SE467498B - PROCEDURES IN VITRO ANALYSIS OF MERCURY SILVER ALLERGIES - Google Patents

Abstract

The invention describes a procedure for the in vitro determination of allergy in vitro with the help of lymphocytes, so-called memory cells, in the blood, in which a lymphocyte fraction is isolated from defibrinated blood and is cultivated in the presence of an antigen with the aim of determination of eventual presence of memory cells for that particular antigen among the lymphocytes in which stimulated memory cells, so-called lymphoblasts, are determined by radioactive precursor incorporation into DNA and by morphological evaluation, which is characterized by the lymphocyte fraction which is repeatedly depleted of monocytes and other adherent cells. The antigen may be metals, organic and inorganic compounds, ceramics, glass ionomers, pigments and composite materials. The invention covers also the use of procedure for study of diseases which may be caused by metal allergy or in which metal allergy acts as promotor (contributing factor).

Description

4 10 15 20 25 30 35 6 O 0 749 2 reactive chemicals on the skin result in a strengthening, "boosting" of the individual's immune system and thus contribute to the worsening of allergy symptoms. Thus patch tests for mercury, cobalt formalin or epoxides can not be used without In addition, the Food and Drug Administration (FDA) has recently received a report of side effects since patch tests were performed to determine hypersensitivity to mercury (Food and Drug Administration, National Center for Devices and Radiological Health, Device Monitoring Branch., Device Experience Network Monthly Report., 08: 11- 12), 1983.

Another method that has been used before is the so-called LTT method, the lymphocyte transformation test, sometimes also called LST, the lymphocyte stimulation test. Transformation means that a small lymphocyte grows and is "transformed" into a lymphoblast. This can be done with the help of surface receptor active substances, so-called mitogens, which non-specifically stimulate lymphocytes from all individuals to mitosis, so-called mitogens. The same process can be distinguished after the addition of a specific antigen to the blood of an animal or human, which has previously been exposed to a foreign substance, the specific antigen, eg a virus or a bacterium. LTT measures the individual's immune response to the antigen examined.

The technology has previously been used with more or less success for the diagnosis of certain types of hypersensitivity reactions, so-called allergies. The technique has mainly been used for allergies of so-called type 4 reactions (delayed type of allergy) which are known to be cell mediated. Such reactions are behind conditions such as contact dermatitis eczema. The most studied antigens have been penicillins and heavy metals, mainly nickel but also cobalt, gold, beryllium and inorganic mercury. Both mitogenic and antigenic properties of such substances have been determined by this technique. The LTT method has thus not been able to distinguish these two properties.

In the usual LTT method, heparinized blood is separated on a density gradient, and the lymphocyte suspension, which also contains monocytes, is recovered. Granulocytes and red blood cells settle to the bottom of the centrifuge tube. The cells in the lymphocyte suspension are cultured with the antigens to be studied in microplates (with 96 holes) in 0.2 ml of medium containing 200,000 cells for 4-5 days. After culture, the presence of lymphoblasts is measured by the addition of radioisotope-labeled thymidine or some other amino acid, which is incorporated into DNA or RNA during cell division.

When previously studying the effects of inorganic mercury compounds such as mercury chloride (HgCl2), it has been widely considered that they act as a mitogen (cell-dividing) substance for human and animal lymphocytes in culture. Schöpf E., Schulz KH, Grom, Naturwissenschaften, 2l: 568-9, 1967.

That is, it has been found in these experiments that lymphocytes from humans with or without a positive patch test against mercury were stimulated. Positive patch test was then considered a measure of the presence of Hg allergy. Because lymphocytes from both allergy and non-allergy sufferers were stimulated, no further studies were performed on the possible use of Hg-specific lymphocytes as indicators of Hg allergy. It should be noted, however, that no studies have ever been published on individuals without amalgam fillings. Schopf and co-workers (Die Naturwissenschaften, pp. 461-465, 1969, Schopf E. Schulz KH, 1 Isensee, Arch. Klin. Exp. Derm., 234: 420-33, 1969), have reported that many organic mercury compounds such as phenylmercury , ethylmercury and methoxyethylmercury did not stimulate lymphocytes in in vitro testing. Some other organic mercury compounds such as dibromohydroxy mercury proved to be stimulating. The previously mentioned "mitogenicity" of HgCl2 has led to the fact that the possibility of using LTT for the diagnosis of mercury allergy in vitro has not been further investigated.

