GB2117514A - Polyvalent antiglobulin reagent - Google Patents

Abstract

A polyvalent antiglobulin reagent containing, in admixture, anti-IgG antibodies and one or more anti-C3d monoclonal antibodies (preferably the reagent also contains one or more anti-C3c monoclonal antibodies and/or one or more anti-C3g monoclonal antibodies) which may be used to identify human blood cells coated with either incomplete IgG antibodies or complement.

Description

SPECIFICATION Polyvalent antiglobulin reagent The present invention relates to a polyvalent antiglobulin reagent and to antiglobulin tests that employ said reagent.

Antibodies to blood group antigens are characterised as either "complete or "incomplete". "Complete" (IgM) antibodies react with red blood cells suspended in saline media to form agglutinates which may be visually identified. By contrast, "incomplete" (IgG) antibodies merely coat red blood cells in saline media without causing agglutination. In order to produce a visual reaction in this latter case a third substance, generally an antiglobulin reagent, must be added to the reaction mixture.

The use of anti-human globulin serum to detect red cell-bound, non agglutinating antibodies (the antiglobulin or Coombs test) is now both widespread and routine. The success of these antiglobulin reagents is not only attributable to their ability to detect IgG however. A later discovery showed that antibodies, present in correctly prepared antiglobulin sera, that had been known up to the date of the discovery as anti-non-gamma, were in fact directed against certain of the components of complement.Thus it was realised that anti-human globulin serum detects not only non-agglutinating antibody molecules but also molecules of complement that have become attached to red cells following in vivo or in vitro antigen-antibody reactions. (Complement is a complex substance, with at least 11 discrete components, which occurs naturally in the blood plasma of man and which takes an active part in both serologic, hemolytic and complement fixation reactions. Complement's components are designated C1, C2, C3, C4 etc, with their numerals referring to their order of discovery. Each component is further sub-divided (eg C3b, C3d, C3g) according to the antigenic determinants present on the molecule). Originally antiglobulin reagents were prepared by injecting human serum (or purified globulin from human serum) into a suitable laboratory animal, generally a rabbit or a goat.The human serum globulin then acted as a foreign antigen and the animal responded by producing antibodies directed against human globulin. After a suitable course of immunisation the animal was bled. Unwanted antibodies (such as anti-species, anti-A, anti-B and anti-H) were removed by suitable absorption and the serum was diluted to a point where several anti-human globulin antibodies reacted optimally. More recentlyantiglobulin reagents have been prepared by first producing anti-lgG and anti-complement (using separate immunisation procedures) and then blending these antibodies in suitable ratio. This latter method of preparation allows the levels of anti-lgG and anti-complement in a given reagent to be controlled. This in turn allows the reagent to be formulated so that every component is present at its optimum dilution.

Once prepared the antiglobulin reagent may be used in 2 types of antiglobulin test: a. the direct test which detects red cells already coated with antibody in vivo, and b. the indirect test, in which the red cells must first be coated with antibody in vitro and then, after suitable preparation, tested with antiglobulin reagent.

The direct test consists of washing the already coated cells, then mixing with antiglobulin serum, centrifuging, and examining for agglutination.

This test is used in the diagnosis of erthyroblastosis fetalis, in which the red cells of the infant or the cells of the cord blood have been coated with antibody in utero; in the diagnosis of autoimmune hemolytic anemeia, in which the red cells have also been coated in vivo; and in the investigation of transfusion reactions, in which an incomplete antibody is suspected and the transfused cells would then have become coated in vivo.

In the indirect test the cells are suspended in saline and incomplete antibody antiserum is added.

After mixing and incubating to allow time for the antibody and the cells to combine, the cells are washed thoroughly to remove all free antibody and protein, and antiglobulin reagent is added, and the test completed by the spin tube ortile procedure as appropriate.

