CHARACTERIZATION OR DETERMINATION OF THE AMOUNT OF MAMMALIAN CELLS BY MEANS OF ANTIBODIES

Abstract: A process for the characterization and/or determination of the amount of mammalian cells by the use of antobodies from birds or reptiles on such cells which may interact with antibodies from mammals in any other way than by the antigen-binding activity of the antibodies.

Description: CHARACTERIZATION OR DETERMINATION OF THE AMOUNT OF MAMMALIAN CELLS BYMEANSOFANTIBODIES TECHNICAL FIELD The present invention relates to a process for the characterization or determination of the amount of mamma¬ lian cells according to the technique previously known per se where antibodies are utilized for said purpose. Up to now antibodies prepared in a mammal, especially rabbit, have been utilized for such purposes, but the present in¬ vention relates to the utilization of other types of anti¬ bodies in these connections, whereby disadvantages in con¬ nection with the prior art are eliminated, even in such a way that the invention enables such characterizations or determinations which would otherwise be troublesome or even impossible. BACKGROUND OF THE INVENTION Although the invention is generally applicable to the characerization or determination of many different types of mammalian cells, which ' will be more specifically exemp¬ lified bejLow, it is of a ver- great value in connection with thrombocytes (blood plarelets), especially with refe¬ rence to the central parts of the thrombocyte functions in different connections. Therefore, a background of the in¬ vention will be given with reference to thrombocytes, as the problems to be s" 'ved by the invention and the advan¬ tages thereof will be most obvious thereby. However, cor¬ responding or similar advantages can be achieved in the determination of other mammalian cells. Thrombocytes have a central part of the haemosta~is. It is well known that low throir. cyte numbers in the blood give rise to bleeding complications. There are also data - which indicate that an increased number of activated thrombocytes and/or a reduced activation threshold may have an importance for the development of thrombi. Also such diseases as preeclampsia (a syndrome of pregnants with the symptoms of high blood pressure and urine pro¬ tein) are associated with such alterations of the normal thrombocyte functions. Examinations of the thrombocyte functions can also be expected to become of great impor- tance for decisions as to which patients should receive thrombocyte concentrates. Haemostasis requires aggregation of thrombocytes. However, said aggregation can take place only if the thrombocytes will expose receptors for fibrinogen on their surfaces. In their condition of rest the thrombocytes have a minor capability of binding fibrinogen and then they can not either form aggregates which are the primary haemosta¬ tic plug (the first blocking of a vascular lesion) or part of a thrombosis (a clot). The activation of thrombocytes means that the thrombocytes will get a significantly in¬ creased capability of binding fibrinogen. In other words there is a great demand of determi¬ ning, in the blood of a patient, the amount or portion of the thrombocytes which are activated, i.e. which are high- ly capable of binding fibrinogen. However, those routine methods which have been utilized for testing the thrombo¬ cyte function, e.g. bleeding time and thrombocyte aggre¬ gation, are complex and insensitive and merely give a non¬ specific information about the function. For instance, they will give no picture of the discrete or separate thrombocytes but merely an average value. Furthermore, there are methods for determining proteins which are libe¬ rated from the thrombocytes, but the results thereof are unreliable as they are influenced inter alia by the meta- bolism of the patient. In recent years fluorescence activated flow cytometry has, however, made it possible to develop new assays of thrombocyte functions. In this context laser beams are utilized which illuminate cells in a passing liquid jet. The light scattering gives information about the sizes of the cells and their structures. Further information will be obtained by the addition of antibodies or other sub- stances which have been linked to different substances which when illuminated are caused to produce light (fluo¬ rescence). These methods are rapid and sensitive and can give a specific information about thrombocyte functions. Moreover, it is possible to study each thrombocyte sepa¬ rately and not only an average of all thrombocytes. For an assay a few microliters of whole blood or thrombocyte-rich plasma will be required. The thrombocytes can be differen¬ tiated from other blood cells by means of a specific anti- body or by their sizes. Commercially available fluorescence-labelled rabbit antibodies have been utilized for the study of the fibri¬ nogen binding of thrombocytes in these connections. This is disclosed in an article by R.M. Hardisty et al. in "Measurement