WO2001034788A1

WO2001034788A1 - In-vitro assay for assessment of cytotoxicity of a candidate agent

Info

Publication number: WO2001034788A1
Application number: PCT/IL2000/000730
Authority: WO
Inventors: Tamar Jehuda-Cohen
Original assignee: Yoreh Biotechnologies Ltd.
Priority date: 1999-11-10
Filing date: 2000-11-08
Publication date: 2001-05-17
Also published as: AU1174401A

Abstract

This invention provides an in-vitro assay method of assessing cytotoxicity of an agent, comprising the following steps: a) obtaining a sample; b) culturing the sample in the presence of a mitogen so as to induce polyclonal and specific activation of lymphocytic cells leading to antibody production; c) contacting the culture of step b) with the agent; d) detecting the level of the antibody in the sample, e) comparing the level of the antibody in the sample, wherein if the level of the antibody in step d) is less than a control level the agent is cytotoxic, thereby assessing the cytotoxicity of an agent.

Description

IN-VITRO ASSAY FOR ASSESSMENT OF CYTOTOXICITY OF A CANDIDATE AGENT

FIELD OF THE INVENTION

The present invention relates to an assay for the determination of the potential cytotoxicity of a candidate agent. The assay is based on a cellular assay in the presence of a mitogen.

BACKGROUND OF THE INVENTION

Todays technology is identifying millions of drug candidates and biochemicals. However, there are inadequate methods to sort through the therapeutic potentials of the drug candidates in a cost effective manner. Currently available for in-vitro drug toxicity testing are two assays: the apoptosis or programmed cell death assay and the cell proliferation assay which is based on thymidine uptake. These assays are not based on, nor provide, the full functionality of the cultured cells and the ability of the (normal) cells to function in the presence of a candidate drug.

SUMMARY OF THE INVENTION

This invention provides cost effective and simple method to determine if a drug is cytotoxic to cells. This invention provides an in-vitro assay method of assessing cytotoxicity of an agent, comprising the following steps: a) obtaining a sample; b) cultuhng the sample in the presence of a mitogen so as to induce polyclonal and specific activation of lymphocytic cells leading to antibody production; c) contacting the culture of step b) with the agent; d) detecting the level of the antibody in the sample, e) comparing the level of the antibody in the sample, wherein if the level of the antibody in step d) is less than a control level the agent is cytotoxic, thereby assessing the cytotoxicity of an agent. BRIEF DESCRIPTION OF THE FIGURES

Figure 1 (A): Effect of different Heparin concentrations on mitogen induced IgM secretion Figure 2(B): Effect of Heparin on antibody production can be removed by heparinase.

Figure 3(c): Inhibition of Heparin on IgM production (but not on proliferation, see figure 4)

Figure 4: Proliferation is not effected by the presence of Heparin. Figure 5: Different effects of natural extracts on IgM production and proliferation.

Figure 6: Different effects of natural extracts on IgM production and proliferation.

Figure 7: IgM secretion and proliferation in the presence of Different natural extracts.

DETAILED DESCRIPTION OF THE INVENTION

As shown herein, the mitogenic effect of mitogens are relatively insensitive to culture media, concentration of cultire media components, and the introduction of inhibitors or toxins. The antibody production from the cells is however very sensitive. Maturation and differentiation of B cells to plasma cells appears even more sensitive. Thus, the present invention provides a low cost and sensitive manner of determining cytotoxicity of a candidate drug or agent.

This invention provides an in-vitro assay method of assessing cytotoxicity of an agent, comprising the following steps: a) obtaining a sample; b) culturing the sample in the presence of a mitogen so as to induce polyclonal and specific activation of lymphocytic cells leading to antibody production; c) contacting the culture of step b) with the agent; d) detecting the level of the antibody in the sample, e) comparing the level of the antibody in the sample, wherein if the level of the antibody in step d) is less than a control level the agent is cytotoxic, thereby assessing the cytotoxicity of an agent. In one embodiment the newly produced antibody is labeled with a marker. In another embodiment, the level of the antibody is detected by exposing the culture of step b) with an antigen so as to form an antibody-antigen-complex and detecting the complex, and the complex is either labeled with a marker, coloremetric, bioluminescen, or chemiluminescent agent, or is detected by any other means for detecting the formation of the Ab-Ag complex or other bindings and interactions used for the detection. The level of antibody also includes any cellular changes that are associated or connected to the antibody productions and/or secretion, such as antibodies, cytokines, other cellular componenets, cell membrane markers; both directly or indirectly.