This test is used in cross-matching to detect incompatibility in which a complete antibody may be involved, in detecting and identifying irregular antibodies, in detecting antigens not demonstrable by other methods and in mixed agglutination tests. The major disadvantage associated with the preparation of past and present antiglobulin reagents is the extensive absorption of the animal serum with human red cells that must be undertaken to remove all traces of anti-human red cell activity. If this absorption is not adequately performed, false reactions may occur. The purification of all the components of an antiglobulin reagent by this adsorption procedure is time-consuming, however it is particularly onerous in the case of anti-C3.For example it is the present inventors experience that in order to arrive at a suitable anti-C3 serum, donations of human red cells of the groups A, B and 0 have first to be washed 9 times to remove all traces of human serum (that would otherwise neutralise the anti-C3 antibodies) and then the animal serum has to be adsorbed until free of false reactions for human red cells. It is one object of the present invention to provide a polyvalent antiglobulin reagent the preparation of which eliminates at least some of the presently required adsorption steps. Other objects and advantages of the present invention will be apparent from the following description thereof.

According to the present invention there is provided a polyvalent antiglobulin reagent comprising, in admixture, anti-lgG antibodies and one or more anti-C3d monoclonal antibodies. Preferably the reagent also comprises one or more anti-C3c monoclonal antibodies and/or one or more anti-C3g monoclonal antibodies.

The anti-lgG antibodies blended into the present reagent must be both potent enough and of broad enough spectrum to detect all clinically significant IgG antibodies by spin tube or tile methods. However the dilution chosen must not produce zoning with any antibody sensitised cell. They may be mixtures of monoclonal antibodies or, which is preferred, conventional IgG antisera prepared by known immunisation techniques. Although the preparation of conventional polyvalent IgG antisera involves the adsorption of the animal serum with human red cells, in this case the number of adsorptions required is small.It follows that, at present, it is more cost effective to employ the well documented animal immunisation techniques to produce IgG antisera for the present reagent than to prepare and blend all of the monoclonal anti-lgG antibodies that would be necessary to provide a reagent of equivalent breadth and potency.

It should be noted that anti-lgG antisera, when prepared by immunisation techniques are often contaminated by a low level of anti-lgA antibodies. This is another reason to prefer, at present, conventional antisera to mixtures of monoclonal antibodies, since to match the breadth and potency of anti-lgA containing antisera the monoclonal mixture would also have to contain monoclonal anti-lgA antibodies.

It is important to note that in view of the above the present invention also encompasses antiglobulin reagents that contain low levels of anti-lgA antibodies. Preferably these antibodies are contaminants in conventional IgG antisera, however they may be monoclonal anti-lgA antibodies added to monoclonal anti-lgG antibodies to provide a reagent with a breadth and potency equivalent to that produced by conventional IgG antisera.

In the present specification an anti-C3d monoclonal antibody is a monoclonal antibody, raised against the C3 component of complement, which reacts with the trypsin resistant C3 component of C3 coated cells. An anti-C3g monoclonal antibody is a monoclonal antibody, raised against the C3 component of complement, which reacts with the trypsin sensitive component of < 2D ( C2D is equivalent to C3g plus C3d). An anti-C3c monoclonal antibody is a monoclonal antibody, raised against the C3 component of complement, which reacts with the component of complement which is found only on freshly prepared C3b and C3bi coated cells, which is absent from t2D and which is equisitely sensitive to destruction by traces of trypsin.

The anti-C3d, anti-C3c and anti-C3g monoclonal antibodies will preferably be of the HL type. Preferably they are derived from hybrid myelomas produced by the fusion of mouse or rat myeloma cells with spleen or other immunocyte cells from mice or rats immunised with human antigen C3d, C3c or C3g. Preferably the myeloma cells are selected from the mouse myeloma cell line NS1 (abbreviated from P3-NS1/1-Ag 4-1; G.

Kohler, S.C. Howe and C. Milstein, Eur. J. Immunol, 1976, 6, 292;) the mouse myeloma cell line NSO/1 (G.

