracted. ~Iypotonic l~sis does not signi-
ficantly remove much membxane bound antibody, and formation
of a second layer can be detected visually or by optical
densitometry by means of its hemoglobin content. Usin~ scanning
-13-
1089;~9
, . .
densitometry, for instance, at 415 nm quantitative answers can
be obtained to ~uestions such as ''Is a second layer formed?",
nHow many cells (in terms of an obvious maximum) does the
second layer` represent?'; "~hat is the distribution of the
second layer?"
To answer the last question, the cell suspension
applied to the antibody-coated monolayer should have just
sufficient concentration to allow settling of a second mono-
layer. The second layer is then washed to remove unbound cells
and, after this washing, the distribution of the bound portions
of the second layer becomes a function of the percentage of
"positive" cells which adhere and of "negative" cells which
do not adhere. The holes or islands which result can be de-
tected in microscopic scanning densitometry, and their freq~ency
and extent can be expressed as a ratio of the scanned surface.
It should be noted that in the present invention,
the binding of antibody by immunoadsorbence to a layer of
red cells provides several important advantages. ~Firstly, sera
with concentrations of antibodies too low for conventional
liquid-phase tests can be used successfully because their
specific antibody content can be~ concentrated as bound anti-
body on the monolayer of cells. Secondly, under circumstances
where undiluted sera cannot be used for liquid-phase tests
because of other interfering serum proteins, such interference
is abolished in solid-phase tests by selective adsorption of
the antibody to be tested and washing to remove the inter-
fering proteins. Thirdly, many sera are useless for liquid-
phase tests because they contain non-removable antibodies
with unwanted specificity. In the presen~ invention, by
appropriate selection of cells to form the first layer, it
-14-
.
I l 22557
1089359
.. . . .
is possible to adsorb only the antibodies which are to be
tested.
The present invention is suited for testing of either
cell type or cell compatibility. For cell typing, for instanca,
the first layer with its bound antibody would be constructed
o~ cells and antibodies of a known type. The typing of unknown
cells of a patient or donor would be determined from their
capacity to form a second cell layer. For purposes of the
carrying out of compatibility tests, donor cells may be used
to form the first monolayer which are then reacted with possible
antibodies in a patient's serum, which would be bound by
immunoadsorption. Thereafter, this can be detected either
through lysis following addltion of complement or determining
the capability of the bound antibodies of the patient's serum
(to the extent that such binding occurs) to form a second
layer of the same donor cells after another application.
Detection methods suitable for use in the present
.
invention will be obvious to those skilled in the art. Erythro-
cytes contain their own label, namely, pigmented protein
- ~ 20 (hemoglobin) which has a maximum absorbence at 415 nm. The
presence or absence of erythrocytes, therefore, will be con-
veniently detected specifically as described above. However,
other labeling techniques can be used equally well if desired.
Such techniques include the use of radioactive labels ~for
example, 51Cr, 125I), biochemical technique tfor example, a
selected intra-cellular enzyme), or fluorescent detection tfor
example, using a molecular probe to identify either a living
or dead cell). All of these approa~hes can be readily instru-
mented and, therefore, made subject to automation. They are
_, ... , ... , . .. .. _ .. .. , ., ., .. , . , . .. _ . . ._ .. _.__ _ . _ ., , ,. _ .. _ . .. . . . _._ _.. . . .... .....
.... . . ~ .. , ... .. ... . . . ~.
~ ~ ZZ557
1(~ 359
equally applicable to blood banking serology, medico-legal
blood testing, tissue typing, and pre-transfusion and pre-
grafting tests for compatibility.
As is well known in the art, there are a n~mber of
procedures used now in liquid-phase tests which will be equally
successful in solid-phase tests. These include the optimal
use of additives that are known to potentiatè agglutination
(e.g., symmetrical and assymetrical hydrophilic colloids,
proteases, ionic concentration, polyelectrolytes, tonicity,
and buffer systems to control pH). These factors are describe~,
for example, by Berkman, et al., Transfusion, 11:317, 1971.