of fibrinogen binding to platelets in whole blood by flow cytometry: a micro method for the detection of platelet activation." Br.J.Hematol 1990, 76, 387-394. GENERAL DISCLOSURE OF THE INVENTION According to the present invention it has unexpected¬ ly been shown that by the choice of another kind or type of antibodies than those mammalian antibodies which have previously been utilized for the characterization or de¬ termination of mammalian cells, essential advantages are achieved relative to the prior art. More specifically it has been found that by selecting antibodies from birds or reptiles one can avoid or elimi¬ nate method artefacts appearing in connection with mamma¬ lian antibodies. Thus, mammalian antibodies form immuno complexes with fibrinogen in plasma, which activate the thrombocytes and give rise to erroneous assay results. Ap¬ parently this is not all or to a very reduced extent the case when utilizing the novel antibodies according to the present invention. Although the invention is not limited to any specific theory in this context, it seems as if the antibodies from birds and reptiles do not provoke physio¬ logical reactions in the formation of complexes with anti- gen and interactions with antibody-specific cell recep¬ tors. In other words the present invention is based on the unexpected discovery that in experiments with antibodies against fibrinogen, which antibodies had been prepared by the immunization of hens with human fibrinogen, fibrinogen could be found on the thrombocyte surfaces without any activations of the thrombocytes, and this in spite of the fact that immuno complexes could be observed between plas- ma fibrinogen and fluorescence-labelled hen antifibrinogen antibody (in the experiments thrombocytes and immuno com¬ plexes could be distinguished from each other due to dif¬ ferent light scatterings or by means of a thrombocyte an¬ tibody labelled in any other way, e.g. with another dye or with a radionucleide). The use of hen or chicken antibodies in an assay of the ELISA type is previously known per se (see e.g. Comp. Immun. Microbiol. Infect. Dis., Vol. 13, No. 4, pp 199- 201, 1990) but what is unexpected in connection with the invention is, thus, that a troublesome influence upon the examined or assayed cells is avoided. This means that the process according to the inven¬ tion is intended for the characterization or determination of such cells which interact with antibodies from mammals, or with their complexes with antigen, in any other way than by means of the antigen-binding capability of the an¬ tibodies. This generally means that such cells are refer¬ red to which are able to undergo a transformation or con¬ version, i.e. a change of the surface structure, and where antibodies from mammals, or their complexes with antigens, induce such a transformation or conversion. The purpose of the process according to the invention is to assay or detect in what condition or state the cells are present or to assay or determine the amount or propor- tion of cells in at least one of said states, method arte¬ facts or conversions of said cells being avoided by the invention, which would otherwise give an overrated or er- roneous value of the proportion of cells in either of said states or conditions. Although the invention has been generally described above in connection with chicken or hen antibodies, it should be useful for antibodies from birds or poultry in general as well as for antibodies from reptiles. Further¬ more, it is not necessary to utilize the antibodies per se, as corresponding or similar results would be obtained by immunologically similar proteins, i.e. such proteins which possess essentially the same or similar antigen- binding activity as the antibodies referred to. For in¬ stance it may be fragments of antibodies and recombinant antibodies. More specifically this means that the process accor- ding to the invention is characterized by utilizing, as the antibodies, antibodies from birds or reptiles or im¬ munologically similar proteins having essentially the same or similar antigen-binding activity or capability as said antibodies and by performing the characterization and/or .termination on such mammalian cells which interact with antibodies from mammals, or with their complexes with an¬ tigens, in any other way than by means of the antigen- binding activities of the antibodies, said characteriza¬ tion meaning that one detects the state or condition in which the cells are present and/or the determination mea¬ ning that or.e determines the number r proportion of the cells in at least one of said stat or conditions. As concerns the expression "characterization" of mam¬ malian cells it should be interpreted in a wide sense, es- pecially as the characterization per se can be performed in accordance with known principles, e.g. to detect cer¬ tain surface structures of the cells. Thus, the invention is applicable to any characterization where the mammalian cells interact in the way