DE2755363A1 - TEST PROCEDURE FOR DIAGNOSIS OF MALIGNOMAS - Google Patents

Description

Applicant: R & Z Vermögensverwaltungsges. mbH. 415 Krefeld

Magdeburger Str. 37

Title: Test Procedure for Diagnosing Malignancies

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The invention relates to an in vitro test method for Diagnosis of malignant tumors, preferably in humans, in which procedure the change in mobility cellular blood components with a migration inhibition factor (MIF) is determined, the MIF being excreted by lymphocytes which are sensitized to tumor tissue. Such a MIF is not eliminated, if the lymphocytes come from patients who do not have a malignant tumor (malignancy), as these lymphocytes are not sensitized and therefore do not release MIF on contact with tumor antigen.

From the literature (Douwes, F. R. et al; Verh. Deutsche Ges. Inn. Med., Volume 82, J. F. Bergmann-Verlag, Munich 1976; there further evidence) it is known that with the help of the so-called electrophoresis mobility test (EMT) early detection of malignant diseases is possible. The principle of the EMT is based on the fact that the migration speed (mobility) of certain, in the electrical field of a special microscope (Cytopherometer) visible bodies (macophages, Granulocytes, other blood cells) only changes when they are sensitized with lymphokines from lymphocytes get in touch. It is assumed that

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that lymphocytes raised against a specific antigen are sensitized (so-called T lymphocytes), release a number of soluble factors, the so-called lymphokines. An important one of these factors is the Migration Inhibition Factor (MIF). It influences the ability to migrate of blood cells, especially macrophages and granulocytes, and ensures that they are locked in place and accumulation at the site of the presence of the sensitizing antigen. In vivo that means the possibility of destroying the tumor.

Lymphocytes of malignancy patients react specifically by releasing the so-called soluble factors, when they come into contact with a basic protein that comes from the brain substance on the one hand or from one Carcinoma tissue, on the other hand, is taken. The basic proteins that control the specific reactions of the Lymphocytes are identified in the literature as encephalitogenic factor (EF) and basic protein called from carcinoma tissue (CA BP). The extraction takes place according to methods known per se (Caspary, E. A. et al., Brit. Med. 1971, pages 613-617 and other publications; see Douwes, F. R. a. a. 0.).

The results of

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Blood tests with the EMT show that with the help of the test a nearly 100% differentiation of the Samples can be made to determine whether they are an organism with malignant (tumor) diseases or a have been taken from a healthy organism or an organism with non-malignant diseases. All examined patients with (malignant) tumors showed a positive lymphocyte response, i. H. it was by the lymphocytes given a factor that leads to a measurable and characteristic change in mobility of indicator cells leads (see. Douwes, F. R., op. cit.) control examinations on patients with non-malignant Diseases showed no such lymphocyte response. Incorrect results could be due to certain causes to be led back. Accordingly, the EMT can be viewed as the most sensitive cancer test currently available.

A disadvantage of using the test is that it is relatively complicated to master operating instrument, namely the cytopherometer, It is assumed that the personnel observing and measuring the speed of migration are very careful train and supervise. Due to the decreasing attention, there is only a limited amount of work possible. The application of the EMT is limited

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therefore to specialized institutes. The evaluation of the test takes place during the movement of the blood cells, so dynamic. It is not possible to "stop" the experiment, so there is no re-measurement perform.

The object of the invention is to provide a test method for diagnosing malignancies which As general and unspecific as possible with regard to the type of malignancy with a high degree of accuracy signaled by malignancies, but in principle applied or carried out in every doctor's laboratory and is particularly suitable for mass screening in cancer screening. The result the test should be readily measurable. The test should be statistically evaluable and without intermediaries Logging can be archived.

These tasks are achieved with a test method of the type mentioned at the outset, in which as a cellular Blood components the lymphocytes to be examined themselves in a known migratron inhibition test in the course of which the lymphocytes are treated with an antigen that stimulates the formation of MIF brought into contact and then incubated

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and, after a fixed period of time, the lymphocyte migration is inhibited and fixed.