It should be noted that the present invention also
has application to other problems invol~ing immuno-specific
cellular reactions. One such are antiglobulin tests to eva-
luate cells for their coating by IgG, IgM, IgA, IgD and IgEimmunoglobulins, and by C3, C4 and other complement components
or bound ac~ivation products (see Rosenfield~ et al., Vox-Sang.,
26:2&9-333, 1974). Another application would be solid-phase
tests, with quantitative evaluation, of both passive and re-
versed passive hemagglutination tests. Passive hemagglutina-
tion assays can ~e used or direct analysis of antibody con-
centration and also for indirect analysis of soluble antigen
- concentration by competitiye binding on the basis of shared
antigens (Nusbacher, et al., J. Immunol., 108:893, 1972). Re-
.
versed passive tests measure soluble antigen directly tCook~
Immunol., 8:74, 1965, and Ju~i and Yokochi, Japan, J. xptl.
Med.; 39:615, lg69); Fur~hermore, just as blood cells can be
tested for their susceptibility to antibody-mediated agglutina-
tion or lysis by solid-phase tests, bacteria, protozoa, fungi,
and cultured cell-1ines ~Fom either tissues or tumors can be
~16-
,
.~ lU89!;~9
analyzed for antigenic constituents on their surface by the
~ solid-phase tests described in this patent. We even anticipate
using solid-phase tests ultimately to solve problems concerned
with molecular antibody concentration, K value of antibody bind-
ing, an~ degree of K value heterogeneity.
It is recognized that many of the underlying chemical
principles utilized in the present invention are, of course,
well known. The invention is unique because it has not pre-
viously been recognized that these princlples could be adapted
for construction of solid-phase cell monolayers for the effi-
cient performance of blood cell typing and tests for the com-
patibility of blood and other cells.
.
Examples
The following are examples of the practice of the
present invention:
Example l
~ .
Immune lysis by human anti-A. Using ~Falcon" poly-
styrene test tubes (10 mm I.D. x 75 mm), we have applie~ 0.2 ml
- fibrinogen solution (0.5 mg/ml) for 2 minutes followed, a~ter
washing, by application of 0.2 ml 140,000 M.W. poly-D-lysine
HBr solution (O.l mg/ml) for 2 minutes. After additional
washing, 0.2 ml 2-5% (v/v~ suspension of type Al cells in
.. . . . . .
; 0.9% NaCl was applied for 2 minutes. This resulted in the
- adherencè of a flat-surface monolayer of red cells with a
density of 2 x lO6/cm2. These red cells remained adherent
despite numerous washings, and despite application of strong
human anti-A. If, after application of anti-A, 0.2 ml comple-
~ ment was appiied, hemoglobin of the adherent red cells was
-' , ,
-17-
,
1 ~ ~Z557
lV893~9
released, and the degree of immune lysis observed was measur-
able, either as retained hemoglobin or as retained or released
raldioactivity in the form of SlCr used to label the cells used
to construct the monolayer. With a standard dose of complement
the lytic potential of anti-A cduld be measured quantitatively.
Alternatively, with a standard dose of anti-A, lytic comple-
mçnt could be defined and measured by titration in toto, via
the classical pathway o~ complement action (using guinea pig
RP [Pillemer, L., et al., Science, 120:279, 1954]), or via the
alternate properdin pathway (using EGTA to chelate the Ca~+
but not the Mg+~ of human serum tunpublished observations3).
None of these approaches to the study of immune A-
anti-A lysis can be performed sensitively and.reproducibly
by usual fluid-phase tests. IgM, IgG, and IgA anti-A are all
very efficient agglutinins of type Al cells, and agglutination
interferes with immune lysis. By solid-phase approach, the
lytic potential of anti-A was not only measurable but detect-
able at a dilution SOX that discernible by fluid-phase tests.
The diagnostic possibilities of this method are, therefore,
enormous.
Example 2
. Blood typin~. The problem of typing human red cells,
and~detecting human anti-red cell antibodies at the time of
pre-transfusion compatibility testing, are considerable. In-
deed, the only means of discerning some human red cell bloodtypes by direct agglutination has been expensive and complicated
instru~entation (Berkman, E. ~., et al., Transfusion, 11:317,
1971). However, instrumented fluid-phase methods have been
adapted to soli~-phase testing where we have succeeded in
,
-18-
~089359
achieving specific typing for Rh, Kell, Kidd, Duffy, Xga,
~ewis, Lutheran, and MNSs, all with a sensitivity exceeding
that obtained with the described instrumented fluid-phase
t~sts.
