CA1089359A - Blood cell typing and compatibility test by solid phase immunoadsorbtion - Google Patents

Abstract

ABSTRACT OF THE DISCLOSURE

A solid-phase blood typing procedure is described based upon either agglutination or immune lysis. In this in-vention, a monolayer of ceils is irreversibly (e.g., covalently) bound to a solid matrix, and thereafter a serum containing antibodies is brought into contact with the bound cell layer.
Immunoadsorption of antibodies by the bound cells occurs where the antigens of the cell membranes and the antibodies in the serum are complementary to each other. This antibody-sensitized monolayer of blood cells can either bind a second layer of blood cells carrying complementary antigen (solid-phase agglutination) or undergo lysis in the presence of serum complement (solid-phase immune lysis). Carrying out these reactions with a mono-layer of blood cells bound to a solid matrix allows quantitative evaluation of results by such standard instrumentable proce-dures as densitometric scanning, radioisotope counting, etc.

Description

~ 22557 . 1L)893~i~
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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.
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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 ~ ~ 22557 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.

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)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 -~

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~ 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-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 .

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 . '

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.

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. ' -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 . . .

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- 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.

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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.

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 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 .

I l 22557 .. . . .
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 _, ... , ... , . .. .. _ .. .. , ., ., .. , . , . .. _ . . ._ .. _.__ _ . _ ., , ,. _ .. _ . .. . . . _._ _.. . . .... .....
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~ ~ 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.

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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 -' , , , 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 , ~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.

, ... . ... . , . . ... ,.. . . _.,. _.. _ . ___ . __ ~ . . _.. _ .. ___.__ . _ . . .. . -- .. ... ... .. .. . _ . .
. . . . .. . .. . .

` 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 ~ 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.

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Claims (55)

WE CLAIM:

1. A method for blood cell typing on a solid matrix comprising the steps of (a) forming a first layer of blood cells irreversibly bound to a solid matrix;
(b) contacting said layer (a) with a first solution containing antibodies, the blood type of the blood cells of said first layer or said antibodies in the solution being known; and (c) thereafter measuring the extent of immuno-adsorption of said antibodies to said blood cells.

2. A method according to claim 1 wherein the extent of immunoadsorption of said antibodies is measured by applying a second solution containing hemolytic complement effective to lyse the blood cells of said layer (a) in the presence of immunoadsorbed antibody and observing the extent of immune lysis which occurs.

3. A method according to claim } wherein the extent of immunoadsorption of said antibodies is measured by applying a second suspension of blood cells of the same type as the blood cells of said layer (a) and determining the extent of the binding of blood cells from said second suspension.

4. A method for measuring blood cell compatibility on a solid matrix comprising the steps of (a) forming a layer of blood cells irreversibly bound to a solid matrix, said blood cells being from a first person;

(b) contacting said layer (a) with serum from the blood of a second person; and (c) thereafter determining the extent to which antibodies from the serum of said second person are immuno-adsorbed by the blood cells from said first person.

5. A method according to claim 4 wherein the extent of immunoadsorption of antibodies from the serum of said second person is determined by applying a solution containing hemolytic complement effective to lyse the blood cells of said layer (a) in the presence of immunoadsorbed antibodies, and observing the extent of immune lysis which occurs.

6. A method according to claim 4 wherein the extent of immunoadsorption of antibodies from the serum of said second person is determined by applying a second suspension of blood cells of said first person and measuring the extent of formation of a second layer of said first person's blood cells.

7. A method of assaying for antigens on the surface of a cell population comprising 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 irreversibly 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 antibodies of known specificity reactive by immuno-adsorption with the antigens to be assayed on said cell surface;
and (c) measuring the extent of immunoadsorption which occurs.

8. A method according to claim 7 wherein the extent of immunoadsorption is measured by determining the extent to which a second layer of cells from said cell population to be tested can be adsorbed onto said first layer after applica-tion of said solution of antibodies.

9. A method for assaying antigens by competitive immunoadsorption comprising the steps of (a) applying to a solid matrix a first suspen-sion of cells known to carry antigens to be tested under con-ditions effective to bind irreversibly a layer of said cells to said matrix;
(b) contacting said layer (a) with a mixture of (i) 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 to which said antibody in solution (i) is bound by immunoadsorption to said layer (a).