It has been found according to the invention that the LTT method gives false positive or false negative 3 H-thymidine incorporation caused by the presence of monocytes or macrophages in the isolated lymphocyte fraction. The read radioactivity therefore does not have to reflect the actual concentration of antigen-specific lymphocytes, so-called memory cells, but there may be a false positive incorporation of the isotope into the nucleus of macrophages, which upon harvesting of the lymphocyte fraction is broken down and stuck together with the stimulated lymphocytes, so-called lymphoblasts, on the filter paper used. Furthermore, macrophages can incorporate cell fragments that contain isotopes (non-specific adherence), so-called phagocytosis.

The amount of isotope available in the culture medium therefore decreases, since phagocytosis is a faster process than incorporation of isotope into the lymphoblasts. This leads to a less effective labeling of the lymphoblasts. The macrophage cytoplasm breaks down at harvest and the isotope is washed away.

Although lymphoblasts may be present in such cultures, they cannot be detected by the isotope label. The present invention, MELISA, relates to a method for the in vitro determination of mercury allergy by means of lymphocytes, so-called memory cells, in blood, wherein a lymphocyte fraction isolated from blood samples grown in the presence of an antigen to determine the possible presence of memory cells for the antigen among the lymphocytes by stimulating them to grow, which is determined, which method is characterized in that the lymphocyte fraction is substantially free of monocytes and macrophages and in that the antigen is one or more organic mercury compounds and low doses of inorganic mercury (HgCl2). In addition to the radioactive technology used to measure memory cell division, direct microscopic assessment of memory cell growth in cultures (presence of lymphoblasts) is also performed. Because the new method according to the invention, in contrast to the lymphocyte transformation test (LTT), safely measures the antigen-specific lymphocytes or the so-called memory lymphocytes, the method has been called MELISA from the English Memory Lymphocyte Immuno-Stimulation Assay. During 10 years of studies of memory cells formed against low molecular weight haptens, no results have been found that would indicate a non-specific mitogenic effect from such haptens.

The procedure is based on venous blood, which has been treated in such a way that coagulation is prevented. This can be done by adding anticoagulants to the blood. However, it has been shown that heparin, EDTA and most citrates interfere with the analysis, probably by inhibiting the hapten presentation. A suitable citrate, however, is ACD solution (Becton, Dickinson Vacutainer Systems, Rutherford, New Jersey 07070). Preferably, the blood is defibrinated by shaking it by hand or in a mechanical stirrer together with particles. Preferably shake for about 5-10 minutes together with plastic or glass balls. In particular, the blood is collected in 10 ml sterile test tubes and shaken with plastic beads (Bekton Dickinson) for about 6 minutes.

The lymphocyte fraction is then recovered by gradient centrifugation.

This can be done by layering the defibrinated blood on Leuco PREP (R) (Bekton Dickinson) and centrifuging at 1300-1600 g for 10-20 minutes, preferably at 1500 g for about 15 minutes. One can also dilute the blood at least twice preferably with isotonic solution, in particular a culture medium such as RPMI 1640 medium (Gibco, UK, Roswell Park Memorial Institute, contains 10 mmol of N'-2-hydroxy-ethylpiperazine- -N ' -2-ethanesulfonic acid and 1 g NaHCO 3 per liter). The medium is preferably supplemented with antibiotics such as gentamycin and with glutamine. Then the mixture is layered over Ficoll-Paque (Ficoll-Paque contains per 100 ml of Ficoll 400 5.7 g of sodium diatrizoate, 9 g of sodium-calcium edetate, Pharmacia) in sterile centrifuge tubes and centrifuged for about 20-45 minutes at about 400- 800 g, preferably at 30 minutes at 600 g. Suitably relative ratio of the RPMI diluted blood to Ficoll is 6: 4. After centrifugation, the intermediate phase containing monocytes and lymphocytes is recovered and optionally washed with isotonic solution or medium such as RPMI 1640 medium.