Galfre and C. Milstein, Methods in Enzymology, 1981,73,3) both available from the MRC Laboratory of Molecular Biology, Hills Road, Cambridge, and the rat myeloma cell line Y3-Ag 1.2.3 (CNCM 1-078) available from the Pasteur Institute in Paris. Thus in one embodiment of the present reagent 2 anti-C3d monoclonal antibodies (derived respectively from the rat x rat hybrid myeloma YB2/3HL and from the mouse x mouse hybrid myeloma BRIC 8) are mixed with IgG antisera (produced by an immunisation procedure). In another embodiment of the present reagent either or both of the anti-C3d monoclonal antibodies (derived respectively from YB2/3HL and BRIC 8) are mixed with an anti-C3c monoclonal antibody (derived from the rat x rat hybrid myeloma YB2/4HLK) and IgG antisera (produced by an immunisation procedure).In yet another embodiment of the present reagent either or both of the anti-C3d monoclonal antibodies (derived respectively from YB2i3HL and BRIC 8) are mixed with the anti-C3c monoclonal antibody (derived from YB2 4HLK), an anti-C3g monoclonal antibody (derived from the rat x rat hybrid myelom YB2/9HLK) and IgG antisera (produced by an immunisation procedure).

The monoclonal antibodies used to form the present reagent may be separated either from the ascitic fluids of tumours produced in animals, generally mice or rats or from tissue culture supernatants. The anti-C3 antibodies in the present antiglobulin reagent detect complement binding antibodies that may be of the IgM type and therefore not detected by anti-lgG (examples of such complement binding antibodies are anti-Lea, anti a and some anti-Kells). Two of them (anti-C3d and anti-C3g) also detectC3gd, previously known as X2D, that is found on the cells of patients suffering from Acquired Haemolytic Anaemia due to cold antibodies of high titre that can result in complement activation and hence red cell destruction.

The primary advantage of the present antiglobulin reagent is that the anti-C3 monoclonal antibodies do not have to be purified by absorption with human red cells. This leads to a considerable saving in both man-hours and materials. A further advantage of the present reagent is that anti-C3 monoclonal antibodies are more specific than their conventional C3 antisera counterparts and this allows standardisation work to be improved so that reagents of clearly defined anti-C3 characteristics are obtained with a consequent improvement in batch to batch reproducibility. A still further advantage of the present reagent is that anti-C3 monoclonal antibodies may define new determinants for use as research tools and/or in diagnosis.

The working dilution of anti-C3d monoclonal antibody is determined by the need to detect the C3d of Cold Agglutinin disease cases (CHAD) without causing false positives. The chosen dilution is therefore a compromise, too little meaning failure to detect CHAD cases, too much meaning an increase in false positives (since normal human red cells do have some C3d on their surface). The proper level can only be found in an empirical manner using a standardisation procedure, however it is the experience of the present inventors that a dilution in the region 200-1000 is particularly advantageous.

Although the presence of anti-C3c and/or anti-C3g monoclonal antibodies in the present reagent is optional, the presence of both is preferred. In particular it has been found that mixtures of anti-C3c and anti-C3d monoclonal antibodies give a better reaction than is achieved by either alone. In the case of anti-C3c and anti-C3g monoclonal antibodies the optimal working dilution is the one that gives the maximum reactions with a range of antibody sensitised cells in a standardisation procedure. For these antibodies the detection of CHAD cases is not relevant to the question of dilution. In the experience of the present inventors a dilution, for both anti-C3c and anti-C3g monoclonal antibodies, in the region of 600-1000 is particularly advantageous.

The present antiglobulin reagent will be used to test for either incomplete (IgG) antibodies or blood cells coated with complement. In order to facilitate this test there is also provided a process for the identification of human blood cells coated with either incomplete (IgG) antibodies or complement comprising adding to a suspension of coated cells in sodium chloride solution, a polyvalent antiglobulin reagent comprising, in admixture, anti-lgG antibodies and one or more anti-C3d monoclonal antibodies. Preferably the reagent also comprises one or more anti-C3c monoclonal antibodies and/or one or more anti-C3g monoclonal antibodies.