10. A method according to claim 9 wherein the extent of immunoadsorption of said antibody is measured by applying a solution containing hemolytic complement effective to lyse the cells of said layer (a) in the presence of immunoadsorbed antibody and observing the extent of immune lysis which occurs.

11. A method according to claim 9 wherein the extent of immunoadsorption of said antibody is measured by applying a second suspension of cells of the same type as the cells of said layer (a) and thereafter determining the extent of immuno-adsorption of cells from said second suspension.

12. A method for assaying antigens by competitive inhibition comprising the steps of (a) applying to a solid matrix a first suspen-sion of cells known to carry antigens to be tested under con-ditions effective to bind irreversibly a layer of said cells to said matrix;
(b) applying to said layer (a) a solution con-taining 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 solution 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.

13. A method of assaying antibodies on the surface of a cell population comprising 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 irreversibly 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 reactive by immuno-adsorption with the antibodies to be assayed on said cell surface; and (c) measuring the extent of immunoadsorption which occurs.

14. A method according to claim 13 wherein the ex-tent of immunoadsorption is measured by determining the extent to which a second layer of cells from said cell population to be tested can be adsorbed onto said first layer after appli-cation of said solution of antigens.

15. A method for assaying antigens in the presence of antibodies on the surface of a cell population by competi-tive immunoadsorption comprising the steps of (a) applying to a solid matrix a first suspen-sion of cells known to carry antibodies under conditions effec-tive to bind irreversibly a layer of said cells to said matrix;
(b) contacting said layer (a) with a mixture of (i) a solution to be assayed for antigen, and (ii) a second solution ox suspension containing antibodies reactive by immunoadsorption with the antigen to be assayed by competitive inhibition; and (c) thereafter measuring the extent to which said antigen in solution (i) is bound by immunoadsorption to said layer, (a).

16. A method according to claim 15 wherein the extent of said immunoadsorption is measured by determining the extent to which a second layer of cells carrying an antibody can be adsorbed onto said first layer after applica-tion of said solutions or suspensions of antigen (i) and antibody (ii).

17. A method for assaying cell type or compatibility on a solid matrix comprising the steps of (a) forming a first monolayer of cells ir-reversibly bound to a solid matrix, said cells having intrinsic or acquired antigens thereon;
(b) contacting said layer (a) with a solu-tion containing antibodies. whereby a layer of antibodies is bound by immunoadsorption, to the extent that the antibodies of said solution are reactive with the antigens of the cells of said first layer;
(c) thereafter applying a suspension of second cells, said second cells having antigens thereon; and (d) subsequently measuring the extent of immunoadherence of said second cells by antibody bound to said first layer of cells.

18. A method according to claim 17, wherein said first layer of cells and said second cells are the cells of a first person and said solution is a serum obtained from a second person.

19. A method according to claim 17, wherein layer (c) is formed by applying cells from a cell population to be tested in a number essentially just sufficient to form a monolayer, and the extent of immunoadherence of said mono-layer is a function of the percentage of tested cells that carry specific antigen.

20. A method according to claim 17, wherein said solution containing antibodies is a mixture of (i) a solu-tion of an antibody reactive by immunoadsorption with an antigen carried by the cells of said layer (a) and (ii) a second solution or suspension to be assayed for antigenic determinants by inhibition of antibody in said solution (i).

21. The method according to claim 17, wherein the antigens of said first cell layer (a) are antigens carried by immunoglobulins or complement components bound on the surfaces of said cell population; the antibodies in said solution used in step (b) are anti-immunoglobulin or anti-complement to the antigenic determinants carried by the immunoglobulins or complement components to be assayed on said first cell monolayer; and the cells of said second layer carry antigenic determinants which are immunoadherent to the antibodies employed in step (b).

22. The method according to claim 21, wherein the immunoglobulins or complement components are bound to the cells of said cell layer (a) after the layer (a) has been constructed.