As defined herein, an "candidate agent" may be any molecule, chemical, drug, compound, composition, protein or peptide which is desired to be assayed for cytotoxicity. Agents include but are not limited to: chemotherapeutic, inflammatory, anti-microbial, anti-viral, anti-cancer, co-enzymes, vitamins, neuropeptides, psychiatric drugs, angiotic drugs, blood thinners and diluants, narcotics and paon relievers, doting and anti clotting reagents. Chemicals produced in the laboatory (such as via combinatorial chemistry for example) defined and undifined natural extracs (such as form plants, yeast, microbial sources, algea.

"Antigens" include but are not limited to: any ligand, immunogens, allergens, carcinogens, alloantigens, self-antigens(auto-anitgens), cancer antigens, cancer associated antigens, transplantation antigens, blood group antigens, or pollutants. As defined herein all antigens could elicit an immune response. Thus looking for an antibody (or antibodies) against it means that you would use the whole or segments (peptides) of it as the antigen for the detection systems and tests. The antigen may be proteins, peptides, fragments, disassociated, peptides (of any length); lipoproteins; carbohydrates, such as glycoproteins and glycolipids (both have representatives on the bacteria walls, blood group antigens, and myeloma structures for example); oligosaccharides/ polysaccharides; lipids/fats; haptens; and chemicals such as TNP (trinitrophenyl), benzene arsenate, and non- organic compounds. Antigens which are used would depend on the antibody for which one is determining. Such antibodies are known to those skilled in the art. For example, Helicobacterium pylori, the bacteria that has been implicated as the cause of a large part of the peptic ulcers. This bacteria has many antigenic/antigenic epitopes or structures. The entire bacteria, or some of its proteins, carbohydrates, mucins, or peptides may serve as an antigen for the antibody detection. The same applies to HCV, HBV, CMV, Tetanus toxoide, rubela, measles (and other pathogens, or allergens, or feta antigens, etc).

As defined herein, "contacting" means that the agent is introduced into the sample culture in a test tube, flask, tissue culture multi well plates or the like, and incubated at a temperature and time sufficient to permit the effect of the agent (and the mitogen) on the cell culture. Methods for contacting the agents with the culture, are known to those skilled in the art and may be selected depending on the type of assay protocol to be run. Incubation methods are also standard and are known to those skilled in the art.

A "control level" means the level of the antibody produced by the cell(s) without the contact with the agent. The control level is indicative of a baseline level of antibodies of the subject or subsequent measurements following determination of the baseline level. In another embodiment, the level is a value reflective of a normal antibody level, or antibody production level . In one embodiment, The system can be calibrated by a predetermined level, or count, or readout for the control value, or a control culture can be run (in parrallel) without he agent.

"Mitogens" as defined herein means any material that activates lymphocytic cells, so as to secrete or produce polyclonal (non psecific) and specific antibodies. In one embodiment the mitogen is a B cell activator, such as pokeweed mitogen, a lectin, a bacterial endotoxin, an antigen, lipid A, or a lymphokine. In another embodiment the mitogen is a superantigen such as a toxin from bacteria which include staphylococci and staphylococci A toxins (30KD toxins), enterotoxins A,B,C1 ,C2,D,E (from Staphylococcus aureus), exotoxins AB,C, and exfoliative toxin AB. In another embodiment, the mitogen is a grain-negative LPS sequence. In another embodiment the mitogen is a peptidoglycan from both gram negative and gram positive bacteria, for example, toxic shock syndrome toxin TSST- 1 , ExFT, MAM, Strep M., or a Gram-negative lipopolysacchride LPS) sequences. In another embodiment the mitogen is herpesviruses such as Epstein-Barr Virus(EBV), a retrorirus, mouse mammary virus (NDATV), picomavirus(rats) Coxsackie virus, mumps and measles viruses and Mtv virus (I- 9,11 ,13,43). In another embodiment the mitogen is heat shock proteins(HSP). In another embodiment the mitogen is an antibody which includes but is not limited to: Anti CD3 antibodies, Anti TCR (T cell Receptor), Anti IgM, Anti IgD, Anti CD28 in both soluble form or bound. It is contemplated that interleukines or cytokines, such as IIL-4, either alone or in conjunction with additional factors may be added. In another embodiment the mitogen is phorbol ester such as phorbol myristate acetate, PMA with calcium ionophore and IIL-4. In another embodiment pharmacological activators (such as diacylglycerol) that work through paths such as the PIP2 derived second messenger path. In another embodiment the mitogen is a lectin including Pokeweed mitogen (PWM) and similar acting mitogens with or without antigens. In the preferred embodiment Pokeweed mitogen is used. Pokeweed mitogen includes pure and crude extracts from Phylotacca Americana. As used herein, "whole blood' means blood collected from an animal or human (and not separated).