Thereafter, monocytes are removed from the lymphocyte fraction by treatment with monocytotoxic substances, for example with lysosome tropical agents (L-leucine methyl ester, or structurally similar substances) or the maturation of monocytes into macrophages is prevented by the addition of maturation inhibitors. substances such as adenosine or structurally similar substances. Preferably, the monocytes are removed by adhesion to surfaces such as plastic or glass. These are suitably sterile. This is done by suspending the lymphocyte fraction in RPMI 1640 medium, and incubating it with a culture medium for 10 minutes - overnight, preferably until monocytes and macrophages have adhered to the surface (can be determined under an inverted microscope), in particular for 1 hour in a plastic vessel. Preferably incubated at 36-38 ° C. During this time, the vast majority of monocytes and other antigen-presenting non-lymphocyte cells will attach to the plastic surface, and in this way they can be easily removed from the lymphocytes. RPMI 1640 medium containing heat-activated serum or plasma, in particular 20% heat-inactivated human AB + serum, can be used as culture medium. Non-adherent cells are diluted in culture medium. The cells are preferably counted by taking a sample thereof and staining with Gentiana violet solution according to Türk KLVI (Apoteksbolaget Malmö) and the number of cells is determined by counting under a microscope in a Bürker chamber (Kebo-lab Stockholm) or in an automatic cell counter. The cells are diluted to a density of 0.5-2.106 cells per ml, preferably 1,106 cells per ml. A culture medium preferably RPMI 1640 medium containing 10% heat inactivated AB + serum was used as diluent.

The lymphocytes are distributed in sterile round-bottomed culture tubes, preferably 10 ml, which are loaded together with the solution to be tested. Plates (preferably sterile) with 48 wells can be used for culture, and the test substance, the antigen, is either added in advance or lyophilized directly on the plate before the lymphocytes are added. The final cell concentration is 0.5-3.106, in particular 1,106 cells per culture. The cultures are incubated for 4 to 8 days, especially 4-5 days.

The lymphocyte transformation is then analyzed by determining whether any DNA synthesis has taken place in the culture and by assessing the morphology under a microscope. Assessment of DNA synthesis when culture tubes were used.

To each well of a 96-well microplate is added 30-60 kBq of labeled thymidine or another functionally similar amino acid, eg preferably 37.0 kBq of methyl 3 H-thymidine (specific activity 3.18 TBg mmol). 100-250 μl of cell suspension in triplicate is added from each culture, preferably 200 μl is used. The plates are then incubated at 36-38 ° C, preferably 37 ° C for 3-6 hours in a CO 2 incubator. After incubation, the cells are harvested. Preferably, an automatic cell harvesting device (INOTECH AG, Wohlen, Switzerland) is used, using distilled water for washing. The plates are harvested directly or stored overnight at 4 ° C before harvest.

The plates can also be frozen for a longer period of time.

Determination of DNA synthesis when 48-well plates are used for culture.

A 48-well plate is provided with 75-150 kBq of methyl 3H-thymidine (specific activity 3.18 TBq / mmol) or another functionally similar amino acid, preferably 111 kBq per well. The cell sediment in the culture plates is resuspended and 300-800 μl, preferably 600 μl of each culture is removed and transferred to the labeled plate. Incubation and harvesting takes place as above.

After harvesting, the filter papers from the automatic cell harvesting device, which filter papers contain the lysed cell filtrate, are placed in micro ampoules and 1 ml of Opti-Scint (LKB) is added. The ampoules are then sealed and the radioactivity is measured in a ß-liquid scintillation counter. He can also measure DNA directly on filters without micro ampoules and scintillating fluid in, for example, the Tracer 96 Counting unit (INOTECH AG).

For morphological evaluation under a microscope, 50-100 pg, preferably 75 μl of cultured cells are taken and centrifuged in a cytocentrifuge at 70-100 g, for 1-10 minutes, preferably 467 at 20 g for 8 minutes. Cell smears are preferably fixed in methanol and stained with May-Grünewald and Giemsa (Diagnostica, Merck). A semi-quantitative determination of the number of lymphoblasts (transformed lymphocytes) and macrophages is done by microscopy. During microscopy, the general condition of the cells is also assessed and the number of macrophages and their degree of stimulation are noted, especially if they deviate significantly. The level of DNA synthesis in the test culture reflects the proportion of cells stimulated with the test substance.

DNA synthesis is expressed as the number of readings per minute (CPM) for each cell precipitate. Stimulation of DNA synthesis by treating the cultures with the antigen is expressed as a Stimulation index, which is defined as Stimulation index (SI) = CPM in treated culture mean for CPM in negative control cultures DNA synthesis is also advantageously expressed as A CPM, which is defined as: A CPM = mean value of CPM in test culture - mean value of CPM in negative control cultures DNA synthesis in the control cultures, which only received solvents without test substance, can vary within wide limits due to varying spontaneous activation of the lymphocytes. CPM values of less than 100 per 200,000 cells indicate suboptimal growth.