S For blood typing, a monolayer of red cells was con-
structed as described in Example 1 for immune lysis. Now,
however, this monolayer was first exposed to known specific
antibody (0.2 ml) and then hypotonically lysed with distilled
water. At this point, a second application of 0.1 ml red cells
in 0.2% ~v/v) strength was allowed to settle gravitationally
as a li~ht second monolayer. It was now possihle to augment
the specific antibody binding of this second monolayer in a
number of ways. For example, the low ionic method of Berkman
et al., was employed successully by, first, replacing super-
natant ~luid with a solution of 5% mannitol and 0.0025% protamine
~ ate at pH 6.0 and, second, after 5 minutes at room tempera-
ture to wash the second monolayer with 0.0014 M phosphate
~buffered 0.85% NaCl at pH 7.3. This removed non-antibody-
bound known negative cells but not antibody-bound known positive
cells. But othe~ methods for augmenting the antibody-binding
of cells proved to be even more advantageous, and some of
these could not be employed successfully by Berkman's liquid-
., phase instrumented method. Thus, we could augment specific
antibody binding of the second monolayer by sequentially re-
placing supernatant fluid ~irst with a buffer at pH 7.0 contain-
ing 2.5~ PVP, and then with a similar buffer at pH 6.0 con-
taining 0.01~ protamine sulfate. These tests were finally
washed with 0.2M phosphate buffer at pH 7.3. Clearly, the
possibilities for augmentation of solid-phase agglutination
tests ar~ numerous because the test procedurè avoids the many
problems associ~ted with liquid-phase tests.
--19--
, ... . ... . , . . ... ,.. . . _.,. _.. _ . ___ . __ ~ . . _.. _ .. ___.__ . _ . . .. . -- .. ... ... .. .. . _ . .
. . . . .. . .. . .
` 10~9359
These solid-phase tests have proven to be extra-
ordinarily sensitive for the detection of IgG Rh antibody.
We have achieved approximately the same sensitivity on an
antibody molecule per red cell ~asis as described previously
S for augmented AutoAnalyzer assaysi, i.e., ~ 10 antibody mole- -
cules per cell at 50% hemagglutination (Rosenfield, et al.,
Ann. N.Y. Acad. Sci., 190:519, 1971). With solid-phase tests,
however, we employ 1/1000 fewer red cells and have 1000 times
the working sensitivity, detecting about 1 pg antibody pro-
tein/ml, which exceeds the sensitivity of any previously
described test including tests for cell survival in vivo.
Tests with anti-Rh were also conducted successfully
with protease-treated xed cells. Such tests performed on flat-
bottom micro-titer polystyrene dishes allowed microscopic
lS examination of specifically adherent red cells. Bound cells -
were present only if they were Rh-positive and, in tests of
artificial mixtures of Rh-posi~ive and Rh-negative red cells,
"holes" from non-adherent cells were observed to correspond
in area to the percentage of Rh-negative cells in the artifi-
cial mixture. -This result indicates that our invention can
quantitatively ascertain the proportion of unbound cells in
a blood sample, which is a crucially important problem both
in assays of transfused cell survival by the Ashby method
(Arch. Int. Med., 35:516, 1925) and in characterization of
human chimeras (~ace and Sanger, "Blood Groups in Man," Davis,
1968, pp. 475-490).
_xample 3
Dixect antiglobulin tests. These tests were per-
formed iike blood typing tests except that washed red cells
-20-
~ 1~8~359 ~ 22557
-
from a patient with suspected acquired hemolytic anemia were
usecl to construct both the first and the second cell monolayers.
First monolayers (bound by polylysine-fibrinogen) were exposed
to 21 single xenogeneic anti-human globulin serum, and seven
specificities were evalu~ted. These sera were individually
specific for IgG, IgM, IgA, IgD, IgE, C3 and C4. The antibody-
coated first monolayer was then hypotonically ~ysed and washed
before applying cells for the second monolayer. Bind ing of
t~e second monolayer was augmented by adding 1% K-90 poly-
vinylpyrrolidone (PVP, average M.W. 300,000) in 0.9% NaCl.
~he second monolayer was finally washed with ptain 0.9% N~Cl.
The procedure closely resembles that described by Hsu et a7.
tVox Sang., 26:305, 1974) but positive results by solid-phase
testing were distinctly superior to those of Hsu's liquid-
phase instrume~ted tests. One pztient with Active acquired
hemolytic anemia who, because of in,tense spontaneous fluid-
phase agglutination in PVP, could not be typed for adh~rent
proteins, was found to be clearly positive for IgG, IgM, IgA,
IgE, C3 and C4.
.
,
.' ' ' ' . ' ,' ' ` , .
.
,
.
-21-
~ -