Suitable samples may be obtained from a veterinary or human donors and include, but are not limited to, whole blood cells, any mononuclear cell, leukocytes, lymphocytes, T-cells, B-cells, monocytes, , neutrophils, or Peripheral Blood Mononuclear cells. Also included are cell lines (such as immortalized B cells of different sorts)

The whole blood contains red blood cells which may be lysed while maintaining the viability of the remaining white blood cells. Alternatively the "buffy coat" can be collected after the RBC sedimetation. Whole blood may be collected with heparin, EDTA, citrate or any other substance that prevents coagulation and clotting. In one embodiment, the optimal concentration of mitogen with or without antigen is easily determined without undue experimentation by one of ordinary skill in the art. With regard to the preferred mitogen, pokeweed mitogen, the preferred concentration range is between approximately 1 :100 and 1 :1600 dilutions of stock PWM. The most preferred concentration range is between approximately 1 :200 and 1 : 1 :400 dilutions of stock PWMThe lyophilized PWM is reconstituted with 5 ml of distilled water to make the stock solution. The concentration of pokeweed mitogen may range from about 0.025-50.0 ul/ml. The concentration range may be from about 0.1-0.5 ul/n-A. The preferred concentration is 0.2 ul/ml. If the mitogen is Wheat germ agglutination the concentration range is about 0. 1-2.5 ul/ml. If the mitogen is Sac Cowan I mitogen the concentration range is 1 :200-1 :2000 dilution. The mitogen can also be a mixture of diferent mitogens (usualy lower concentrations are than used) .As defined herein, "culture medium" means any medium than can be used to sustain a sample to practice the present invention, including but not limited to RPMI 1640 (GEBCO, New York, New York) with or without fetal calf serum, preferably supplemented with appropriate antibiotics and glutamine. Other culture media which may be used in practicing the present invention include, but are not limited to, Eagles, Dulbecco's, McCoy's, Media 199, Waymouth's media, and serum free medium with or without supplement. In another embodiment the mitogen is without media.

The antigens (and the antibodies produced could act as antigens in the detection system) or antibodies may also be labeled using fluorescent labels, enzyme labels, free radical labels, or bacteriophage labels, using techniques known in the art. Typical fluorescent labels include fluorescein isothiocyanate, rhodamine, phycoerythrin, phycocyanin, alophycocyanin, and Texas Red. Since specific enzymes may be coupled to other sequences by covalent links, the possibility also exists that they might be used as labels for the production of tracer materials. Suitable enzymes include alkaline phosphatase, beta-galactosidase, glucose-6-phosphate dehydrogenase, maleate dehydrogenase, and peroxidase. Types of enzyme immunoassay are the enzyme-linked immunosorbent assay (ELISA), ELISPOT, and the homogeneous enzyme immunoassay, also known as enzyme-multiplied immunoassay (EMIT, Syva Corporation, Palo Alto, CA). In the ELISA system, separation may be achieved, for example, by the use of antibodies coupled to a solid phase. The EAET system depends on deactivation of the enzyme in the tracer-antibody complex; the activity can thus be measured without the need for a separation step. Additionally, chemiluminescent compounds may be used as labels. Typical chemiluminescent compounds include luminol, isoluminol, aromatic acridinium esters, imidazoles, acridinium salts, and oxalate esters. Similarly, bioluminescent compounds may be utilized for labeling, the bioluminescent compounds including luciferin, luciferase, and aequorin. The antibody binding can also be detected by other means such as conformatinal changes in the antibody or the binding mollecule (Ag), change in conductivity, charge shift . Furher, such detection means may determine the level of amount of DNA RNA of the antibody or the change in such level or amount before and after the candidate agent.

