SAMPLE PRETREATMENT IN HISTOCOMPATIBILITY IMMUNOASSAY

Abstract: A pretreatment for use in an assay for antigenic material in body fluids in which the body fluid is treated with a reducing agent comprising a compound containing an -SH group. The invention is particularly useful as a pretreatment for use with an assay for Class II antigens in body fluids (especially synovial fluids) of patients with rheumatoid arthritis.

Description: SAMPLE PRETREATMENT IK HISTOCOMPATIBILITY IMMUNOASSAY This invention relates to a pretreatment for use with assays for antigenic material in human or animal body fluids; to an assay including the pretreatment; to the use of the assay in the diagnosis of a disease and to a diagnostic kit for conducting the assay. In particular the invention relates to a pretreatment for use with a quantitive assay for Class II antigens in the body fluids of patients with rheumatoid arthritis. Class II antigens are produced by activated cells which are a feature of a number of diseases including certain leukaemias (Herlyn M. et al, (1984), Leuk. Res. 8, 323: Stevenson F.K. et al (1986), J. Immunol. Meths. 86, 187) and inflammatory diseases, in particular rheumatoid arthritis (Duke et al, (1982), Clin. Exp. Immunol., 49, 22) . There exists at present an urgent need to develop a reliable assay which can quickly distinguish rheumatoid arthritis from other forms of arthritis and in particular from spondylarthritis. At present there are considerable problems in diagnosing the cause of early synovitis and correctly treating it. If a patient's swelling is caused by rheumatoid arthritis then early treatment is very important. If however the swelling is due to another arthritic condition then it requires a different approach and may resolve without treatment. Since the treatment for rheumatoid arthritis can itself be destructive it is undesirable that it should be used unnecessarily to treat other arthritic conditions. Thus it is important at an early stage to distinguish rheumatoid arthritis from other forms of arthritis. Present diagnostic methods are based on a set of clinical criteria including patients' own descriptions of the effects of their condition. An objective method would be very useful in place of this. In rheumatoid arthritis, activation of various cell populations occurs in synovial tissue and an enhanced amount of Class II antigen has been een in tissue sections (Duke et al, (1982), Clin. Exp. Immunol., 49, 22) . The source of this may include interdigitating cells which are known to increase in number in this disease, macrophages and possibly B and activated T lymphocytes. It has been reported that Class II antigen was present in a soluble form (Stevenson et al, (1986), J. Immunol. Meths., 86, 187) but it was found to be difficult to extend the study because of interference from rheumatoid factor (RF) . Complexes which commonly interfere with antigen capture assays are RFs which are pentameric IgM molecules which have affinity for IgG of both human and animal sources. By binding to the capture antibody on a microtitre plate, the pentamer can form a direct bridge with an enzyme-conjugated detecting antibody thereby giving a false positive result. Such interference presents a problem in all such assays and is not confined to patients with overt autoimmune disease (Courtney-Luck N.S. et al Cancer Res., 47, 4520 (1987)). Treatment of serum with dithiothreitol to eliminate IgM has been reported by Palosuos and Milgrom (J. Immunol. Meths., 47, 171-181 (1981)) but this was prior to assay for IgG rheumatoid factor. We have now found a method to overcome the problem in assays for other antigenic material; this has allowed analysis of fluids for the presence of the material and further investigation of its molecular form. According to the present invention we provide a pretreatment method for use in an immunoassay for histocompatibility antigens characterised in that a body fluid is treated with a reducing agent comprising a compound containing an -SH group which compound causes a reduction in the level of rheumatoid factor (RF) in the fluid without reacting to a significant extent with antigenic material in the fluid. Further according to the present invention we provide an immunoassay for histocompatibility antigens in a body fluid characterised in that it comprises a pretreatment step in which the body fluid is treated with a reducing agent comprising a compound containing an -SH group which compound causes a reduction in the level of rheumatoid factor (RF) in the fluid without reacting to a significant