In an indirect antiglobulin test the cells are coated by IgG or complement in vitro. In a direct antiglobulin test the cells are coated in vivo.

The preferred antibodies, the preferred methods of preparation and the preferred dilutions are as listed above. Once again the reagent may contain a low level of contaminating anti-lg A antibodies. The present blended antiglobulin reagents are as stable as the antibodies used in the blend when stored at temperatures of 4"C and below.

Once the present reagent has been added to the coated blood cell suspension in sodium chloride solution, the test is generally continued by observing whether or not agglutination of the cells takes place. Generally if the present reagent contains an antibody specific for an IgG antibody or complement component(s) coated onto the test cells, then agglutination will take place. Such agglutination is a visible reaction and therefore may be followed visually in a qualitative or quantitative manner. On the other hand if the present reagent does not contain an antibody specific for the IgG or complement coated to the cell then, generally, unless a cross reaction takes place, no agglutination will occur. (Cross reaction refers to both false reactions and auto antibody sensitised cells).For convenience the present antiglobulin reagent may be provided for use in the above process in the form of a reagent kit. In one embodiment of this kit the anti-lgG antibodies, the anti-C3d monoclonal antibodies and, optionally, the anti-C3c monoclonal antibodies and/or anti C3g monoclonal antibodies are provided in the form of a mixture. In a second embodiment the anti-lgG antibodies, the anti-C3d monoclonal antibodies and, optionally, the anti-C3c monoclonal antibodies and/orthe anti-C3g monoclonal antibodies are provided in separate containers together with instructions on the method of mixing.

The reagents, processes and kits of the present invention will now be described by way of example only.

MATERIALS AND METHODS Analysis to determine antibody heavy and light chains The myelomas NS1 and Y3-Ag 1.2.3 each produces K light chains which can be mixed with the light chains of spleen cell origin and the antibody secreted as mixed chain types called HLK. Full antibody activity requires both light and heavy chains, hence HLK antibody molecules are defective as some of the light chains are non-antibody myeloma K chains and thus reduce the antibody binding efficiency of the molecules. Therefore it is obviously desirable to select HL variants to obtain the best agglutinating antibodies. Recently myelomas which do not produce K chains have become available (eg NS0/1) and this eliminates the HLK problem.Composition is established by radio-autograph of electrophoretic runs detecting the C14 lysine incorporated into the antibodies secreted by the hybrid-myeloma cells. There are 2 types of procedure for separating the antibody compartments in polyacrylamide gel slabs for susequent radioautograph analysis: a. Isoelectric focussing (IEF). The separation is based on pH gradient separating components having different isoelectric points with prior reduction by 2-mercaptoethanol to break the interchain links.

b. Electrophoresis of the reduced antibodies with sodium dodecyl sulphate polyacrylamide slab gel electrophoresis (SDS-PAGE). The separation is based on molecular sieving of the different sized components that have a similar negative charge by being coated with SDS.

Standard reagents Rabbit antiglobulin reagent CBT-14 is obtained by immunisation procedures and is available on request from Dr D. Voak, National Blood Transfusion Service, Regional Transfusion and Immuno-Haematology Centre, Long Road, Cambridge CB2 2PT.

Antiglobulin tests These were either spin tube or tile tests.

Spin tube method standard method) a. Mix 2 volumes serum and 1 volume 5% cells.

b. Incubate at 370C for lhr c. Wash4timesand remove all supernatantwash.

d. Add 2 volumes of antihuman globulin (AHG) reagent and mix by shaking the tubes.

e. Spin for 1 min at 900 rpm in a centrifuge of radius 13cm or using any centrifuge giving an RCF of 100 to 120.

f. Read on a slide by the normal technique for reading tube haemagglutination reaction.

NB: The use of Sorvall Machines. Experience has shown that in the final spin with AHG, the low speed setting of 1,200 rpm for 20-30 seconds gives excellent results. Longer spin times at 1,200 rpm do not increase the sensitivity of the test and may cause false positives due to overspin effects.