Various migration inhibition tests are known which are basically suitable for the investigation according to the task. Is known under among other things the so-called capillary technique according to S0BORG and BENDIXEN. To realize the idea of the invention it is necessary to increase the mobility of the indicator cells by "freezing" one after a certain To measure the time reached migration status.

According to the invention, the spread or change in mobility of the lymphocytes within

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a very fine, approximately ipm thick "open" capillary layer measured. A test arrangement that consists of a two-layer element has proven to be particularly suitable consists, namely of a carrier made of an inert medium, e.g. B. glass, and an agar that comes with Nutrients for blood cells is fortified. A particularly suitable agar is an agarose commercially available agar manufactured by Behring-Werke, Marburg.

The intermediate layer between agar and carrier layer

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is preferably partially filled with liquid. It has a thickness of about 1-20 μm. Preferably an incubation time for MIT between 10 and 30 hours is chosen.

The test is carried out with a sample with leukocyte-rich supernatant ("buffycoat") obtained from human blood with a fixed number of leukocytes between

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10 and 10 performed per sample. These standard values are tried and tested values that are a Do not require excessive blood draw from the patient.

An antigen solution in a ratio of 1:50 to 1:10 is added to the buffy coat sample. Antigen solutions can be used which are obtained according to a procedure specified by CASPARY and FIELD or were obtained with the aid of a 3 m KCl extraction according to MELTZER. Human beings serve as the starting material Malignant tissue from surgery or human brain matter. However, it is also possible to use antigen solution from tumor cells grown in vitro.

The test method according to the invention can not only be

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apply a non-specific malignancy examination, but also extend it to an organ-specific examination. If antigens are used which have been obtained from carcinoma tissue of certain organs, lymphocytes from patients who are afflicted by the same organ carcinoma react in the manner described. In the literature it is described that e.g. B. Antigen solution obtained from breast cancer according to CASPARY and FIELD or MELTZER result in a specific reaction exclusively for lymphocytes that come from patients with breast cancer.

It is also possible, if there is a positive immune response, in a general test, i. H. at Suspected carcinoma, do a series of tests with organ-specific antigens and thus find out in a relatively short time which organs could be affected by a carcinoma.

It is very important that the so-called immune response, i. H. causing the slowing effect in the Surprisingly, movement of the indicator cells in the present tests can be increased by that a solution with a so-called transfer factor is added to the sample solution. This factor is off

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known in immunology (see e.g. M. Rosenthal; The transfer factor and its therapeutic application; Switzerland, rad. Wschr., Vol. 104, 1974; Pp. 1501 ... 1506). The transfer factor can be produced from normal blood leukocytes. It represents a subcellular, dialyzable, non-immunogenic leukocyte factor responsible for the T-lymphocyte-dependent reaction is. Transfer factor preparations are available (SCHURA, Krefeld), which are converted into liquid by means of a fraction Oxygen and subsequent stepwise ultrafractionation are produced. One in the trade Unit contains a transfer factor of approx. 5. 10 Leuko- V cytes. The preparation is with 1% human alumumin solution stabilized.

It turns out that when transfer factor is added a Enhancement of the immune response is to be achieved. The addition of the transfer factor therefore results in the discrimination of the individual test tests has been significantly improved.

Furthermore, the invention relates to a test plate for performing the method, which consists of a flat support plate made of inert material, e.g. B. glass, on which a 0.5 ... 5 mm thick solidified

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Agar layer is applied, the at least two substantially circular punch holes with a diameter between 0.5 and 5 mm (preferably 2.2 to 3.0 mm). Each punch hole has about a capacity from 5 ... 20, preferably 8 µl test substance.

It is important that the punch holes are cut so that the uniformity of the capillary migration layer is also given in the area of the transition from the punched hole to the layer.