extent with antigenic material in the fluid. Further according to the present invention we provide an immunoassay for histocompatibility antigens in a body fluid which comprises the following steps: 1. Pretreating the body fluid with a reducing agent comprising a compound containing an -SH group which compound causes a reduction in the level of rheumatoid factor (RF) in the fluid without reacting to a significant extent with antigenic material in the fluid; 2. Contacting the pretreated body fluid with a first antibody specific for the antigenic material to be determined by the assay; 3. Contacting the body fluid and first antibody directly or indirectly with a labelled antibody; and 4. using the labelled antibody to determine the amount of antigenic material in the body fluid. Further according to the present invention we provide an assay kit for diagnostic, monitoring and/or testing purposes, which comprises a reducing agent, a first antibody and a labelled antibody all as described above together with appropriate packaging and instructions for performing a method as described in this specification according to any feature of the present invention. Further according to the present invention, we provide the use in the diagnosis of a disease, in particular rheumatoid arthritis, of a pretreatment step in which a body fluid is treated with a reducing agent comprising a compound containing an -SH group which compound causes a reduction in the level of rheumatoid factor (RF) in the fluid without reacting to a significant extent with antigenic material in the fluid. The invention is used to assay for histocompatibility antigens, in particular for Class II antigens, in body fluids. Suitably the pretreatment method causes a substantial reduction in the level of RF in the body fluid which is treated, preferably removing substantially all the RF from the fluid. The invention is suitably used in an enzyme linked immunoassay (ELISA assay) . The antibodies employed are preferably monoclonal antibodies. Preferably the first antibody is rat antibody which is attached to a plate or particle. The labelled antibody may be applied directly to an antigen attached to the first antibody, ie as a second antibody, but can also be applied indirectly, ie being attached to a second, third, etc, antibody which is itself connected directly or indirectly to an antigen. Suitably the labelled antibody is a mouse or rat antibody. The invention can be used in an assay of the type described by Stevenson et al, Clin. Exp. Immunol. (1990), 80, 32-37, the content of which is incorporated herein by reference. The reducing agent is suitably one which effects a mild reduction without any significant effect upon the antigenicity of the antigen to be assayed. Preferred reducing agents comprise compounds containing a single -SH group in their structures. A very suitable reducing agent is 2-mercaptoethanol. Preferably released -SH groups are blocked at the end of the reduction with a suitable blocking agent, eg iodoacetamide. The body fluids which may be treated include serum, urine and particularly synovial fluid. Any suitable labelled antibody may be used including radiolabelled, fluorescence-labelled and, very suitably enzyme-labelled antibodies. A preferred enzyme-labelled antibody comprises a peroxidase which causes a suitable substrate, eg o-phenylenediamine/hydrogen peroxide to undergo a colour change from colourless to brown. A typical assay method according to the invention may comprise the following steps: a) taking a sample of body fluid from a patient (this may be stored for some time before the assay, particularly if it is synovial fluid which is very stable); b) conducting the pretreatment step; c) adding the pretreated body fluid to a first antibody on, eg a plate; d) washing; e) adding an enzyme labelled antibody to the mixture on the plate; f) washing; g) adding a substrate which gives a colour change on interaction with the enzyme label; and h) measuring the extent of the colour change using a spectrophotometer. If the enzyme-labelled antibody is used indirectly then the above typical assay method will contain additional steps and the steps after step d) will be as follows: c) adding a second antibody, preferably a mouse antibody; f) washing; g) adding an enzyme-labelled antibody against the second antibody; h) washing ; i) adding a substrate which gives a colour change on interaction with the enzyme label; and j) measuring the extent of the colour change using a spectrometer. A typical kit according to the invention may comprise the