This can be confirmed under a microscope. Higher values (eg Patient L 16, Table 3A) indicate spontaneous activation, but both of these conditions must be confirmed by morphological study under a microscope. As a positive control of lymphocyte transformation in the assay, 0.01-20 pg of PPD (Purified Protein Derivative - Tuberculin, Statens Serum Institute, Copenhagen, Denmark) can be used, preferably in a concentration of 0.1-1 pg / ml, or any other antigen to which all humans have an immune response 10 15 20 25 30 35 4e7 49s 9 against, eg SK-SD (Streptokinase - Streptodormas) or tetanus toxoid. The degree of response to PPD varies between different individuals. However, a significant increase in DNA synthesis should be obtained in cultures treated with PPD. There are rare cases of non-response from PPD due to, for example, genetic factors or due to certain diseases. If a low PPD response is obtained, it is important to find out if the patient may not have been immunized with Tuberculin or if the patient is "genetically non-responsive" (cannot be immunized). This can be found out by asking if the patient reacted in a patch test with Tuberculin. A lack of Tuberculin response may also indicate an immunological defect or disease.

Low growth and thus low control value can lead to false positive DNA results. Strong spontaneous blastogenesis (lymphocyte transformation) contributes to low sensitivity of the test. The patient should be tested again to confirm data if there is any uncertainty.

The evaluation of the results in relation to the stimulation of DNA synthesis is based on the following new criteria. Stimulation responses that are at least 2 times higher than the background are considered to be slightly positive if they are achieved with concentrations that are known to be optimal for the tested compound. The more subsequent concentrations that the lymphocytes respond to, the more certain the positive response is considered to be. 2. Stimulation response 2 3 times higher than the background is considered positive. Positive responses (paragraphs 1 and 2) should preferably show a dose-response relationship.

A semi-quantitative analysis of the proportion of lymphoblasts is performed with morphological assessment under a microscope. The number of lymphoblasts on the entire slide is indicated by a number of up to 100 or 10 15 20 25 30 35 467 498 10 is indicated by 2,100 when there are more. Other observations that may be important for the evaluation of the results, such as spontaneous transformation of untreated cells, the presence of large numbers of macrophages in the culture, or a decrease in the viability of the cells (broken, destroyed lymphocytes) should be observed.

Morphological examination of cytological preparations of cell cultures provides a confirmation of data from the analysis of DNA synthesis. Thus, a correlation between the increase in the number of lymphoblasts and DNA synthesis should be observed.

Morphological information also provides facts about the general state of cell culture after incubation. Activated macrophages can sometimes produce a false positive response due to the incorporation of 3H-thymidine. Cultures containing only activated macrophages and not lymphoblasts should be considered negative despite 3 H-thymidine incorporation.

Activated macrophages can also, due to phagocytosis, contribute to false negative results.

By repeatedly using the new method to actively remove monocytes and macrophages, however, the risk of false positive and negative results is reduced.

According to the present invention, most monocytes and other adherent cells are removed at an early stage as the recovered lymphocyte fraction is incubated without the presence of test substance so that monocytes and other adherent cells can attach to the walls of the culture vessel. (1st adherence step).

MELISA is also preferably carried out by culturing in the presence of the test substance or the so-called antigen on a plate with wells or in test tubes in a step which is separate from the step in which the culturing takes place in the presence of the radiolabeled amino acid. Thus, a transfer of the cells from the first plate or test tube, where the culture takes place together with the test substance, to the plate or test tube, where the culture takes place together with radiolabeled amino acid, is performed. (2nd adherence step). During culture (4-5 days), a number of macrophages have developed in the culture (this is clearly visible in cell smears) despite the fact that a 1st adhesion step was performed. This means that a number of monocytes do not attach during the 1st adhesion step but later. However, according to the known LTT method, the culture is carried out together with antigen and in one and the same vessel in which incubation with radiolabeled amino acid takes place later (96-hole microplates). In addition, no additional culture step is often performed to attach monocytes and other non-lymphocytic antigen-presenting cells to the walls of the culture vessel before culturing in the presence of test substance.

Thus, with the present method, monocytes and macrophages are preferably removed at least twice by transferring the cells between different vessels so that monocytes and macrophages on at least two occasions have the opportunity to attach to the vessel wall and be separated in this way.

Between the two adherence steps (or between the latter if more adherence steps are used), the cells should be grown on medium until any non-adhering monocytes mature into macrophages. This maturity can be determined by microscopy.

Cultivation one day to 5 days is suitable.

The invention is further elucidated by means of the accompanying drawings, in which Fig. 1 shows the lymphocyte proliferation against inorganic and organic mercury compounds in patients with oral lichen, expressed with maximum stimulation indices, Fig. 2 shows maximum stimulation indices in individuals tolerating amalgam, Fig. 3 shows the lymphocyte proliferation against inorganic and organic mercury compounds in amalgam-free donors, expressed with maximal stimulation indices, Fig. 4 shows the lymphocyte proliferation against mercury in a patient with oral lichen, expressed with radioactivity rash (A CPM).