described or are present in a convertible form and where the convertion could influence upon the measured values or results. According to a preferable embodiment of the invention there are used as said bird antibodies antibodies from hens or chickens, such as from chicken blood. Furthermore, it can often be advantageous rather to utilize hen or chicken antibodies from eggs, as the amount of antibodies thereby obtainable is considerably larger than if chicken blood were used as the source. Especially with reference to what has been mentioned above in connection with thrombocytes it may also be pre- ferable to utilize chicken antifibrinogen antibodies (throughout the specification and claims the term chicken is used to include also hen). In connection with all preferable embodiments concer¬ ning chicken antibodies it should be understood that said applications also comprise uses of the corresponding immu- nologically similar proteins. The process according to the invention is applicable to the characterization or determination of the amount or number of any mammalian cells, e.g. blood cells, such as thrombocytes and white blood cells, or leucocytes, or en- dothelial cells, etc., but for the above-mentioned reasons the characterization and/or determination of thrombocytes is of course especially interesting, the purpose generally being to detect thrombocytes in activated forms or to de- termine the proportion of activated thrombocytes. In this context activation primarily means the capability of bind¬ ing fibrinogen. Furthermore, it can be added that the process accor¬ ding to the invention is preferably performed in the form of fluorescence activated flow cytometry (FACS = Fluore¬ scent Antibody Cell Sorter), especially with reference to the fact that said method is rapid, sensible and gives specific information. EXAMPLES For the purpose of comparing the invention with the use of antibodies according to prior art thrombocytes in plasma were admixed with antibodies from chicken and rab- bit, respectively, directed against the human plasma pro¬ teins fibrinogen, α-2-macroglobulin and IgA. The concen¬ trations of said antibodies were selected in such a way that immuno complexes would be formed to a great extent. The degree of activation was measured by means of fluo- rescence-labelled chicken antibodies directed against fib¬ rinogen. As a negative control there was added merely a saline and as a positive control the known thrombocyte- activating substance adenosine diphosphate. A detailed method description will follow below and the results are presented in the accompanying figure, from which it can be gathered that a strong activation was ob¬ tained with all immuno complexes containing rabbit anti¬ bodies. On the contrary, the only combination with chicken antibodies for which an activation was obtained was in the complex with IgA, but in this specific case the human im- munoglobulin IgA in itself should be responsible for said activation. DETAILED METHOD DESCRIPTION Reagents Normal mouse IgG, adenosine-5-diphosphate (grade 1) and fluorescein isothiocyanate (FITC) were purchased from Sigma (St Louis, Mo, USA). Sephadex G-25 and DEAE-Sepha- rose were purchased from Pharmacia (Uppsala, Sweden). Chicken antibodies, IgG-fractic .; purified from egg yolks, directed against human fibrinogen, human IgA, human alfa- 2 macro' Lobulin and mouse immunoglobulin were obtained from Immunsystem AB (Uppsala, Sweden). The antifibrinogen antibodies were affinity purified on insolubilized human fibrinogen (Larson A. et al J Immunol Meth 1988, 113:93- 99). Some of these were conjugated with FITC after having been dialysed during a night against 0.1 mole/L of NaHCO.,, pH 9.5. The conjugation ratio was 11:1, i.e. 11 parts by mole of FITC per 1 part by mole of antibody. The mixture was incubated at room temperature in darkness for three hours. Conjugated antibodies were separated from free FITC by gel filtration in 0.01 mole/L of Tris, pH 7.3, by gel filtration on Sephadex G-25. Antibodies having the proper F/P-ratio were separated by means of ion exchange chroma- tography on DEAE-Sepharose CL 6B. The column was washed with the Tris-buffer with an addition of 0.1 mole/L of NaCl and the antibodies were eluted by means of a saline gradient to 0.25 mole/L. Non-co jugated and FITC-conju- gated rabbit antibodies against human fibrinogen, human IgA, human alfa-2-macroglobulin and mouse immunoglobulin were purchased from Dakopatts AS (Glostrup, Denmark). Blood testing Venous blood was taken with an open needle without stasis from healthy volunteers who had not taken any medi¬ cine during the last 2 weeks. 