Tile method a. Mix 2 volumes of serum and 1 volume 50% cells.

b. Incubate at 37C c. Wash 4times and remove all supernatantwash.

d. Place 1 volume of AHG reqgent onto the tile, add 1 volume 50% cells, mix, rock the tile gently for 10 seconds, then leave the tile on the bench for 5 minutes before reading the test over a light source.

Preparation oflgG antisera The IgG fraction was prepared from whole human serum by ion exchange chromatography using DEAE Sephacel on a 26 x 400 mm column, (K26/40, Pharmacia Fine Chemicals). Rabbits were then immunised with IgG in Freunds Complete Adjuvant as shown in Table 1.

TABLE 1 Primary Animals Immunisation Booster Bleed Rabbits (80% 10-12 mg/ml 5.0 mg/ml 40 ml on success). IgG in IgG in 2 5 Reject Freunds ml saline successive animals not Complete days up to Adjuvant Test Test standard bleed bleed after2 5 x 0.2 ml after8 after boosters site sub- weeks IV 8 cutaneous weeks along back Note: a. Standard required tile tests with titred anti-lgG.

1. 5 + reaction to a dilution of 1/320 x strong anti-D sensitised cells. (IgG anti-D Alb titre 16 x R1r).

2. ++ reaction to a dilution of 1/160 x anti-Fya sensitised cells.

The rabbit anti-igG serum containing added sodium azide as a preservative (0.001%) was then heated at 56"C for 20-30 minutes to destroy complement and thus avoid haemolysis during adsorption of the serum.

The heated anti-lgG serum diluted with an equal volume of phosphate buffered saline was absorbed with well washed (10 x) human red cells of the groups A1, B and 0 to remove antibodies to human red antigens.

The optimum dilution of the adsorbed anti-lgG serum was then selected by a standardisation procedure. In one preferred procedure doubling dilutions of the anti-lgG were first tested against a range of anti-D sensitised cells to ensure that the working dilution will not "zone" due to anti-lgG excess with weak anti-D sensitised cells. Then the working dilution was shown to have adequate avidity (using both spin tube and tile methods) for a wide range of antibodies, paying particular attention to anti-Fya antibodies. Using this procedure dilutions of up to about 200 are often found to be the optimum.

EXAMPLE 1 Preparation of anti-C3d monoclonal antibodies frat x rat) A. The antigen used was prepared as follows. A suspension of Inulin (British Drug Houses) 100 mg/ml in phosphate buffered saline was sonicated for 1 min. 0.5 ml was treated with 10 ml of normal human serum (made to 10 m M with EGTA and 7 m M with Mg) for 15 min at 37"C. This allows activation of the alternative complement pathway and fixation of C3 to the inulin. The suspension was washed in physiological saline, in 2M NaCI and again in physiological saline before being made up to the original volume. AO rats were given 0.5 ml of antigen emulsified in Freund's complete adjuvant in multiple intramuscular sites. Three weeks later each rat was given 0.5 ml antigen (without Freund's adjuvant) ip with 5 x 109 B. pertussis. Four weeks later 0.5 ml was given iv to each rat with no further adjuvant and the suceeding month 5 iv injections were given.

Fusion was carried out on the third day following the last iv injection.