Diagnosis of mercury allergy Patient and control material Patients with lichenoid changes in the oral mucosa located next to amalgam fillings (group I) and other patients who suspected mercury allergy as a cause of their diseases (L 16, S 31, S 92 and Table 1) were investigated with patch tests and with ig_giL; g The MLISA test of the present invention. The MELISA test was performed according to Examples 1 and 2 using phenylmercury acetate, HgCl 2 and methylmercury chloride and ethylmercury chloride as test substance. At the same time, trouble-free amalgam carriers (group II and, for example, blood donors SK 45) and amalgam-free people (group III) were examined.

Specific memory cells against the phenylmercury acetate were detected in most patients with lichenoid changes localized to amalgam fillings (10 of 13 in Fig. 1). 3 patients lacked such memory cells and therefore their oral problems must be due to toxic-irritating effect rather than an allergy to mercury. Healthy amalgam carriers and amalgam-free individuals lacked such memory cells (Figs. 2 and 3). All patients with oral mucosal problems showed memory cells against HgCl2. Since 12 of 15 hassle-free amalgam carriers and 7 of 12 amalgam-free individuals reacted similarly, the presence of memory cells against inorganic mercury indicates an immunological response to mercury and not to mercury allergy. Positive patch test against phenylmercury and Timerosal (ethylmercury thiosalicylate) was detected in 6 of 13 patients with oral lichen. Many of these patients also responded to Timerosal in MELISA. Both tests showed clinical relevance to amalgam seals. Because memory cells (as opposed to mucosal changes) are present in the blood throughout the body, they can be used to advantage for the diagnosis of systemic mercury allergy. Patients' symptoms disappeared or improved significantly after amalgam removal (so-called clinical relevance).

Since the reactivity of memory cells to phenylmercury was a hallmark only in patients with pathological changes caused by amalgam and not in healthy amalgam-exposed and non-exposed individuals, phenylmercury and structurally similar compounds can be used for a relevant diagnosis of human mercury allergy.

This very unexpected finding can be explained if it was also possible to find cross-reactivity to other organic mercury compounds which, according to published work, can occur in the oral cavity and gastrointestinal tract. One such compound is methylmercury. According to Heinze (Scand. J. Dental Res., Vol. 91, pp. 150-152, 1983), certain strains of oral microorganisms, such as Streptococus mitior, mutans and sanguis, can convert metallic and inorganic mercury to methylmercury. Some microbes in the human gastrointestinal tract have similar properties (Abdullah, M., Arnesjö B. and Einsi I.

Scand. J. Gastroenterology, pp. 565-567, 1973). Preliminary results show that lymphocytes from patients with oral problems who react to phenylmercury also cross-react with methylmercury derivatives, eg methylmercury chloride and to a lesser extent with ethylmercury compounds (Table 1, Table 3AB). Positive responses to methylmercury and ethylmercury were not found in the other two groups (Table 1, Table 4).

Together, these results show that human lymphocyte activity against phenylmercury and structurally related organic mercury compounds can be used for objective diagnosis in gigs of mercury allergy in humans. In this way, one can distinguish the "real" mercury allergy sufferers from people who show a false positive skin test or whose oral or other health problems are not caused by an allergic reaction to mercury. One can further investigate whether a mercury allergy is behind certain autoimmune nerve diseases, for example multiple sclerosis (MS), acute lateral sclerosis (ALS), Parkinson's disease, Alzheimer's disease, acrodynia and others. Since some patients with oral lichen also have other problems, for example inflammation in the joints, diabetes, Sjögren's disease, fatigue, fever, etc., the connection to mercury allergy can be studied. By identifying the cause of these diseases, it is possible to treat them appropriately.