23. The method according to claim 21, wherein the cells bound in step (a) carry or are suspected to carry immunoglobulins or complement components to be assayed.

24. The method according to claim 17, wherein the cell monolayer (a) is formed by applying a suspension of said cells to said solid matrix to be coated, there being, on the surface of said matrix, reactive chemical groups effective to couple said cells to said matrix.

25. The method according to claim 24, wherein said reactive chemical groups are formed by successively treating a solid matrix of a polystyrene with a fibrinogen solution and a polylysine solution under conditions effective to form said reactive chemical groups on the surface of said matrix.

26. 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, said erythrocytes having intrinsic or acquired antigens thereon;
(b) contacting said layer (a) with a solu-tion containing antibodies, whereby a layer of antibodies is bound by immunoadsorption to the extent that the antibodies of said solution are reactive with the antigens of the erythrocytes of said first layer;
(c) thereafter applying a suspension of second erythrocytes, said second erythrocytes having antigens thereon; and (d) subsequently measuring the extent of immunoadherence of said second erythrocytes by antibody bound to said first layer of erythrocytes.

27. The method according to claim 26, wherein said first layer of erythrocytes is lysed prior to application of the second erythrocytes.

28. The method according to claim 27, wherein the erythrocytes of said first layer are lysed after application of said solution (b).

29. A method according to claim 26, wherein said first layer of erythrocytes and said second erythrocytes are the erythrocytes of a first person and said solution is a serum obtained from a second person.

30. A method according to claim 26, wherein layer (c) is formed by applying cells from an erythrocyte population to be tested in a number essentially just sufficient to form a monolayer, and the extent of immunoadsorption of said second monolayer is a function of the percentage of tested erythrocytes that carry specific antigen.

31. A method according to claim 26, wherein said solution containing antibodies is a mixture of (i) a solution of an antibody reactive by immunoadsorption with an antigen carried by the erythrocytes of said layer (a), and (ii) a second solution of suspension to be assayed for antigenic determinants by inhibition of antibody in said solution (i).

32. The method according to claim 26, wherein the antigens of said first erythrocyte layer are antigens carried by immunoglobulins or complement components bound on the surfaces of said erythrocyte population, the antibodies in said solution used in step (b) are anti-immunoglobulin or anti-complement to the antigenic determinants carried by the antibody or complement to be assayed on said first erythrocyte monolayer, and the erythrocytes of said second layer carry antigenic determinants which are immunoadherent to the anti-bodies employed in step (b).

33. The method according to claim 32, wherein the immunoglobulins or complement components are bound to the erythrocytes of said layer (a) after said layer (a) has been constructed.

34. The method according to claim 26, wherein the erythrocyte monolayer (a) is formed by applying a suspension of said erythrocytes to said solid matrix to be coated, there being, on the surface of said matrix, reactive chemical groups effective to couple said erythrocytes to said matrix.

35. The method according to claim 34, wherein said reactive chemical groups are formed by successively treating a solid matrix of a polystyrene with a fibrinogen solution and a polylysine solution under conditions effective to form said reactive chemical groups on the surface of said matrix.

36. A method for assaying agglutinins in a serum comprising the steps of (a) forming a first monolayer of cells irre-versibly bound to a solid matrix, said cells being known to have intrinsic or acquired antigens corresponding to the agglutinin to be assayed;
(b) contacting said layer (a) with a solution to be assayed for agglutinins;
(c) thereafter applying a second suspension of cells, said second cells known to have or to carry antigens to the agglutinins to be assayed; and (d) subsequently measuring the extent of immunoadherence of said second cells to said first cell layer.

37. A method according to claim 36, wherein the first cell layer is lysed prior to application of said second cells.

38. A method according to claim 37, wherein the erythrocytes of said first layer are lysed after application of said solution (b).

39. A method according to claim 36, wherein said cell monolayer (a) is formed by applying a suspension of said cells to said solid matrix to be coated, there being on the surface of said matrix reactive chemical groups effective to couple said cells to said matrix.

40. A method according to claim 39, wherein said reactive chemical groups are formed by successively treating a solid matrix of polystyrene with a fibrinogen solution and a polylysine solution under conditions effective to form said reactive chemical groups on the surface of said matrix.