B. The spleen of a chosen rat was removed under sterile conditions, placed in 5 ml fresh medium without serum (Dulbecco's MEM) in a small petri dish on ice. After some cuts were made in the spleen, it was transferred to a 10 ml plastic round-bottomed tube containing 4 ml medium without serum and disrupted with a teflon pestle (loose fit, ie 2 mm clearance). It was left on ice for 1 min and the top 3.5 ml transferred to a plastic universal tube. The remainder of the spleen was washed with 5 ml medium without serum, and combined with the other cells after 1 min to allow major debris to settle. The universal tube was topped up and spun at 600 rpm for 7 min on a bench centrifuge. The cells were resuspended, washed and resuspended again.From this, 108 cells were taken and mixed with 107 Y3 Ag 1.2.3 washed cells in a 50 ml conical centrifuge tube. (In more recent experiments with this line 5 x 107 Y3 cells have been used with 105 spleen cells). Medium without serum was added and the cells were centrifuged. The tube containing the drained cell pellet was put in a 37"C water bath and tapped gently. 0.8 ml PEG solution kept at 37"C was added to the cells over a period of 1 min. A pipette was used during addition for gentle mixing. After further mixing for another minute, 2 ml of medium without serum were added over a period of 2 min, 8 ml over 3 min and a further 10 ml added dropwise. The volume was then made up to 45 ml.The cells were spun, resuspended very gently first in a few drops and then in 25 ml pre-warmed medium (Dulbecco's MEM + 20% FCS). The cells were distributed in 48-96 wells in Limbro plates. About 105 washed spleen cells were added to each well (in more recent experiments a monolayer of irradiated 3T3 fibroblasts has been used instead), and incubated at 37 C.

Next day, 1 ml medium was removed and 1 ml HAT medium containing 20% heat-inactivated FCS added and the operation repeated on the next 2 days and every 2-3 days thereafter.

C. The culture supernatants were tested 2 weeks post fusion and those cells with binding activity on C3 coated cells were cloned on soft agar and grown to larger volumes (as described in R.G.H. Cotton etal; Eur J Immunol, 19733, 136). They were then recloned and regrown twice more and finally divided into aliquots for larger scale antibody production and liquid nitrogen storage of the selected cell line(s).

An anti-C3d secreting clone (YB2/3HL) was identified by ability of its antibody to react with the trypsin resistant C3 component of C3 coated cells and by the negative response of its antibody to C4 coated blood cells. The chain composition of the antibody was HL.

D. About 108 cells of the established anti-C3d secreting clone (YB2/3HL) were injected into AO/LOU rats.

Solid tumours developed after about 3 weeks. The serum derived from these animals contained in all cases about 5-10 mg/ml of anti-C3d monoclonal antibodies.

E. As an alternative to (D) above, the establishd anti-C3d secreting clone (YB2/3HL) was grown in Dulbecco's modified Eagle's medium (DMEM, Gibco Biocult) supplemented with fetal calf serum (FCS, Seral-Lab). The concentration of FCS is gradually reduced from 15 to 5%. The tissue culture supernatants were centrifuged, to remove cells and debris, supplemented with 10 m M Hepes and 0.1% sodium azide and stored at -20 C.

EXAMPLE 2 Prepration of anti-C3d monoclonal antibodies (mouse x mouse) The method of preparation was essentially that described by G Kohler and C Milstein in Nature, 1975,256, 495. The hybridoma cell line was prepared by fusing spleen cells from a mouse immunised with purified C3d protein with a mouse myeloma cell line (P3-NSI/1-Ag 4-1). The cells from the hybridoma cell line thus produced (BRIC 8) were injected into mice to produce tumours and the monoclonal anti-C3d antibody was present in the ascitic fluids.

EXAMPLE 3 Preparation of anti-C3g monoclonal antibodies (rat x rat) The method described in Example 1 was repeated except that an anti-C3g secreting clone (YB2/9HLK)was identified by the reaction of its antibody with the trypsin sensitive component of 2D.

The chain composition of the antibody was HLK.

EXAMPLE 4 Preparation ofanti-C3cmonoclonalantibodies frat x rat) The method described in Example 1 was repeated except that an anti-C3c secreting clone (YB2/4HLK) was identified by the ability of its antibody to react with the component of complement which is found only on freshly prepared C3b and C3bi coated cel Is, which is absent from oc2D and which is destroyed by trypsin.

The chain composition of the antibody was HLK.

Characterisation of anti-C3 monoclonal antibodies produceed by hybrid myelomas i. The monoclonal antibodies formed by the methods of Examples 1,3 and 4 were characterised by their reaction with C3 coated red cells produced by the following methods or from the following sources: a. The low ionic, low temperature method of M.J. Fruitstone, Transfusion, 1978, 18, 125, using 7.5 x 10-4M Mg2+.

b. The low ionic method of D.E. Jenkins Jr etal, Transfusion, 1975, 15,402.

c. Cold Haemagglutinin Disease (CHAD) cells, and d. The low ionic, low temperature method of Fruitstone followed by treatment of the cells with 0.05% trypsin for 5 mins.