The invention is illustrated by means of examples. The following figures are also attached:

1 shows a test plate according to the invention in plan view;

Fig. 2 shows a test plate in section.

example 1

A Obtaining the leukocyte suspension

3O ml of human blood are mixed with 3OO USP units of Na

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Heparinate heparinized. The heparinized human blood (Patient's blood) is in a plastic tube with 8 ml 6 wt .-% dextran solution in physiological Saline solution (molecular weight of the dextran: 75,000) underlayed. The erythrocytes are then left sediment at 37 C in the oven for about 45 min. The leukocyte-rich supernatant (Buffycoat) is siphoned off and mixed with the same volume of 0.9% strength by weight NaCl solution, then Centrifuged for 15 min at an acceleration of 750 g. That which is deposited in the centrifuge The pellet is washed two more times in 10 ml of Hank's solution (pH 7.2) each time and in a Tc 199 medium (Manuf .: Serya, Heidelberg) suspended. When washing and suspending the cells become with a Pipette whirled up until macroscopically no more cell clumps can be seen. The leukocytes will counted after staining with Türk's solution in the Neubauerkammer and on 2. 10 cells / ul in Tc 199 medium set. On average, the sample yields 25% mononuclear cells (lymphocytes and monocytes) and 75% granulocytes. The erythrocyte contamination is less than 1 to 1.

B Preparation of the antigen solution

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To prepare the antigen solution, the encephalitogenic factor (EF) is | um | the basic protein Isolate carcinoma tissue (CaBP).

The tumor or the brain will be under sterile conditions as soon as possible after the operative Distance edited. Connective tissue and healthy tissue are removed from the tumor. After that, will cut the tumor or brain tissue into small pieces with sterile scissors (scalpel) and further comminuted in the homogenizer in an ice bath. The Zumor tissue must not move during this process heat. The homogenate is suspended in four times the volume of distilled water and then in Centrifuged 23,0OO g. The resulting pellet is resuspended in four times the volume of 0.9% NaCl, homogenized and centrifuged at 23,000 g for 30 min. Which in turn is distilled in four times its volume Water resuspended pellet is freeze-dried. The freeze-dried powder is im Ratio 1:10 with a chloroform / methanol mixture (2: 1) and stirred for 30 min. That Mixture is centrifuged at 600 g for 10 minutes. The supernatant is discarded.

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This operation is repeated twice. The pellet is air dried. The dry pellet is resuspended in five times the volume of a 5% NaCl solution in water and 30 min at 23,0OO centrifuged g. The supernatant is discarded. With the addition of n / 100 NaCl, this is again five times as high Volume of distilled water resuspended pellet adjusted to pH 3.5. The suspension is gently shaken for 30 min, then centrifuged again for 30 min at 23,000 g. The survived is discarded. The pellet is again resuspended in five times the amount of distilled water, which is adjusted to a pH of 2.6 with n / 100 NaCl and left to stand for 3 to 18 hours will. The pH value must not change during this time. The suspension is at 23,000 g for 30 min centrifuged for a long time. The supernatant is kept, the pellet is again in n / 100 NaCl at pH 2.6 washed, centrifuged. The overhang that arises here is canceled. The latter two Supernatants are pooled and then dialyzed against distilled water. Then takes place a freeze drying.

An antigen test solution is made by dissolving

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0.1 mg / ml of the freeze-dried substance obtained in distilled water. In principle The same manufacturing process also applies to a solution of the basic protein from tumor tissue (CaBP).

Manufacture of the test plate
(See also Figs. 1 and 2)

0.5 g of a special culture agar (agarose; manufacturer Behring-Werke, Marburg) are in 50 ml Erlenmeyer flask weighed in. To this end, 22.5 ml of sterile double-distilled water are added.

In a second Erlenmeyer flask, 22.5 ml of the double-concentrated Tc 199 medium and 5 ml given plasma obtained from human blood. The agarose is placed in a holder while gently rotating Shake dissolved in a boiling water bath. The Erlenmeyer flask is covered with a foil or closed with a stopper, otherwise part of the water would evaporate.

After the agarose has completely dissolved, it is allowed to cool to 47 ° C. in a water bath. In the same bathroom

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the medium-plasma mixture is then preheated and added to the agarose with shaking, so that 50 ml of a 1% agarose in simple Tc 199 medium with 10% plasma are now available.