41. A method for assaying cell type or com-patibility on a solid matrix comprising the steps of (a) forming a first monolayer of washed erythro-cytes irreversibly bound to a solid matrix, said erythrocytes having intrinsic or acquired antigens thereon;
(b) contacting said 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 said first monolayer of erythrocytes to the ex-tent that the antibodies of said solution are reactive with the antigens of the erythrocytes of said first layer;
(c) thereafter applying a suspension of second erythrocytes, said second erythrocytes having antigens thereon, and allowing said second erythrocytes to settle on said solid matrix as a second monolayer over said first layer of erythro-cytes;
(d) after the said second monolayer has formed, adding protamine sulphate to said second suspension in an amount sufficient to promote immunoadsorption of said second erythrocytes to antibodies bound to said first monolayer; and (e) thereafter washing to remove second erythrocytes which are not immunobound, and measuring the extent of immunoadherence of said second erythrocytes to anti-bodies bound to said first layer of erythrocytes.

42. The method according to claim 41 wherein between steps (a) and (b) and between steps (b) and (c), the first monolayer is washed to remove nonadherent cells and unwanted reagents.

43. The method according to claim 41 wherein following step (b) said first monolayer of erythrocytes is hypotonically lysed with distilled water.

44. 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 irre-versibly adhered to said face, said red blood cells having been lysed to render the cells transparent, said cells carrying the antigenic determinants to be assayed.

A substrate according to claim 44 having in addition, antibodies immunoadsorbed by the antigenic determinants to be assayed on said monolayer of red blood cells.

46. 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 said face having reactive chemical groups thereon, effective to bind cells to said substrate;
(c) a monolayer of red blood cells adhered to said face by said layer (b), said red blood cells having been lysed to render the cells transparent, said cells carrying the antigenic determinants to be assayed.

47. A substrate according to claim 46 having, in addition, antibodies immunoadsorbed by the antigenic determinants to be assayed on said monolayer of red blood cells.

48. A substrate for assaying cell type or com-patibility consisting essentially of (a) a light transmitting solid support member of polystyrene with a face adapted to permit measure-ment of a surface layer thereon;

(b) a layer of fibrinogen bound to said face of the polystyrene substrate; and (c) a layer of polylysine bound to said fibrinogen, said polylysine layer being capable of irre-versibly binding a monolayer of cells to be assayed thereto.

49. A substrate according to claim 48 having a monolayer of cells irreversibly bound to said polylysine layer, said monolayer of cells carrying an antigenic de-terminant to be assayed.

50. A method for assaying cell type or compati-bility on a solid matrix comprising the steps of (a) forming a first monolayer of cells irreversibly bound to a solid matrix, said cells having or carrying antigens thereon;
(b) contacting said layer (a) with a solu-tion containing antibodies capable of forming a layer of antibodies bound by immunoadsorption to said first layer of cells to the extent that the antibodies of said solution are reactive with the antigens of the cells of said layer;
and (c) thereafter measuring the extent of im-munoadsorption of said antibodies by applying a second solu-tion containing lytic complement effective to lyse the cells of said layer (a) in the presence of immunoadsorbed antibody and observing the extent of immune lysis which occurs.

51. A method according to claim 50 wherein the cells of said first layer are cells of a first person and said solution containing antibodies is a serum obtained from a second person.

52. The method according to claim 50 wherein said cell layer (a) is formed by applying a suspension of cells to said solid matrix to be coated, there being, on the surface of said matrix, reactive chemical groups effec-tive to couple said cells to said matrix.

53. The method according to claim 52 wherein the cells of said first layer are erythrocytes.

54. The method according to claim 52 wherein said reactive chemical groups are formed by successively treating a solid matrix of a polystyrene with a fibrinogen solution and a polylysine solution under conditions effective to form said reactive chemical groups on the surface of said matrix.

55. The method according to claim 51 wherein the cell monolayer (a) is formed by applying a suspension of said cells to said solid matrix to be coated, there being, on the surface of said matrix, reactive chemical groups effective to couple said cells to said matrix.