following components: a) a coated plate, particles or beads (the coating possibly comprising the first antibody); b) a tube containing the first antibody (if not coated onto the plate or particle) ; c) a tube containing an enzyme-labelled antibody; d) a substrate for the enzyme label; e) a tube containing the reducing agent for the pretreatment step; and f) packaging and instructions. The pretreatment method of the invention leaves the antigen, particularly a Class II antigen, substantially unaffected and increases the accuracy and sensitivity of the subsequent assay. This gives significant benefits in the study of rheumatoid arthritis, for example, leading to potential improvements in diagnosis, monitoring and treatment of the condition. It provides a very useful means for distinguishing rheumatoid arthritis from other forms of arthritis, in particular spondylarthritis. The invention will now be further described with reference to the following example and accompanying Figures 1 to 3. In the Figures: Fig 1 Illustrates the first investigation and shows a typical elution profile from a column separation; Fig 2 Illustrates the second investigation and shows the levels of HLA Class II antigens for different categories of arthritis. Fig 3 illustrates the second investigation and compares the level of Class II antigen in synovial fluid of patents with RA in 2 groups. EXAMPLE Two investigations were carried out involving different groups of patients. Materials and Methods In the first investigation, results of which are given in the Tables and in Fig. 1, the method of the invention was employed in assays performed on synovial fluid from twenty four patients with classical or definite rheumatoid arthritis according to ARA criteria (Ropes et al, Bull. Rheum. Dis. 9, 175, 1958). Their mean age was 60 years (range 24.5-79) and the mean duration of disease was 10.5 years (range 0.33-16.0). The majority were seropositive for IgM rheumatoid factor (RF) . All patients were taking non-steroidal anti-inflammatory medication and most were also undergoing therapy with one of the following disease-modifying anti-rheumatic drugs: gold, sodium thiomalate, D-penicillamine, azathioprine or hydroxychloroquine. Each patient had synovitis of at least one knee joint considered sufficiently severe to require aspiration of the effusion and intra-articular injection of a corticosteroid. No patient had received such an injection in the three months prior to aspiration for the assay. Synovial fluids were sent directly to the laboratory in a sterile container together with a serum sample taken at the same hospital visit. Fluids were centrifuged at 1500xg and the supernatant stored at 4°C with sodium azide (lOmM) as preservative. In some cases the pH of the fluid was adjusted to 5.8 and was treated with testicular hyaluronidase (Sigma Chemical Co. Ltd., Poole, Dorset) at 20 ug/ml for 30 min at 37°C to reduce viscosity. Cultured human B cell line (FLE) An FLE cell line used for comparative studies, was derived from human B cells by EBV transformation and is used as a homozygous typing cell (HLA-DR4, DQw3,Dwl3) in the laboratory. The cells express HLA-Class II strongly at the cell surface and were used to assess release of this material in short-term culture following exposure to various kinds of stress. These included inhibition of protein synthesis by cycloheximide (5mM), irradiation (2000 rads for 40 min) and water lysis. Cells were cultured at 107/ml for 6 hours with gentle swirling, centrifuged and supernatants analysed by immunoassay for HLA-Class II. Monoclonal antibodies Ascitic fluid containing a rat monoclonal antibody against human HLA-DR (Class II) antigens (MAS 054) obtained from Sera-Lab (Crawley Down, Sussex) was precipitated with 50% saturated ammonium sulphate solution, spun and redissolved at 1 mg/ml in 0.02M Tris-HCl-O.lM NaCl-0.02% NaN3, pH 8.0. The antibody recognises a monomorphic determinant of both the DR and DP subloci. The second antibody with Class II specificity (WR 18) was raised in the Tenovus Laboratory, Southampton General Hospital, Southampton S09 4XY, UK, through which samples may be obtained and recognises the B-chains of DR, DQ and DP antigens (Reference 23, Stevenson et al., J. Immunol, Meths, 86, 187, 1986). Monoclonal antibodies against the MHC Class I (HB 95), CD3 (OKT 3, CRL 8001), CD2 (OKT 11, CRL 8027) and CD1 (OKT 6, CRL 8020) were all obtained from the American Type Culture Collection. Monoclonal antibodies against CD37 (WR 17) and CD45 (WR 6) were raised in the Tenovus Laboratory, referred to above, and assigned at the 3rd International Conference on Human Leucocyte Differentiation Antigens. Enzyme-linked immunosorbent assay (ELISA) Class II antigens A double-determinant assay already described was used to measure levels of Class II antigen (Stevenson et al., J. Immunol. Meths. 