With a preheated dispensing pipette each 5 ml of the agar are filled into round tissue culture dishes (Petri dishes) 10 with a diameter of 50 mm, which are shown in Fig. 1 in plan view. After the agar has solidified, use a telescopic punch cannula (Behring-Werke, Marburg) with the aid of a template, holes 11 with a diameter of 2.5 mm punched into the agar. When punching out, make sure that the agar layer is in the punching area is not detached from the glass layer so that the capillary layer does not enlarge and so does the Migration of the lymphocytes is no longer guaranteed.

The arrangement of the holes 11 results from the Fig. 1. Of course, other figurations are conceivable. FIG. 2 shows an enlarged section through the lower part of the dish 10. It is indicated that the agar layer 1 from the glass layer the shell 10 through a thin capillary layer 3

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is separated. The individual rows of holes are intended, for example, for: A Control (standard) B Test suspension with general antigen C Test suspension with organ-specific

antigen

D Test suspension with organic antigen and transfer factor.

D reaction, incubation and fixation

The following approaches are used for a test:

1. each with 100 μΐ leukocyte suspension according to A and 8 ul antigen suspension according to B;

2. each with 100 μl leukocyte suspension and 8 μl n / 100 NaCl solution (for the reference value).

The mixed batches 1 and 2 are incubated for 60 min at 37 ° C. in a heating cabinet with 100% air humidity. Then 8 ul of the incubated solution or substance containing leukocytes and antigen is poured into each punch hole. Filled in standard substance. For the evaluation of the leukocyte migration and the influence by the Migration-slowing factors produced by the lymphocytes are available depending on the configuration

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2-5 measurement results and 2-5 reference values are available on the test plate (number of punched holes). The cells contained in the suspension that were inserted into the punch holes at the start of the experiment, migrate into the intermediate layer 3 between agar and the surface of the dish. By incubating the Plates at 37 ° C in a heating cabinet for 18 hours, this hike over a defined span maintain. The leukocytes do not migrate into the agar, but simply draw from it the nutrients. The incubation is carried out at a relative humidity of 100% in a humidity cabinet carried out.

After incubation, the plates are washed with methanol for 15 minutes and then with formalin solution for 10 minutes (37 wt .-% methanal) overlaid. The agar is then carefully removed as a skin from the bottom of the dish deducted. The cells that have migrated in the intermediate layer are now fixed to the surface of the shell. After drying, the cells are stained according to Pappenheim, with dark purple halos ^ (wandering courtyards) result on the plate surface (see. Fig. 1).

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E measuring and determining the results

The diameter is measured with a measuring magnifier (Bausch & Lomb) of the migration halo. The polymorphonuclear ones lying in the periphery are isolated To capture cells with. The arithmetic mean is calculated from several measurements.

The quotient of the migration area F ₁ with antigen and the migration area F ₂ without antigen gives a "migration index" MI:
MI = F ₁ ZF ₂ .

A migration index <0.85 means inhibition of the leukocytes to a significant extent and thus a positive cell response. The following table shows a measurement example:

line 0 (mm) F. (mm ² ) 8th MI B. 6, 75 F. 1 = ³⁵ ' 75 0.80 A. 7, 55 F. ₂ = 44, (D

Initial studies on a total of 97 patients should determine the effectiveness of the migration inhibition

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Testes in breast cancer. 27 of 46 examined patients with breast cancer show one Inhibition greater than 15% (= 58.7%), i.e. H. the migration index MI is below 0.85. Both In 23 examined patients with mastopathy, only four of 23 showed such an inhibition (= 17%) In patients with fibroadenoma, one patient out of five (= 20%) showed inhibition. The following table gives a general overview:

diagnosis Patient number
(n) Migration index
(inhibited) (normal) (0%) 2O (100%) Controls 20th 0 (17%) 19th (83%) Mastopathia cystica 23 4th (20%) 4th (80%) Fibroadenoma 5 1 (O%) 4th (100%) Breast cysts 4th 0 (33%) 6th (67%) other malignant
Diseases 9 3 (58.7%) 19th (41.3 Mammary carζinom 46 27

Example 2

Use of the transfer factor to strengthen the immune response.