4.5 mL of blood were collected in plastic tubes con¬ taining 0.5 mL of 3.8% sodium citrate. Thrombocyte-rich plasma was prepared by centrifugation for 10 minutes at 140 x g. Preparation of samples for flow cytometry 5 μL of thrombocyte-rich plasma were added to plastic tubes containing 50 or 55 μL of HEPES-buffer (137 mmole/L of NaCl, 2.7 mmole/L of KC1, 1 mmole/L of MgCl 2 , 5.6 mmole/L of glucose, 1 mg/mL of bovine serum albumine and 20 mmole/L of HEPES, pH 7.40) and for some experiments 5 μL of antibodies. In some cases ADP had been added to the HEPES-buffer. The samples were carefully mixed once and were left to stand at room temperature for exactly 10 minutes. Then 10 μL of FITC-conjugated chicken antifibri- nogen antibody (0.35 g/L) were added and the test tubes were left to stand for additionally exactly 20 minutes. The reaction was terminated by 500 μL of icecold PBS with or without 1% of paraformaldehyde. No washing steps were needed. The samples were kept in darkness on ice until they were analyzed in the flow cytometer, which was always performed during the same day. Flow cytometry The fluorescence-labelled thrombocytes in the buffer were assayed by means of a FACScan cytometer (Becton Dickinson, Mt View, California, USA) equipped with a 15 mW air cooled 488 nm argon laser with light scattering for- wardly (FwSc) and laterally (SSc), and green (FITC)- and red (PHYCO)-signals were collected with a logarithmic amp¬ lification with one 530/30 nm and one 585/42 nm filter for a green and red signal, respectively. Collections from 10000 cells per sample and computer processings thereof were made with a Consort 30 program (also Becton Dickin¬ son) on a Hewlett Packard 310 PC. Based on the light scat¬ tering properties each cell was represented by a dot in a rectangular system of coordinates. A collection window is placed around the crowd of dots representing the thrombo¬ cytes. Controls have been made with fluorescence-labelled antibodies against the thrombocyte receptors GPIIIa and GPIb. The instrument gives the percentage of positive cells (for fibrinogen generally thrombocytes in a buffer with the addition of adenosine and theophyllamine or EDTA have been chosen as the negative control, for the above- mentioned glycoproteins a fluorescence-labelled antibody against the leucocyte surface antigen CD-3), the average fluorescence intensity, the complexity (SSc) and the ave- rage particle volume (FwSc) of the cell population within the analysis window. Other experimental results The activation of immuno complexes can at least part- ■ ly be counteracted if the thrombocytes are firstly mixed with a monoclonal antibody against the Fc-receptor. Immuno complexes between mouse antibodies and rabbit antibodies activate thrombocytes but not complexes between mouse antibodies and chicken anti-mouse-antibodies. At least some monoclonal antibodies against thrombocyte surface antigen may give some activation without the addition of any other antibody. Said activation is strongly enhanced by rabbit-anti-mouse-antibodies but not chicken-anti- mouse-antibodies.

Claims: CLAIMS 1. A process for the characterization and/or deter¬ mination of the amount of mammalian cells by means of antibodies, characterized by utilizing, as said anti¬ bodies, antibodies from birds or reptiles or immunologi- cally similar proteins having essentially the same or similar antigen-binding activity as said antibodies, and by performing the characterization and/or determination on such mammalian cells which interact with antibodies from mammals, or with their complexes with antigen, in any other way than by the antigen-binding activity of the an¬ tibodies, said characterization meaning that one deter¬ mines in what condition or state the cells are present and/or said determination meaning that one determines the proportion of cells in at least one of said conditions or states. 2. A process according to claim 1, characterized by performing said characterization and/or said determination on such mammalian cells which are able to be transformed or converted and on which antibodies from mammals, or their complexes with antigens, induce such a transforma¬ tion or convertion. 3. A process according to claim 1 or 2, characterized by utilizing chicken antibodies or proteins immunologi- cally siir 'ar therewith. 4. A process according to claim 3, characterized by utilizing chicken antibodies from eggs. 5. A process according to claim 3 or 4, characterized by utilizing chicken antifibrinogen antibodies. 6. A process according to anyone of the preceding claims, characterized in that it is 3rformed on blood or endothelial cells, preferably on thrombocytes or leuco¬ cytes. 7. A process according to claim 6, characterized in that it is performed on thrombocytes. 8. A process according to claim 7, characterized in that it is performed on thrombocytes for the purpose of detecting thrombocytes in activated form and/or determi¬ ning the proportion of activated thrombocytes. 9. A process according to anyone of the preceding claims, characterized in that it is performed in the form of fluorescence-activated flow cytometry.

Inventors: LARSSON ANDERS, LINDAHL TOMAS, LARSSON, ANDERS, LINDAHL, TOMAS
Applicants: LARSSON ANDERS, LINDAHL TOMAS, LARSSON, ANDERS, LINDAHL, TOMAS
Application year: 1992
Main classification: G01N33
IPC codes: G01N33