86, 187, 1986). Briefly, the rat anti-Class II antibody (MAS 054) was used at 2 ug/ml to coat the wells of a microtitre plate. After washing, antigen-containing fluids were added and incubated for 1.5 hr at 37°C. Plates were washed again and exposed similarly to the second (mouse) anti-Class II antibody (WR 18) at 2 ug/ml. Bound mouse IgG was detected by HRP-rat anti-mouse IgG (Jackson ImmunoResearch Labs. Inc., Avondale, PA), at 1:500 dilution. Controls were carried out either by using no mouse antibody or by using a subclass-matched (IgG2a) mouse antibody of irrelevant specificity, an anti-idiotypic antibody against a guinea pig lymphoma. Colour was developed with the substrate o-phenylenediamine. The assay had previously been standardised by using a lysate of cells from a patient with a B-cell leukaemia and it was possible then to use one of the positive synovial fluids as a secondary standard. More details are given in the Stevenson et al reference above including a description of units used herein and in the accompanying graphs and tables. Investiqation of other molecules associated with Class II Molecules which may be linked to Class II antigen were investigated by a modification of the assay above. The Class II-containing antigen was first bound to the antibody on the microtitre plate. Associated antigens such as Class I were then detected by exposure to an appropriate mouse monoclonal antibody in the same way as for the double determinant assay described. Concentrations of antibodies were saturating for cell antigens as judged by immunofluorescence and were usually 2-10 ug/ml of IgG. Detection and removal of rheumatoid factor activity RF activity in this system was defined as the colour yield obtained in the assay when only coating rat anti-Class II antibody, patient fluid and detecting HRP-rat anti-mouse antibody were used. Since the two rat antibodies do not interact, no colour should be developed unless there is a bridging molecule such as RF in the patient fluid. The only other possible interfering molecule would be the pre-existing human anti-rat antibody in the patient and, although this was detected in some fluids, it was rare ( 1% of patients) and those with significant levels were eliminated from the study. In contrast, RF was found frequently but was removed from synovial fluids or sera by incubation with 2-mercaptoethanol (20mM final concentration) in 0.5M Tris-EDTA buffer, pH 8.0, for 30 min at 37°C. Released -SH groups were blocked after reduction, by incubation with iodoacetamide (22mM) for 10 min at 37 C after which the material could be stored for assay when desired. Gel filtration of synovial fluids This was carried out on synovial fluids precipitated in 50% ammonium sulphate in which the Class II material was insoluble. After precipitation it was redissolved in Tris buffer and loaded (1ml) onto a column of AcA 22 (Ultrogel) (1.6x80cm) equilibrated with 0.2M Tris buffer, pH 8.0. Fractions (2.5ml) were collected and assayed for Class II antigen. RESULTS Measurement of Class II antigen Levels of HLA Class II antigen are expressed herein in units as outlined above and in more detail, by Stevenson et al, J. Immunol. Meths. 86, 187. The results of measuring Class II antigen in untreated synovial fluids (SF) from 11 patients with rheumatoid arthritis are shown in Table 1. In the fluids which contained RF activity, the levels of Class II antigen in untreated fluids had to be calculated from the difference in colour yield obtained in the ELISA in the presence and the absence of the second mouse anti-Class II antibody as described in Methods. Exposure of the fluids to reducing agent, according to the method of the invention, generally removed this "background" activity without affecting net levels of Class II antigen. The actual levels of RF before and after reduction are also shown in Table 1 and indicate almost total removal by this procedure. In a minority of cases, such as Dou in Table 1, a high percentage of apparent RF persisted after reduction and other factors such as IgG RF or antibody against rodent IgG may be involved. Similar results showing almost complete loss of RF after reduction were obtained for serum samples. As seen in Table 1, the levels of Class II antigen are very variable between patients. Similarly