The antibody may be labeled directly with a marker or an antigen may be complexed to the antigen. Fluorescent markers include but are not limited to: fluorescein, rhodamine and auramine. Colorimetric markers include, but are not limited to: biotin, and digoxigenin. Methods of producing the polyclonal or monoclonal antibody are known to those of ordinary skill in the art, Further, the antibody or nucleic acid sequence complex may be detected by a second antibody, or complemetary sequence (for mRNA), or non specific binders which may be linked to an enzyme, such as alkaline phosphatase or horseradish peroxidase. Other enzymes which may be employed are well known to one of ordinary skill in the art. Also radioactive markers and compounds can be used.

Once labeled, the antibody may be employed to identify and quantify immunologic counterparts (antibody or antigenic polypeptide) utilizing techniques well-known to the art. Antibodies include but are not limited to: IgG and subsets, IgA, IgE, IgM, IgD.

The phrase "specifically binds to an antigen " or "specifically immunoreactive with", when referring to the antigen, refers to a binding reaction which is determinative of the presence of the antigen. Thus, under designated immunoassay conditions, the specified antigen binds to the antigen and does not bind in a significant amount to other antibodies present in the sample. Specific binding to an antigen under such conditions may require an antigen that is selected for its specificity for a particular protein. For example, The binding can be done also in a non specific manner such as sephadex A or G (for total antibidy production).

Examples of detectable moieties include fluorescein and rhodamine (for fluorescence microscopy), horseradish peroxidase (for either light microscopy or electron microscopy and biochemical detection), biotin-strepavidin (for light or electron microscopy) and alkaline phosphatase (for biochemical detection by color change). The detection methods and moieties used can be selected, for example, from the list above or other suitable examples by the standard criteria applied to such selections (Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1988 ).

Labels are known to those skilled in the art. For example, labels which, are included but not limited to, include aphycoerythrin, or fluorescien isothiocyanate. Examples of types of labels encompassed by the present invention include, but are not limited to, radioisotopic labels (e.g., .sup.3 H, .sup.125 I, .sup.131 I, .sup.35 S, .sup.14 C, etc.), non-radioactive isotopic labels (e.g., .sup.55 Mn, .sup.56 Fe, etc.), fluorescent labels (e.g., a fluorescein label, an isothiocyanate label, a rhodamine label, a phycoerythrin label, a phycocyanin label, an allophycocyanin label, art O-phthaldehyde label, a fluorescamine label, etc.) for example, as in peridinin chlorophyll protein (PerCP), chemiluminescent labels, enzyme labels (e.g., alkaline phosphatase, horse radish peroxidase, etc.), protein labels, labels useful in radioimaging and radioimmunoimaging.

Further, as used herein, the term "label" refers to a molecule, which may be conjugated or otherwise attached (i.e., covalently or non-covalently) to a binding protein such as an antibody as defined herein. Particularly suitable labels include, e.g., fluorochromes. Preferred fluorochromes include phycoerythrin (P.E., Coulter Corp., Hialeah, FL), phycoerythrin-cyanin dye 5 (PECyδ, Coulter), and fluorescein isothiocyanate (FITC, International Biological Supplies, Melbourne, FL). Other suitable detectable labels include those useful in colorimetric enzyme systems, e. g., horseradish peroxidase (HRP) and alkaline phosphatase (AP). Other proximal enzyme systems are known to those of skill in the art, including hexokinase in conjunction with glucose-6-phosphate dehydrogenase. Chemiluminescent labels, such as green fluorescent proteins, blue fluorescent proteins, and variants thereof are known. Also bioluminescence or chemiluminescence can be detected using, respectively, NAD oxidoreductase with luciferase and substrates NADH and FNIN or peroxidase with luminol and substrate peroxide. Other suitable label systems useful in the present invention include radioactive compounds or elements, or immunoelectrodes.