Table 1 Lymphocyte proliferation against acrodynorganic 10 mercury compounds in patients with oral lichen (L + S) and in healthy blood donors (SK) Stimulation index Phenylmercury Lympho-Methylmercury Lympho- Patient A CPM SI blaster A CPM SI blaster L 16 19 665 4.5 + 15 573 3.8 + S 32 6 434 4.3 + 2 527 2.3 + S 34 1 009 2.4 + 1 580 3.2 + S 93 3 294 2 + 22 819 4.3 + SK 45 * 524 2 - 439 1.3 - SK 46 ** 140 1.1 - 871 1.8 - * sensors that tolerate amalgam ** amalgam-free sensors SI = stimulation index As can be seen from Figures 2 and 3, a lot of healthy amalgam carriers and amalgam-free individuals did not respond to any of the 30 mercury compounds examined. This fact can be explained in two ways, 1) some people are genetically "non responders" to mercury, 2) some people are desensitized by constant exposure to mercury (for example, dental nurses often do not respond to mercury in 35 MELISA). This shows that inorganic mercury is not mitogenic to human lymphocytes as previously thought, but quite surprisingly, acts as an antigen. The reactivity to high doses of HgCl2 in many donors could then reflect the immune response to mercury and, indirectly, the mercury exposure.

Another distinguishing feature of mercury allergy sufferers is their response to low concentrations of inorganic HgCl2 (<0.5 pg / ml media). Such answers are found only in mercury allergy sufferers and not in healthy amalgam carriers or in amalgam-free people. Thus, another property of mercury allergy sufferers may be the presence of mercury-specific memory cells, which can be determined with low doses of inorganic mercury (<0.5 pg HgCl 2 / ml and 1,106 cells).

The invention will now be further elucidated with the aid of some embodiments.

Example 1 Culture in tubes 50 ml of blood were diluted with 50 ml of RPMI 1640 and stored over Ficoll-Paque in a ratio of 2: 3 in 50 ml of sterile plastic tubes (Bekton Dickinson) and separated by gradient centrifugation at 600 g for 30 minutes. The intermediate phase (the opaque white layer) containing mainly lymphocytes and monocytes was removed and washed once with RPMI 1640 medium. The cells were then resuspended in RPMI 1640 medium containing 20% heat-inactivated human AB + serum, glutamine and gentamicin were transferred to a plastic cell culture flask and placed in a CO 2 incubator with 5% CO 2 / air, 100% humidity and 37 ° C for 2 hours. Monocytes and other adherent cells adhered to the plastic surface and were removed by diluting the non-adherent cells twice with RPMI 1640 medium.

The cells were counted by removing x μl of the cells and staining with Türk, and the number of cells was determined by microscopic counting in a Bürker chamber, after which the cells were diluted to a density of 0.5.106 cells / ml. 2 ml of lymphocyte suspension in RPMI 1640 containing 10% heat-inactivated AB + serum was distributed in 10 ml round-bottomed test tubes, which were filled with 100 μl of a solution containing different concentrations of HgCl 2 or phenylmercury acetate. The tubes were grown in CO 2 incubator and 100% humidity for 6 days. A 96-well plate was loaded with 10 μl of methyl 3H-thymidine (37 kBq per well), specific activity 3.18 TBq / mmol, and 200 μl of the cell suspension in triplicate was transferred from each culture. The plates were then incubated at 37 ° C for 4 hours in a CO 2 incubator. After incubation, the cells were harvested in an automatic cell harvesting device (INOTEX) using distilled water for washing. The plates were harvested immediately.

The filter paper from the automatic cell harvesting device, which contained filtrates of the lysed cells, was placed in micro ampoules, and 1 ml of Opti-Scint (LKB) was added. The ampoules were sealed and the radioactivity was measured in a ß-liquid scintillation counter. The results are shown in Fig. 4.

Another sample was taken from each tube for morphological evaluation under a microscope. 75 μl was taken from each tube and centrifuged in a cytocentrifuge at 90 g for 2 minutes.

Cell smears were fixed in methanol and stained with May-GrÜnewald and Giemsa. The following results were obtained (Table 2).

Table 2 shows that patient 31 shows positive results in MELISA with HgCl2 and with phenylmercury acetate. The results indicate mercury allergy. 467 498 17 TABLE 2 PATIENT S31 - ORAL LICHEN - MORPHOLOGICAL EVALUATION Antigen ng / m1 Lymphobiaster per prep.

Kontro11 - 1, 0 HgC12 1 23 0.5 100 0.25> 100 0.12> 100 0.06> 100 Phenykvicksiïver- 1 - toxic acetate 0.5 7 0.25> 100 0.12 53 0.06 39 Tubercu11n (PPO) 1> 100 Evaluation l. Positive response to all concentrations of Hgßïg 2. Positive response to phenylmercury 3. MELISA result shows mercuryaivergy A 10 15 20 25 30 35 67 498 18 Example 2 Culture on 48-well microplate 50 ml of blood was diluted with 50 ml of RPMI 1640 medium and stored over a column packed with Ficoll Isopaque (Pharmacia) in sterile plastic centrifuge tubes and centrifuged for 30 minutes at 600 g as in Example 1.