the RF levels are variable and there is no apparent correlation between the amounts of Class II and RF detected. Comparison of SF and serum Matched sera and synovial fluids were obtained for 10 patients and were examined for Class II antigen after removal of RF. As shown in Table 2, no significant amounts of this material could be detected in the serum samples even when levels were high in synovial fluids. Association of Class II antigen with other molecules By manipulating the ELISA it was possible to examine the association between Class II antigen and other molecules detectable with suitable monoclonal antibodies. Class II antigen was first allowed to bind to the anti-Class II antibody attached to the microtitre plate. Associated antigens were then detected by adding a second monoclonal antibody as described in Methods and profiles obtained are listed in Table 3. The antigen found regularly complexed to Class II was MHC Class I antigen and the ratio of Class II : Class I varied between 1.7:1 to 6:1 with a mean of 3.5. Other common cell surface antigens were also sought with the results given in Table 3: the T-cell antigens CD3 and CD2 and the B-cell associated antigen CD37 were not found in significant amounts; CD1 antigen, found on interdigitating cells, was also very low and variable; CD45, the leucocyte common antigen, was present at levels 2U/ml in 6/12 cases and in only 2 of these did it reach 20% of the total detectable Class II material. Thus the only consistently associated antigen was Class I. Molecular size In order to estimate molecular size of the Class II- containing complex, synovial fluids were first precipitated with 50% saturated ammonium sulphate solution and then passed down an AcA 22 sizing gel column. All the Class II material was precipitable with the ammonium sulphate solution and this provided a simple procedure for partial purification and concentration. A typical elution profile obtained from the column separation is shown in Fig. 1, which shows separation of synovial fluid by gel filtration. Synovial fluid was precipitated with ammonium sulphate (50% saturation) and passed through a column of AcA22 (1.6 x 80cm). Fractions (2.5ml) were monitored for protein (% transmission) at 280nm (* - *), and assayed for HLA Class II ( - ) and Class I (+ - +) by ELISA. The Class II antigen was eluted at the void volume ahead of pentameric IgM, indicating a molecular weight of 1000 KD. The peak from six fluids was similar in being slightly assymetric with a trailing shoulder, and as shown in Fig. 1, the Class II and Class I antigens eluted together. No effect of reduction with 2-mercaptoethanol on the elution profile of either antigen was noted. Release of Class II antigen from a cell line In order to gain insight into the ability of dispersed cells of known phenotype to release Class II antigen into the surrounding fluid, a cell line (FLE) was studied. It is an EBV-transformed human B cell and, as is typical of such lines, expresses large amounts of Class II antigen at the cell surface when examined by immunofluorescence. Analysis of cells, lysed in 0.5% NP 40, by the ELISA gave a figure of 173 U/107 cells for total cellular Class II antigen. Cells were incubated in medium alone with swirling for 6 or 21 hours and the amount of MHC antigen in the supernatant measured by immunoassay. As shown in Table 4, little material was released spontaneously during this time even though the viability fell to 70% at the cell density used (107/ml). If the protein synthesis inhibitor, cycloheximide, was included in the culture medium, viability after 21 hours was essentially zero, but there was still no significant release of MHC antigens. Similarly, prior irradiation of the cells which induced an early death did not bring about production of soluble antigen. Finally, complete lysis of cells by exposure to water did not generate soluble MHC material. Thus there was little spontaneous release of MHC antigens by this cell line and it was not possible to induce release by various kinds of cell trauma. In the second investigation, results of which are given in Figs. 2 and 3, over 100 samples of synovial fluid from 78 patients (some patients had both joints evaluated or repeat evaluations) were assayed for HLA Class II antigen. Breakdown of patients was as follows: Rheumatoid arthritis (RA) 55 Osteoarthritis (OA) 9 Spondyl/Psoriatic arthritis SP/PA 6 Reactive arthritis (RE) 3 Others 5 In Fig. 2, level of HLA Class II antigens are plotted for these different categories. It is clear that rheumatoids tended to show a much