A variety of immunoassay formats may be used to select specific antibodies specifically immunoreactive with a particular protein. For example, solid-phase ELISA immunoassays are routinely used to select monoclonal antibodies specifically immunoreactive with a protein. See Harlow and Lane [Harlow and Lane, (1988) Antibodies, A Laboratory Manual, Cold Spring Harbor Publication, New York.] for a description of immuno assay formats and conditions that can be used to determine specific immunoreactivity. A description of a radioimmunoassay (RIA) may be found in Laboratory Techniques in Biochemistry and Molecular Biology [LaboratoLy Techniques in Biochemistjy and Molecular BiologY (1978) North Holland Publishing Company, New York.], with particular reference to the chapter entitled "An Introduction to Radioinimune Assay and Related Techniques" by Chard, T., incorporated by reference herein Where the antigens are labeled, the labels can include radioisotopes, fluorophores, enzymes, luminescers or particles. These and other labels are well known in the art and are described, for example, in the following U.S. Pat. Nos. 3,766,162; 3,791 ,932; 3,817,837; 3,996,345; and 4,233,402. Assays employing the antigen associated antibodies from the cell lines can be heterogenous, i.e., requiring a separation step, or homogenous. If the assay is heterogenous a variety of separation means can be employed, including centrifugation, filtration, chromatography or magnetism.

A description of general immunometric assays of various types can be found in the following U.S. Pat. Nos. 4,376,110 (David et al.) or 4,098,876 (Piasio) and 4,870,003 (Kortright) are incorporated by reference. The presence or concentration of this antigen-antibody complex is determined to detect or quantitate the presence of antigen associated antibodies in the biological sample.

Enzyme immunoassays such as immunofluorescence assays (IFA), photometric assays, enzyme linked immunosorbent assays (ELISA), ELISPOT, and immunoblotting can be readily adapted to accomplish the detection of the specific antibodies. An ELISA method effective for the detection of the antigen associated antibodies can, for example, be as follows: (1) bind the antigen to a substrate; (2) contact the bound antigen with a biological sample, such as a bodily fluid before and after culture or tissue sample or lymphocytes before and after culture containing the antibody; (3) contact the above with a secondary antibody bound to a detectable moiety (e.g., horseradish peroxidase enzyme or alkaline phosphatase enzyme); (4) contact the above with the substrate for the enzyme; (5) contact the above with a color reagent; (6) observe the color change.

Another method of immunoenzymatic detection of the presence of antibodies is the Western blot. The antigens are separated electrophoretically and transferred to a nitrocellulose membrane or other suitable support. The body fluid to be tested is then brought into contact with the membrane and the presence of the immune complexes formed is detected by the method already described. In a variation on this methods, purified antigen associated antibodies is applied in lines or spots on a membrane and allowed to bind. The membrane is subsequently brought into contact with the body fluid before and after culture to be tested and the immune complexes formed are detected using the previously described techniques.

The use of multiple antibodies, and other specific binding components, is contemplated herein. In particular, the ability to simultaneously measure the levels of more than one antibody subunit and/or other cellular components enables determination of drug cytotoxicity.

The antibody used for the determination according to the present invention may be directly labeled with the preferred fluorescence label, or may be indirectly labeled with the preferred fluorescence label. In the last case, the preferred fluorescence label is conjugated to a secondary antibody, which is directed against the first antibody, such as an anti species Ig antibody. Specifically, contemplated herein one may use IgG or IgM.

The ability to perform cell analysis according to the present invention using cytometric cell analysis requires the ability to render the cell in patient sample permeable to both small molecules such as dye molecules, nucleotides and the like, and large molecules such as antibodies. Permeation methods suitable for flow cytometric analysis are described by Lanza et al, Comparative Analysis of Diferent Permeation Methc ds for The Flow Cytometry Measurements of Cytaplasmic Myeloperoxidase and Lysosyme in Normal and Leukemic cells, Cytometry, (1997), 30, 134-144. Another example for combined multiparameter flow cytometry analysis of both extracellular and intracellular cell components is that of Toba, et al. Methods for automatic autometric analysis of cellular components in whole blood are descibed in US Patent Nos. 5,776,709, and 5,631 ,165. A method for whole blood image analysis of cellular components using non-flow cytometry is described in US Patent No. 5,557,849.