After centrifugation, the white opaque intermediate phase containing mainly lymphocytes and monocytes was separated and washed once with RPMI 1640 medium. The cells were then suspended in RPMI 1640 medium containing 20% heat-inactivated human AB + serum, glutamine and gentamycin were transferred to a plastic cell culture flask and placed in a CO 2 incubator with 5% CO 2 / air, 100% humidity and 37 ° C for 1 hour.

During this time, monocytes and other adherent cells adhered to the plastic wall, and the lymphocytes could be easily removed and diluted twice with RPMI 1640 medium. Cell counting was done as in Example 1. The cells were diluted to a concentration of 1,106 per ml. 1 ml of the lymphocyte suspension in RPMI 1640 containing 10% heat-inactivated AB + serum, glutamine and gentamicin was added to each well of a 48-well plate.

In each well, 100 μl of an antigenic solution of various mercury compounds was added. The cultures were incubated for 5 days using a CO 2 incubator as in Example 1.

After 5 days, the contents of each culture plate have been resuspended 3 times. 600 μl of each culture was taken out and transferred to a new 48-well plate, in which each well was supplemented with 10 μl of methyl 3H-thymidine with a specific activity of 3.18 Tbq / mol 111 kBq per well. Incubation was at 37 ° C for 4 hours in a CO 2 incubator.

After incubation, the cells were harvested in an automatic cell harvesting device as in Example 1. The filter papers containing the lysed cell filtrate were placed in micro-ampoules and 1 ml of Opti-Scint (LKB) was added. The ampoules were sealed and the radioactivity was measured in a β-liquid scintillation counter. The following results have been obtained. (Table BAB and Table 4).

Table 3AB shows lymphocyte profiling against various mercury compounds expressed by radioactivity rash and morphological assessment.

Patient L 16 (Table 3A) shows a positive result in MELISA with all three tested mercury compounds indicating mercury allergy.

Patient 392 (Table 3B), on the other hand, shows a rash only with a high dose of HgCl2 but not with phenylmercury acetate. The result does not indicate mercury allergy.

Table 4 shows lymphocyte proliferation against various mercury compounds in a healthy amalgam carrier, expressed with radioactivity rash and with morphological assessment.

MELISA with HgCl2 is positive only with high doses of HgCl2 (2.25 and 4.5 pg / ml) but not with phenyl, ethyl or methylmercury chloride.

This is a relative pattern that occurs in healthy amalgam carriers.

TABLE 3A PATIENT WITH SUSPECTED MERCURY ALLERGY Patient L 16 - Plate culture Antigen Concentration Number of pg / m1 Acpm SI lymphoblasts Q Control - (16864i5407) 1.0 34 - 46 - 18 Positive control PPD 1.0 299779 18.8> 100 Test substances l.HgC00 4.0 22079 2.3> 2.0 32753 2.9> l00 1.0 32696 2.9> l00 0.5 30975 2.8> l00 0.25 59201 4.5> l00 0.125 50262 4.0> l00 2.Feny1kvick- 2.0 -16531 <0.1 - toxic silver acetate 1.0 -11131 0.3 - toxic 0.5 -1180 0.9 22 0.25 15151 1.9> l00 0.125 58994 4.5> l00 0.062 33939 3.0> l00 3.Methylmercury- 2.0 -16535 <0.1 - toxic silver chloride 1.0 -9520 0.4 20 0.5 -11112 0.3 24 0.25 4056 1.2 90 0.125 18262 2.1 90 0.062 46720 3.8> l00 EVALUATION : 1) Positive proliferation against all concentrations of H (high and low). 2) Positive proliferation towards phenyl-Hg and methyl-Hg. 3) The results indicate a mercury allergy. 9C12 k IN TABLE 313 467 498 PATIENT WITH SUSPECTED MERCURY ALLERGY Patient S 92 - Plate culture Concentration Number Antigen ug / ml ßcpm SI lymphoblasts Control (907i273) * 1.0 l, 0.0 0,0,0 Positive control PPD 0.1 606793 670> 100 0.01 468957 S18> 100 HgCl2 9 -794 0.1 - toxic Mercury- 3 S620 6.8 4 chloride 1 399 1.4 0 0.33 226 1.2 1 0.11 6 1.0 1 0.037 289 1.3 2 0.012 -98 0.9 0 0.004 368 1.4 0 Phenylmercury 0.5 -128 0.8 0 acetate 0.25 -21 1.0 0 0.125 100 1.1 0 0.062 -205 '0.8 0 0.031 -315 0.6 0 0.015 12 1.0 0 0.007 -47 0.9 0 EVALUATION: 1) Positive response only to high dose of HgCl2. 2) Negative response to phenylmercury. 3) The results do not indicate mercury allergy. 467 498 TABLE 4 HEALTHY AMALGAMBER (SK 45) - PLATE CULTIVATION Antigen Concentration Number of pg / ml Acpm SI lymphoblasts Control - (l527i359) * 1.0 0 - 0 - 0 Positive control PPD 1.0 30422 20.9> 100 Test substances l.HgCl2 9.0 -1096 0.3 - 4.5 2442 2.6 10 2.25 3642 3.4 10 1.12 207 ll 2 0.56 -289 0.8 2 0.28 -529 0.7 1 2.Phenylkvick- 0.5 -ll3l 0.3 toxic silver acetate 0.25 56 1.0 0 0.125 21 1.0 2 0.062 l574 2.0 0 0.031 -163 0.9 2 0.015 -513 0.7 4 3.Ethylquick- 0.5 -1100 0.3 0 silver chloride 0.25 -667 0.6 0 0.125 -24 1.0 0 0.062 1485 2.0 0 4.Methylquick- 0.5 -573 0 6 0 silver chloride 0.25 -135 0.9 0 0.125 439 1.3 0 0.062 422 l EVALUATION: l) Weak proliferative response to high concentrations of HgCl2 indicates a weak immunological response to mercury. 2) Negative response to phenyl, methyl and ethyl Hg.