wider variation whilst the spondyl/psoriatic and reactive groups were all below 15. It should be noted that the rheumatoid patient at 223 units was receiving interferon gamma. Fig. 3 compares the level of Class II antigen in synovial fluid of patients with RA in two groups - on the right (Class A) those merely on nonsteroidal anti-inflammatory drugs or none and those on the left receiving a second line agent or disease-modifying drug (Class B) . Roughly the latter group would have had more severe disease. Clearly those with more active disease have higher HLA Class II antigen expression. A further series of assays was performed on samples from a patient with extremely severe reactive arthritis. No Class II antigen expression was observed on three consecutive days rising to a low level of less than 2 after four days. Findings may be summarised as follows: 1. Overall, patients with rheumatoid arthritis have a higher Class II antigen expression than the other disorders. 2. Strikingly, spondylarthritis and reactive arthritis have negative or low expression. 3. A joint with extremely severe reactive arthritis had no expression on three consecutive days. 4. Overall, CA has more active expression than seronegative groups. 5. The higheε- expression was in a patient receiving interferon gamma. 6. More severe RA is associated with higher expression than less severe disease. 7. In patients who had Class II antigen titres measured on the same day from two different joints, the results were comparable, e.g. 2.0 and 2.5, 11.5 and 21.1, 23.5 and 26.0, 23. c and 40.4, 2.0 and 7.5, 5.2 and 6.4, 2.7 and 22.7 and 0 + 0.1. In one patient who had the same joint aspirated on two consecutive days the figures were 5.2 and 6.4 respectively, while in the second patient the results were , 0, 0, and 1. There is considerable interest in the apparent ability of cells to release molecules, usually identified as integral membrane proteins, into the surrounding fluids. Since some of these molecules have described functions as cellular receptors, the potential biological importance of this "soluble" material is clear. Thus one of the polypeptide chains of the interleukin-2 (IL-2) receptor is found in supernatant fluids from activated T lymphocytes and is able to bind IL-2 thereby reducing its availability to local cells (Reference 19, Robb & Kutny, 1987). The molecular nature of the released receptor, however, is not clear since a variety of molecular weights have been reported suggesting either polymerisation or binding to other molecules, and at least some of the released protein appears to arise by proteolysis. Since several types of receptor may be produced during cell activation, measurement of "soluble" receptors could provide some index of disease activity in inflammatory disorders such as rheumatoid arthritis. The availability of monoclonal antibodies against a variety of receptors has allowed the establishment of immunoassavE where a "capturing" antibody is coated to a plastic pla e and the bound antigen, which could be a soluble receptor molecule, is then detected with a second enzyme-labelled antibody. The major problem with this technique is that IgM RF, found frequently in serum and fluids of patients with autoimmune diseases, will link together the capturing antibody and detecting antibody in the complete absence of putative antigen. The presence of these RFε in normal subjects or in patients has been pointed out in relation to problems consequent on administration of xenogeneic monoclonal antibodies, particularly for tumour localisation (Courtenay-Luck et al, Cancer Res. 47, 4520, 1987). However, the potential interference in the multiplicity of assays now available for various serum markers has received little attention, apart from ε few cautionary tales on specific assays (De Clerck et el, Clin. Exp. Immune!. 72, 32, 1988). Thus, unless steps are taken to remove RF or to control the assay by leaving out antigen, results will be misleadingly positive and may well show correlation with disease as can levels of RF. We describe a simple procedure, which is a modification of that used previously to detect IgG RFs (Carson, "Rheumatoid Factor", Textbook of Rheumatology, 2nd Edition, 1985), which removed the problem. Another potential source of interference is the presence of antibody against rodent IgG in patients fluids. Antibodies against mouse IgG have been reported to interfere with commercial kits designed to measure human serum choriogonadotrophin (Clark, Raggatt & Price, 1985, Clin*. Chem. 31, 1962, 1985) . In our experience with patients with rheumatoid arthritis, this has been only a minor problem. One group of cell surface glycoproteins which is released by activated cells is that associated with the MHC. Class I and II antigens have been found in supernatants from activated cells of both mouse and human origin (Emerson, Murphy & Cone, J. Exp. I-ied. 152, 783 1980; Capobianchi et al, Human Immunol. 