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It has already been pointed out that adding a solution with a transfer factor (manufacturer: SCHURA, Krefeld) an enhancement of the immune response can be induced. It turns out that with Addition of factor MI is more pronounced than compared to test example 1.

The following test approaches are made:

1. 100 μl leukocyte suspension + 8 μl antigen solution

2. 1OO μΐ leukocyte suspension + 8 μΐ antigen solution + 20 m1 solution transfer factor (SCHURA preparation; 1 μΐ corresponds approximately to the transfer factor of 5. 10 leukocytes)

3. 100 μΐ leukocyte suspension

4. 100 μΐ leukocyte suspension + 20 μΐ transfer factor solution as with approach 2.

Incubation and preparation of the dish are carried out as in Example 1. It is a dish with four times five punch holes (2.5 mm 0) made. A row with 5 holes is filled with 10 μΐ of the test approaches 1 to 4 loaded.

Results (each averaged from five values):

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approach line 0 (well) F (mm ² ) MI 1 B. 6th , 90 37.4 0.79 2 C. 6th , 05 28.7 0.61 (incl.
Transfer factor) 3 A. 7th , 75 47.2 1.0 standard! 4th D. 7th , 70 46.6 0.99

The low MI of rows B / C indicates a manignoma case.

Results with the migration inhibition test using the transfer factor in the determination of gastrointestinal tumors:

Measurements were carried out using the measurement procedure according to Example 2. In a (healthy) patient control group there was no significant inhibition, the migration index was 1.02 +0.1. The transfer factor influenced these results not. In the group of those suffering from colon carcinoma In patients, the migration index was 0.65 + 0.2. Of the 15 cases examined showed at least 11 had a migration index that was less than 0.85. After adding transfer factor, the Migration index at 0.61 + 0.09. Furthermore

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In 14 of 15 patients the MI after the addition of transfer factor was below 0.85, so that here, too, one significant improvement in the test result had been achieved. In other groups studied no significant inhibition could be found.

Example 3

An antigen solution can also be made from tumor cells that grown in a fermentation culture in vitro, with a neutral buffer under repeated Refraction of the tissues to be extracted.

Cells of the brain tumor are grown in vitro as follows: Care should be taken to ensure that ei, e sterile working method is used. The cells are preferably placed in a Hank's equilibrium solution washed for about 30 seconds. Avoid stirring. All walls and surfaces of the Flasks, preferably 250 ml glass flasks, are carefully washed. The solution is from the cells cleared by centrifugation in the cold for a period of 10 minutes. The medium becomes poured into a beaker. A small amount of buffered proteinase enzyme solution is added

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and rinsed quickly to avoid degradation of the cells. The preferred method is to use in Add up to two ml of trypsin solution (EDTA) to a flask. The trypsin solution is poured off after Flushing of 10 seconds.

An equal volume of fresh trypsin solution is then added. It is incubated until it can be seen by microscopic observation that the cells are separating from the walls of the chamber split off. This usually takes 5-10 minutes. A suitable growth medium, e.g. B. 50 ml added to a solution of a 7-10% solution of the serum of calf fetuses in 100 ml of F-10 nutrient medium.

25 ml of the fresh medium with the cells are transferred to a new growth chamber (flask) for reproduction convicted. Both chambers are placed in a 35 C incubator for approximately 7 days. Following the procedure of this example to this point, a culture of the artificial tumor cells is created divided into two fresh cultures approximately every 7 days. The procedure can take seven days Repeated at intervals. The number of cells growing in vitro doubles

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accordingly every seven days.