Results are given in Table 2.

TABLE 2 Characterisation of monoclonal antibodies produced by hybrid myelomas Method of C3 coat HybridMyeloma C3 coated on red cell production blood red YB2/3HL YB219HLK YB2/4HLK Low ionic, Low temperature (Fruitstone) 3b + Nega + Low ionic 37"C (Jenkins) 3bi + + + CHAD oc2D + + Neg Trypsin treated C3 coated cells 3d + Neg 0 Note: a. In C3b the C3g component is concealed. It is revealed by the action of Factor 1 (KAF).

ii. The monoclonal antibodies produced in Examples 1,3 and 4were also characterised by their reaction with a. C3b coated red cells formed by the method of Fruitstone, b. C3b coated red cells treated with 6.4 x 10-5% trypsin, and c. C3b coated red cells treated with 0.05% trypsin.

In each case the monoclonal antibody was enhanced by sheep anti-rat IgG (1 in 200).

Results are given in Table 3.

TABLE 3 Characterisation of monoclonal antibodies produced by hybrid myelomas A. Monoclonal antibody produced by hybrid myeloma YB2/3HL Dilution 320 640 1280 2560 5000 10000 20000 40000 80000 160000 Trypsin level (%) Nil W GW (+) + ++ ++ ++ ++ + (+) 6.4x10-5 W GW (+) + ++ ++ ++ ++ + (+) 0.05 W GW (+) + ++ ++ ++ ++ + (+) B. Monoclonal antibody produced by hybrid myeloma YB219HLK Dilution 320 640 1280 2560 5000 10000 20000 40000 80000 160000 Trypsin level (%) Nil W W (+) + + + + (+) GW W 6.4x10-5 W W (+) + + + + (+) W W 0.05 W GW GW 0 0 0 0 0 0 0 C.Monoclonal antibody produced by hybrid myeloma YB2/4HLK Dilution 320 640 1280 2560 5000 10000 20000 40000 80000 160000 Trypsin level (%) Nil W + ++ ++ ++ ++ ++ + GW W 6.4x10-5 W 0 0 0 0 W W W (+) (+) 0.05 0 0 0 0 0 0 0 0 0 0 From the results given in Tables 2 and 3 it can be seen that hybrid myeloma YB2/3HL produces anti-C3d, hybrid myeloma YB2/9HLK produces anti-C3g and hybrid myeloma YB2/4HLK produces anti-C3c.

EXAMPLE 5 Blending anti-lgG with monoclonal antibody from YB2/3HL and from BRIC 8 An antiglobulin reagent was prepared by mixing in final concentration (in a 1:1:1 (v/v/v ratio) anti-lgG (80 fold dilution), anti-C3d supernatant(l50 fold dilution, Example 1) and anti-C3d supernatant (1000 fold dilution Example 2).

EXAMPLE 6 The reagent of Example 5 was prepared except that the anti-C3d from Example 1 was diluted 300 fold.

EXAMPLE 7 Blending anti-lgG with monoclonal antibody from BRIC 8 An antiglobulin reagent was prepared by mixing in final concentration (in a 1:1 (v/v) ratio) anti-lgG (80 fold dilution) and anti-C3d (1000 fold dilution, Example 2).

EXAMPLE 8 Blending anti-lgG with monoclonal antibody from YB2/4HLK and from YB2/3HL An antiglobulin reagent was prepared by mixing in final concentration (in a 1:1:1 (v/v/v) ratio) anti-lgG (80 fold dilution), anti-C3c (600 fold dilution, Example 4) and anti-C3d (1000 fold dilution, Example 1).