13.1, 1985), and similar material has been detected in serum (Allison et al, J. Immunol. 118, 1004, 1977). Actual membrane vesicles have been identified in the blood of patients with various types of leukaemia (Carr, Dvorak & Dvorak, Cancer Res., 45, 5944, 1985) and ve and others have reported the presence of Class II antigen in the serum of some patients v.ith ALL (Stevenson et al, J. Immunol. Meths, 86, 187 1986; Herlyn et al, Leuk. Res. 8, 323, 1984) . This might reflect the laroe numbers of blast cells in the circulation of such patients which may be shedding cell surface components, since in normal subjects clearance of injected synthetic vesicles is rapid (Lopez-Berestein et al, Cancer Res., 44, 375, 1984). For patients with RA, no Class II could be detected in serum and it is possible that locally produced activation products either do not enter the blood, or that they are rapidly cleared. Thus the Class II material does not present a useful monitor of disease in the serum but the variable levels in joint fluid might provide an indicator of the inflammatory process. Correlations of levels in SF with clinical status are being carried out both on defined patient groups and on individual patients during various therapeutic schedules and preliminary results are reported above. The large size of the Class II-containing material suggests that it is shed from cells as has been observed for other cell types (Emerson, Murphy & Cone, J. Exp. Med., 152, 783, 1980). Previous reports have indicated that the process of release is an active one which ceases in dying cells (Sachs, Kiszkiss & Kim, J. Immunol. 124, 2130, 1980; Black, Advanc. in Cancer Res. 32, 75, 1980), and also that certain cell surface components are released preferentially. Although it was not possible to mirror the complex cell populations in the synovium, the failed attempts to bring about release of Class II antigen from a B-cell line by applying various forms of stress supports the view that cell death does not give rise to "soluble" material. It also appears that released Class II is not as random cell surface vesicles, carrying multiple cell surface antigens, but is selective for the Class II antigen together with some Class I. A physical association of Class II with some Class I antigen at the cell surface has been indicated by co-capping experiments and this may have functional significance (Keppert & Mueller-Eckhardt, Tissue Antigens, 24, 187, 1984). The physiological importance of the shed material is no * ; yet understood although its ability to induce allogenei- cytotoxic T cell responses has been described (Meeusen, Immunology 61, 321, 1987). The "soluble" Class II antigen could have a role in presenting antigen to T cells and it has been established using labelled antigen that release can occur from macrophages in a vesicular form (Harding, Leyva-Cobian δ* Unanue, Immunol. Rev., 106, 77, 1988) . This it can be conjectured that the presence of large amounts of available Class II antigen in the rheumatoid synovial fluid might be involved in the apparent immune stimulation which occurs in this tissue. Table 1 Levels of HLA-Class II antigen and rheumatoid factor in synovial fluids of patients with rheumatoid arthritis: effect of reduction Untreated Reduced* Patient Class II RF Class II RF U/ml Reduction was with 2 - mercaptoethanol (20mM) , 30 min, 37°C Below assay limits Table 2 Levels of HLA-Class II antigen in matched synovial fluids and sera of patients with rheumatoid arthritis Class II synovial fluid serum U/ml Lon 170 0 Con 91 1.3 Mou 3. 1.0 Cut 67 0 Bos 48 0 Lov 284 0 Bui 17 0 Bla 52 0 Dri 54 0 She 62 1.6 Table 3 Association of other cell surface molecules with Class II antigen in synovial fluids of patients with rheumatoid arthritis Table 4 Effects of stress on release of HLA-antigens from a human B-cell line Soluble Stress Incubation Class II Class I Viability time* (hr) U/10 cells 6 0.8 0 80 None 21 1.7 0 70 0.2 80 Cycloleximide 21 1.7 0.2 0.4 50 Irradiation 21 2.4 0.9 30 Water lysis Cells were incubated at 37 C for the times indicated in medium alone, or in the presence of cycloleximide, or following irradiation. Cells exposed to water lysis were not incubated.