The medium with the cells is transferred to a centrifuge tube and kept in the cold for 10 min Centrifuged 3,000 rpm. The medium is discarded. Those left in the growth chamber Cells are scraped from the chamber walls and placed in the centrifuge tubes with a neutral buffer solution washed into it. The cells are washed twice with a neutral buffer solution, again centrifuged in the cold at 3,000 rpm. The medium is discarded. The washed cells are suspended in 10 ml neutral phosphate buffer until ready for extraction. Then the washed cells are treated with a sonic device in the cold for 20 seconds to remove them to break. However, denaturation of the proteins must be avoided as far as possible. After the sound the cell debris is centrifuged for 30 min at 30,000 rpm. The protruding product will decanted. 10 ml of the buffer solution are used to wash the remaining cell debris. It will sonicated and centrifuged again and the supernatant products are combined. This method is repeated once. The combined

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supernatant product is pre-evaporated, the volume from about 30 ml to about 6 - 7 ml. A corresponding part becomes the total protein analysis removed.

With the help of a liquid column chromatograph and filtration through Millipore filters, the
The preparation is then freed from impurities. Details of this process can be found in DT-OS 26 06 257, Example 2. A polypeptide with a molecular weight of approximately 10,000
to win. An antigen solution is prepared
which contains about a concentration of 100 mg of the antigen per liter.

The experiments according to Examples 1 and 2 are carried out with the artificially obtained antigen. It
it turns out that the artificially obtained antigen produces the same effect as that obtained from body tissue.

Example 4

Use of an organ-specific antigen,

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A The leukocyte suspension is obtained as in Example 1 - A.

B Preparation of an organ-specific antigen solution

As freshly obtained tumor tissue of a Mayiunas carcinoma as possible is taken from the blood, adipose tissue and connective tissue freed and cut into small cubes or strips. All subsequent steps will be carried out at 4 C. The pieces of tissue are placed in a three-fold volume of a 3 molar potassium : Chloride solution crushed into pea-sized pieces. The pH of this coarse suspension is 7.4 set. The tissue is then comminuted with an Ultraturrax® homogenizer (20,000 rpm). The homogenate is left to stand for 24 hours. It is then centrifuged. The one after 60 minutes Centrifugation at 4,000 g is obtained from the supernatant for 2 hours against a 10-fold volume of aqua destilata and then dialyzed against 50 times the volume of 0.9% NaCl solution for a further 24 hours. The dialysate is made by adding 2 molar ammonium sulfate over a period of 1 hour Proteins precipitated and separated by centrifugation at 4,000 g for 20 minutes. After suspension

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the proteins in a 0.9% NaCl solution are dialyzed for one hour against 5 times their volume Aqua distillata and another dialysis against 50 times the volume of 0.1% NaCl. After sterile filtration The antigen extract is filled into small ampoules through a 0.45 milipore micropore filter and stored at -20 ° C. Before use, the protein content of each batch is checked using the biuret method determined after previous TCA precipitation.

C The same test plate as in Example 1 is used.

Three approaches are made for the test:

1. each with 1OO μl leukocyte suspension from patient's blood according to example 1 - A + 8 μΐ n / 100 NaCl solution (for row A, standard value)

2. each with 100 μl leukocyte suspension + 8 gl Antigen suspension according to Example 4 - B,

3. each with 1OO ul leukocyte suspension + 8 μl Antigen suspension according to Example 4 - B + 15 iil transfer factor solution.

The test plate with a hole diameter of 2.5 mm

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8 pi each of the test substance 1-3 incubated according to Example 1 are charged. The results, respectively determined from five values are:

approach line 7th 0 F. (well ) MI 0 1 A. 7th , 70 46 , 5 1, 82 2 C. 6th , 00 38 ,1 0, 63. 3 D. , 10 29 , 3 0,

The result suggests breast cancer, because the MI is well below 0.8 and the result when using the transfer factor is still is reinforced.

Further studies show that the results of patients, as mentioned in Example 1, are change even more significantly. Patients with breast cancer show when using the Method with the help of the transfer factor at over 90% a migration index that is smaller is than 0.80. The migration index is greater than in only 10% of patients with breast cancer 0.85.