Claims (7)

10 15 20 25 30 35 4e7 49s 23 Patent claims A method for the in vitro determination of mercury allergy by means of lymphocytes, so-called memory cells, in blood, wherein a lymphocyte fraction isolated from blood samples is cultured in the presence of an antigen for determining the possible presence of memory cells for the antigen among the lymphocytes by stimulating growth. which is determined, characterized in that the lymphocyte fraction is substantially free of monocytes and other adherent antigen-presenting cells and in that the antigen is one or more organic mercury compounds and low doses of inorganic mercury compounds. Process according to claim 1, characterized in that the organic mercury compound is selected from phenyl, methyl or ethyl mercury or a structurally similar organic mercury compound. A method according to claim 1, characterized in that blood is defibrinated or collected with anticoagulant which does not interfere with the presentation of mercury compounds and then gradient centrifuged to recover the fraction containing lymphocytes, monocytes and other cells, after which adherent non-lymphocytic antigen presenting cells are removed by incubating lymphocyte suspension in a culture vessel, preferably of plastic, wherein some of the cells attach to the vessel wall by adhesion, and the lymphocytes can be removed, or by adding the lymphocyte fraction with monocytic eg leucine methyl ester or structurally similar substances or by adding the lymphocyte fraction with substances which prevent the maturation of monocytes into macrophages, such as adenosine or structurally similar substances, and by removing the monocytes and other adherent cells in at least two steps repeated. Method according to claim 3, characterized in that monocytes and other adherent cells are removed by culturing the lymphocyte fraction in a culture vessel, preferably of 467 498 10 15 20 25 30 24 plastic, until it can be determined by microscopic examination that the cells have adhered to the surface, preferably at least 10 minutes, after which the lymphocytes are transferred to another culture vessel and the procedure is repeated at least once. 5. A method according to any one of claims 1-4, characterized in that the defibrination takes place by shaking a mixture of blood and particles and the supernatant is recovered, preferably by mixing blood and particles and preferably by shaking for about 5-10 minutes, or by the addition of anticoagulants which do not interfere with the determination of memory cells. Method according to claims 1-5, characterized in that the lymphocyte-containing fraction is grown in a culture vessel, preferably of plastic in a culture medium 0.5-3 hours, wherein monocytes and other non-lymphocytic antigen presenting cells adhere to the wall of the culture vessel, after which the lymphocytes are transferred to test tubes or wells in a plate and, grown together with antigen in a CO 2 incubator with 5% coz / air, 10% relative humidity and 37 ° c for 4-5 days, after which the cells are transferred to test tubes or wells. in a plate and incubated with radioactive amino acid, preferably 3H-thymidine is incubated at 31 ° C for 3-4 hours in the same coz incubators and by taking another sample from the culture vessel in parallel for morphological evaluation of lymphoblasts after fixation with methanol and dyeing with May-Grünewald and Giemsa. Use of the method according to any one of claims 1-6 for the analysis of diseases which may be due to mercury allergy.