Claims: Claims 1. A pretreatment method for use in an immunoassay for histocompatibility antigens characterised in that a body fluid is treated with a reducing agent comprising a compound containing an -SH group which compound causes a reduction in the level of rheumatoid factor (RF) in the fluid (without reacting to a significant extent with antigenic material in the fluid) . 2. A pretreatment method according to claim 1 characterised in that the reducing agent comprises a compound containing a single -SH group. 3. A pretreatment method according to claim 2 characterised in that the compound containing a single -SH group is 2-mercaptoethanol. 4. A pretreatment method according to any one of claims 1 to 3 characterisd in that it is used in an assay for Class II histocompatibility antigens. 5. An immunoassay for histocompatibility antigens in a body fluid characterised in that it comprises a pretreatment step in which the fluid is treated with a reducing agent comprising a compound containing an -SH group which compound causes a reduction in the level of rheumatoid factor (RF) in the fluid (without reacting to a significant extent with antigenic material in the fluid) . 6. An immunoassay for histocompatibility antigens material in a body fluid which comprises the following steps: i) pretreating the body fluid with a reducing agent comprising a compound containing an -SH group which compound causes a reduction in the level of rheumatoid factor (RF) in the fluid (without reacting to a significant extent with antigenic material in the fluid) ; ii) contacting the pretreated fluid with a first antibody specific for the antigenic material to be determined by the assay; iii) contacting the body fluid and first antibody directly or indirectly with a labelled antibody; and iv) using the labelled antibody to determine the amount of antigenic material in the body fluid. 7. An assay according to claim 5 to claim 6 characterised in that it is used to assay for Class II histocompatibility antigens. 8. An assay according to any one of claims 5 to 7 characterised in that the reducing agent comprises a compound containing a single -SH group. 9. An assay according to claim 8 characterised in that the compound is 2-mercaptoethanol. 10. An assay according to any one of claims 5 to 9 characterised in that either or both of the fir: - and the labelled antibodies are monoclonal antibodies. 11. An assay according to claim 10 characterised in that the first antibody is a rat antibody. 12. An assay according to claim 10 or claim 11 characterised in that the labelled antibody is a mouse antibody. 13. An assay according to any one of claims 5 to 12 characterised in that the labelled antibody is indirectly linked to the antigen through a third antibody. 14. An assay according to any one of claims 5 to 13 characterised in that the labelled antibody is an enzyme-labelled antibody. 15. An assay according to claim 14 characterised in that the enzyme label effects a colour change and the amount of antigenic material is determined spectrophotometrically. 16. An assay according to any one of claims 5 to 15 characterised in that it is used in the diagnosis of arthritis. 17. An assay kit for diagnostic, monitoring and/or testing purposes, which comprises a reducing agent, a first antibody and a labelled antibody all as hereinbefore described together with appropriate packaging and instructions for performing a method as described in this specification according to any feature of the present invention. 18. An assay kit according to claim 17 characterised in that the reducing agent comprises a compound containing a single -SH group. 19. An assay kit according to claim 18 characterised in that the compound containing a single -SH group is 2-mercaptoethanol. 20. An assay kit according to any one of claims 17 to 19 characterised in that either or both of the first antibody and the labelled antibody are monoclonal antibodies. 21. An assay kit according to claim 20 characterised in that the first antibody is a rat antibody. 22. An assay kit according to claim 20 or claim 21 characterised in that the labelled antibody is a mouse antibody. 23. An assay kit according to any one of claims 17 to 22 characterised in that it contains a third antibody in addition to the first antibody and the labelled antibody. 24. An assay kit according to any one of claims 17 to 23 characterised in that the labelled antibody is an enzyme-labelled antibody. 25. The use in the diagnosis of a disease, of a pretreatment step in which a body fluid is treated with a reducing agent comprising a compound containing an -SH group which compound causes a reduction in the level of rheumatoid factor (RF) in the fluid (without reacting to a significant extent with antigenic material in the fluid) . 26. The use according to claim 25 of a pretreatment step in the diagnosis of arthritis. 27. The use according to claim 25 or claim 26 of a pretreatment step characterised that in the reducing agent comprises a compound containing a single -SH group. 28. The use according to claim 27 of a pretreatment step characterised in that the compound is 2-mercaptoethanol. 29. The use in the diagnosis of a disease of an assay for antigenic material according to any one of claims 5 to 16. 30. The use in the diagnosis of arthritis of an assay according to any one of claims 5 to 16. 31. The use in the diagnosis of a disease of an assay kit according to any one of claims 17 to 24. 32. The use in the diagnosis of arthritis of an assay kit according to any one of claims 17 to 24.

Inventors: STEVENSON FREDA KATHRYN, STEVENSON, FREDA, KATHRYN
Applicants: 3I RES EXPL LTD, 3I RESEARCH EXPLOITATION LIMITED
Application year: 1990
Main classification: G01N33
IPC codes: G01N33