Example 5

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The change in mobility of lymphocytes, Foreign macrophages and other indicator cells in a capillary test according to S0BORG and BENDIXEN measured. A 10 μm thick open capillary is used here. The results correspond those of Examples 1 and 2, the same approaches being used in principle.

closing remarks

The test procedure cannot be used for certain clinical pictures. False negative results were obtained with all so-called leukoses. An explanation for this can not be given for a while, since lukemic cells in particular contain very abundant basic proteins. The cellular immunity in these patients is also not so noticeably impaired that this result could be explained by a disturbance of the immune system. It is therefore also noticeable that chronic lymphatic leukemia, like acute lymphatic leukemia, show no reaction, while lymphosarcomas and lymphogranulomatoses behave like other malignancies. Before chemotherapy, however, pointed

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Patients showed a significant inhibition. This inhibition already occurs after the first chemotherapy cycle however canceled and converted to a false positive value in the second cycle. Still surrender wrong results in patients in tumor cachexia. Patients with rapidly progressive growing Tumors or patients in the tumor cachexia sometimes show incorrect results, because here one Störuny of cellular immunity is present. Attempts to answer this question are still ongoing.

False positive results were primarily found with inflammatory and degenerative neurological Diseases such as multiple sclerosis were found. The explanation for this was given by Caspary and Field in a cross reaction between the EF and the sensitization of the lymphocytes with brain tissue seen.

However, the wrong results can easily be eliminated by a preliminary examination, so that the aforementioned Illnesses do not prevent the test from being used.

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Claims (12)

-Al - claims 1.Jin vitro test method for diagnosing malignant Tumors, preferably in humans, in which test the change in mobility of cellular Blood components that come into contact with a migration inhibition factor (MIF) are determined where the MIF is excreted by lymphocytes that are sensitized to tumor tissue, characterized in that as cellular blood components, the lymphocytes to be examined are themselves known to a person Migration inhibition test are subjected, in the course of which the lymphocytes with a to form brought into contact by MIF stimulating antigen and then incubated, and after a fixed period of time the lymphocyte migration is inhibited and fixed. 2. Test method according to claim 1, characterized in that that the migration speed of the lymphocytes in a capillary intermediate layer of 1-20 pm thickness takes place, which is at least partially liquid-filled is. 909824/0532 original inspected - P 2 - 3. Test method according to claim 2, characterized in that that the intermediate layer on the one hand from an inert medium (z. B. glass) and on the other hand from a nutrient-enriched agar layer is limited. 4. Test method according to claim 1, characterized in that that the test with a sample with leukocyte-rich supernatant ("buffycoat") obtained from human blood with a fixed number of leukocytes between 10 and 10 per sample. 5. Test method according to claim 4, characterized in that the sample is an antigen solution according to CASPARY and FIELD or a 3mKCl extract from tumor tissue according to MEIlZER in the ratio (volume sample to antigen solution) from 1:50 to 1: 10 is added. 6. Test method according to claim 1 and 5, characterized in that the sample is an organ-specific Antigen solution is added. 7. Test method according to claim 4, characterized in that that the sample is one grown from in vitro 909824/0532 - A 3 - Antigen solution obtained from tumor cells is added, which contains between 50 and 200 mg of tumor antigen contains per 1,000 ml of a suitable aqueous solvent. 8. Test method according to claim 1 and 4, characterized in that the sample in addition to the antigen solution additional transfer factor solution is added. 9. Test method according to claim 8, characterized in that that the number of leukocytes from which the added amount of transfer factor is obtained increases the number of leukocytes present in the sample, as 1: 1 to 1: 100. 1O. Test method according to one of the preceding claims, characterized in that the incubation time in the migration inhibition test is between 10 and 30 hours at a temperature of 35 to 37.5 ° C with a relative humidity of 90 - 100%. 11. Test plate for performing the method according to claim 1 and 4, characterized by a flat one Θ0982Α / 0532 - A 4 - Carrier plate or tray (1O) made of inert material (e.g. glass) on which a 2-5 mm thick solidified Agar layer is applied, the at least two substantially circular punched holes (H) with a Has a diameter between 0.5 and 5 mm, preferably 2.5 mm. 12. Test plate according to claim 11, characterized in that the punch holes are cut so that the Uniformity of the capillary migration layer also in the area of the transition from the punched hole to the capillary layer given is. 909824/0532