Using this method and several others, the "Ab production level" and the increase in it can be measured (total or specific) as the cells' number with activation or differntiation markers, or via the number of plasma cells, sesity of Igm or IgD etc on the cell membrane, the apearence of IgG. All the aboe can be achieved using direct measure via systems such as the FACS or or through serogate markers such as RNA/DNA status or detecting cytokines' secreation or pruduction, or other cellular functions that are associated or connected to the mitogen stimulation .

Further, cells may be detected using standard flow cytometry analysis using FACscan analyzer (Becton Dickinson, San Jose, Calif.). Cytometric techniques are known to those skilled in the art. For example the following describe such techniques and are hereby incorporated by reference in their entirety; US Patent No. 5,298,426 Method of differentiating erythroblasts from other cells by flow cytometry; US Patent No. 5,296,378 Method for classifying leukocytes by flow cytometry; US Patent No. 5,270,548 Phase-sensitive flow cytometer; US Patent No. 5,247,340 Flow imaging cytometer; US Patent No. 5,179,026 Method of classifying leukocytes by flow cytometry.

EXPERIMENTAL DETAILS SECTION

Heparin at high concentrations inhibits the antibody production that is enhanced by the mitogen.lt is the heparin itself that inhibits, and not impurities in the solution, as Heparinase (the enzyme that breaks down heparin) removes that inhibition. Under the same experimental conditions antibody production is suppressed and proliferation is not effected at all. Different effects on antibody production and on proliferation when different plant extracts are used, in the same system. The fact that stimulated antibody production is much more sensitive, as a cellular function parameter, than proliferation can be seen also from the effect of changes in pokeweed mitogen (PWM) concentration on these parameters. The active range of PWM for antibody production is narrow while much higher concentrations (that are inhibitory to antibody production) are still effective as mitognes for cellular proliferation.

Materials and Methods:

Figure A:

PBMC from a healthy donor were incubated at 10 million cells per ml, in RPMI

1640 supplemented with 0% FCS and in the presence of Pen-Strep antibiotics, for 5 days in 37c CO2 incubator. The control tube had PWM alone the other culture tubes had different cone, of Heparin added to the culture upon its initiation. After the 5 (or 6) day incubation the supernatant fluids form the

*^• cultures were tested for IgM in a quantitative ELISA using a standard curve for calculating the actual concentration of IgM.

Figure B:

PBMC from a healthy donor were incubated at 10 million cells per ml, in RPMI 1640 supplemented with 10% FCS and in the presence of Pen-Strep antibiotics, for 5 days in 37c CO2 incubator. The control tube had PWM alone the other culture tubes had a fixed cone, of heparin. Some of the tubes got Heparine after a pre-treatment with heparinase (at two cone.) The heparin (treated or untreated) was added to the culture upon its initiation. After the 5 (or 6) day incubation the supernatant fluids form the cultures were tested for IgM in a quantitative ELISA using a standard curve for calculating the actual concentration of IgM.

Figure C: PBMC from a healthy donor were incubated at 10 million cells per ml, in RPMI 1640 supplemented with 10% FCS and in the presence of Pen-Strep antibiotics, for 5 days in 37c CO2 incubator. The control cultures had PWM alone the other cultures had heparin added to the culture upon its initiation. After 5 days incubation the supernatant fluids form the cultures were tested for IgM in a quantitative ELISA using a standard curve for calculating the actual concentration of IgM.

Figure D: PBMC from a healthy donor were incubated at 10 million cells per ml, in RPMI 1640 supplemented with 10% FCS and in the presence of Pen-Strep antibiotics, for 5 days in 37c CO2 incubator. The control cultures had PWM alone the other cultures had heparin added to the culture upon its initiation. After 4 days incubation BrdU was added to the cultures for overnight, using a commercial kit (Cy-Quant) for the determination of the proliferation.

Figures E, F, G: PBMC from a healthy donor were incubated at 10 million cells per ml, in RPMI 1640 supplemented with 10% FCS and in the presence of Pen-Strep antibiotics, for 5 days in 37c CO2 incubator. The control cultures had PWM the other cultures had different natural extracts added to the culture upon its initiation. For determination of IgM levels supernatant fluids form the cultures were tested after a 5 day incubation for IgM in a quantitative ELISA using a standard curve for calculating the actual concentration of IgM. For the determination of proliferation separate yet parallel cultures were set up. After 4 days incubation BrdU was added to the cultures for overnight, using a commercial kit (Cy-Quant) for the determination of the proliferation.