EXAMPLE 9 Blending anti-lgG with monoclonal antibody from YB2/4HLK, from YB2/3HL and from YB219HLK An antiglobulin reagent was prepared by mixing in final concentration (in a 1:1:1:1 (v/v/v/v) ratio) anti-lgG (80 fold dilution), anti-C3c (00 fold dilution, Example 4), anti-C3d (1000 fold dilution, Example 1) and anti-C3g (600 fold dilution, Example 3).

EXAMPLE 10 Spin tube antiglobulin tests A range of cells either uncoated or coated with one of IgG, complement or antibody were tested by the spin tube method using a variety of antiglobulin reagents. A comparison of the results for a conventional rabbit reagent (CBT-14) prepared by immunisation techniques with those of 5 reagents prepared by mixing anti-lgG antibodies and various anti-C3 monoclonal antibodies is given in Table 4.

TABLE 4 Example Reagent Type of Coated Cell Non Sensitised EWeak D EC4 EC3/C4 EC3d EALea ECJKa Anti-IgG - ++ - - - - Rabbit antiglobulin reagent CBT-14 - ++ + +++ +++ + + Anti-lgG (dlution 80), 5 YB2/3HL(150), Mic(1000) - ++ - +++ ++ + ++ 6 Anti-lgG(80), YB2/3HL(300), Mic(1000) - ++ - +++ ++ + ++ 7 Anti-lgG(80), Mic(000) - ++ - ++ + + + 8 Anti-lgG(80), anti-C3c(600), anti-C3d(1000), - ++ - +++ +++ +++ +++ 9 Anti-lgG(80), anti-C3c(600), anti-C3d(1000), anti-C3g(600) - ++ - +++ +++ +++ +++ NB: E = erythrocyte (red cell), EC=red cell + complement, EA= red cell + antibody

Claims (14)

1. A polyvalent antiglobulin reagent comprising, in admixture, anti-lgG antibodies and one or more anti-C3d monoclonal antibodies.

2. A polyvalent antiglobulin reagent according to claim 1 further comprising one or more anti-C3c monoclonal antibodies.

3. A polyvalent antiglobulin reagent according to either claim 1 or claim 2 further comprising one or more anti-C3g monoclonal antibodies.

4. A polyvalent antiglobulin reagent according to any one of claims 1 to 3 wherein the anti-lgG antibodies are in the form of polyvalent IgG antisera prepared by immunisation techniques.

5. A polyvalent antiglobulin reagent according to any one of claims 1 to 4 further comprising anti-lgA antibodies.

6. A polyvalent antiglobulin reagent according to any one of claims 1 to 5 wherein the monoclonal antibodies are derived from hybrid myelomas prepared by fusion of spleen cells with either mouse myeloma cells, selected from NSI cells or NSO cells, or rat myeloma cells desginated CNCM 1-078.

7. A polyvalent antiglobulin reagent according to claim 1 substantially as herein before described with particular reference to any one of Examples 5 to 9.

8. A process for the indentification of human blood cells coated with either incomplete IgG antibodies or complement comprising adding to a suspension of coated cells in sodium chloride solution, a polyvalent antiglobulin reagent according to any one of claims 1 to 7 and detecting the agglutination or non-agglutination of the cells within the solution.

9. A process for the identification of human blood cells coated with either incomplete IgG antibodies or complement according to claim 8 substantially as hereinbefore described with particular reference to Example 10.

10. A polyvalent antiglobulin reagent kit for use in the process of claim 8 comprising a polyvalent antiglobulin reagent according to any one of claims 1 to 7.

11. A polyvalent antiglobulin reagent kit for use in the process of claim 8 comprising, in separate containers.

(a) anti-lgG antibodies, and (b) one or more anti-C3d monoclonal antibodies.

12. A polyvalent antiglobulin reagent kit according to claim 11 further comprising one or more anti-C3c monoclonal antibodies.

13. A polyvalent antiglobulin reagent kit according to either claim 11 or claim 12 further comprising one or more anti-C3g monoclonal antibodies.

14. A polyvalent antiglobulin reagent kit according to any one of claims 10 to 13 sustantially as hereinbefore described.