Measuring the produced antibodies in culture: When using PBMC or lymphocytes, there is no real need to measure a specific antibody for increased levels as the total Ig or the total IgG, IgM, IgE, IgA, IgD antibodies can be measured by simple techniques. This is so since the high level of antibodies in the serum has been removed and washed away. The increased in antibody production can be seen by a single measurement, or when compared to a base line with no agent additive with or without mitogens to the culture medium.

When using a whole blood sample, or buffy coat, there is a high level of antibodies in the mixture even prior to culture. In those situations the measurement needs to be directed at the newly produced antibodies only. Thus options include, but is not exclusive to, labeling (radioactively for example) the new proteins, or measuring the appearance of, or the significant increase in levels of, specific antibodies in the culture fluid for example:

1. Antibodies to old immunizations both natural and induced, such a CMV,

Tetanus toxoide, Hepatitis B (when relevant), etc.

2. Antibodies from new immunizations that have not yet come up in the serum yet (or for which antibodies are at very low levels). 3. Antibodies against any epitope (none of them need to be relevant to the screened agents or drugs in the assay).

Other options include measuring the appearance of Ig mRNA, or the increase in plasma cells etc. Surrogate markers to the effect of the agent on the cells in vitro can be used such as the appearance (in protein or mRNA) and/or the secretion of different cytokines or lymphokines, or any other factor that are associated (or can be associated) with the cell activation towards antibody production (and the cell differentiation and maturation that accompanies it).

As demonstarted herein, the activation of IgM production, in PBMC cultures containing PWM can be inhibited by heparin at concentrations that have no effect on proliferation. Also the sensitivity of the culture to different concentrations of heparin has been shown. When incubating the cultures in the presence of different natural extracts there is a differential effect on the proliferation and the IgM production in the parallel cultures.

In other experiments (results not shown here) the IgM and IgG production under PWM was tested in whole blood cultures that contained varying levels of heparin in the blood collection tubes. The tubes that had higher levels of heparin had lower levels of IgM or IgG produced in culture. In those experiments the IgM produced during culture was labeled with ³⁵S-methionin and the ³⁵S-methionin levels were measured in the bead bound IgM or IgG (after several rinses of unbound ³⁵S-methionin).

Claims

What is claimed is:

1. An in-vitro assay method of assessing cytotoxicity of a candidate agent, comprising the following steps: a) obtaining a sample; b) culturing the sample in the presence of a mitogen so as to induce polyclonal and/or specific activation of lymphocytic cells leading to antibody production; c) contacting the culture of step b) with the candidate agent; d) detecting the level of the antibody in the sample, e) comparing the level of the antibody in the sample, wherein if the level of the antibody in step d) is less than a control level the agent is cytotoxic, thereby assessing the cytotoxicity of a candidate agent.

2. The method of claim 1 , wherein the antibody is labeled with a marker.

3. The method of claim 2, wherein the marker is an enzyme, fluorophore, chromophore, radioisotope, dye, colloidal gold, latex particles, coloremetric, bioluminescence, or chemiluminescent agent.

4. The method of claim 1 , wherein the level of the antibody is detected by exposing the culture of step b) with an antigen so as to form an antigen-antigen-complex and detecting the complex,

5. The method fo claim 4, wherein the complex is lebeled with a marker.

6. The method of claim 2, wherein the marker is an enzyme, fluorophore, chromophore, radioisotope, dye, colloidal gold, latex particles, coloremetric, bioluminescence, or chemiluminescent agent.

7. The method claim 1 , wherein the antiody is a monoclonal or polyconal.

8. The method of claim 1 , wherein the antibody is selected from the group consisting of F(ab')2 fragments, Fab' fragments, or Fv fragments of said antibodies.

9. The method of claim 1 , wherein the antibody is selected from the group consisting of antibodies of IgG, IgM or subunits thereof, F(ab')2 fragments,

Fab' fragments, or Fv fragments of said antibodies.