DD235337A1 - METHOD AND DEVICE FOR IMPLEMENTING IMMUNOLOGICAL PROVISIONS - Google Patents

Abstract

The invention relates to a method and a carrier for carrying out immunofluorescence microscopic investigations for the detection of cellular antigens. Field of application is immunology. The aim is to develop an easily manageable method for carrying out immunofluorescence optical examinations, which makes it possible to manage with a low cell number. Another object of the invention is to provide a reusable carrier for carrying out the method. The task is solved by centrifuging the samples, the cells are attached so firmly to the bottom of the carrier, that they do not dissolve in later washing processes. The advantageous dimensioning of the carrier allows on the one hand a fast and quantitative leaching of unbound antibodies and on the other hand an uncomplicated evaluation.

Description

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The dimensioning of the wells allows a fast and quantitative leaching of unbound antibodies without a detectable cell detachment takes place. The evaluation with a fluorescence microscope is then carried out so that the carrier is inversely with the supports on the microscope table, which prevents leakage of the samples. The liquid surfaces of the samples in the wells form a plane with the carrier surface. These planar liquid surfaces remain stable due to the favorable dimensioning of the wells even when turning the plate, whereby a uniform illumination of the visual field is maintained and unwanted diffraction of light is avoided. The individual reaction fields are easy to find because the plastic plate is opaque.

After completion of the experiment, the carrier is purified by placing in an enzyme-containing cleaning solution, then with water and distilled water. is then rinsed and is then suitable for a new test.

A preferred use of the method and the associated carrier is the testing of hybridoma supernatants for the production of monoclonal antibodies. Due to the similar arrangement of the wells in cell culture plates and the carrier according to the invention a rapid transfer of the supernatants in the corresponding wells of the carrier is possible. Thus, the complex labeling of the individual samples is superfluous, since the position of each sample is identical on both plates. Thus, on the one hand the time required is reduced and on the other hand, comparable conditions for the testing of all samples are guaranteed.

embodiment

Per well 5000 to 20000 rat mast cells, contained in 50 .mu.l isotonic phosphate buffer (PBS) pipetted. The slides are centrifuged at 150 g for 15 min. Subsequently, 50 .mu.l clone supernatants are transferred from the cell culture plate to the test plate per well and incubated for 60 min at 37 ⁰ C or 4 ⁰ C in a humid chamber. The plates are gently rinsed in PBS (immersion of the plates and horizontal movement in the solution). By sweeping over the rows of wells with a Pasteur pipette connected to a water-jet vacuum, the supernatant is aspirated off with 25-30 μΙ. Thereafter, 20 μΙ fluorescein isothiocyanate-labeled goat anti-mouse globulin is pipetted. The carriers are further incubated for 30 min at 37 ⁰ C and 4 ° C in a humid chamber. The quantitative elimination of unbound antibody is carried out by rinsing the plates in PBS (when changed 3 times) in the manner described above.

Subsequently, the surface of the carrier is sucked liquid-free by means of a Pasteur pipette via a water jet vacuum, without removing the PBS supernatant from the wells. The liquid surface of the wells is then filled up with the aid of a 200 μΙ microliter syringe so that it forms a plane with the surface of the plate. The surface of the liquid remains stable and planar when the plate is inverted, thereby maintaining a uniform illumination of the visual field. The evaluation is carried out by a fluorescence microscope, with the glass surface of the plate pointing upwards.

Claims (5)

Invention claim: 1. A method for carrying out immunological determinations, characterized in that cells are pipetted into the wells of a carrier, attached by centrifugation to the bottom of the carrier and subsequently evaluated by reversing the carrier. 2. The method according to item 1, characterized in that the sample surface with the support surface forms a plane and this planar surface is maintained even when evaluating the samples, which is done by reversing the carrier. 3. The method according to the items 1 and 2, characterized in that per sample 5,000 to 20,000 cells are used. 4. support for carrying out the method according to points 1 to 3, characterized in that it consists of a 1 mm thick glass plate, on which an opaque, 5 mm thick plastic plate with 12 times 8 holes, each having a diameter of 5 mm, and at the longitudinal and lateral sides, as well as in the middle of the pads are attached, which prevent a minimum distance of the support from the microscope table and thus a leakage of the samples from the wells during the sample evaluation when reversing the plate. 5. carrier according to item 4, characterized in that it is reusable. For this 1 page drawing Field of application of the invention The invention relates to a method and a carrier for carrying out immunofluorescence microscopic investigations for the detection of cellular antigens. Field of application is immunology. Characteristic of the known technical solutions For the detection of antibodies to cellular antigens, the indirect immunofluorescence test is generally used. The cells are incubated with the serum to be tested. If the serum contains the antibodies to be tested, these are bound to the antigen and made visible in a second step with a fluorescently labeled antiserum against the antibodies to be detected. The cells thus labeled are then placed on a slide and evaluated for their fluorescence. It is also possible to fix the cells on the same slide and perform the entire test on the support (DD-PS 115 388). Various proposals have already been made for the rationalization and standardization of the immunofluorescence technique, but a uniform procedure has not yet been established. Thus, DD-PS 115 388 proposes to use a support made of glass or plastic with 1 to 8 wells, wherein the reactive immunological material is fixed by drying and subsequent lyophilization. This is u. a. disadvantageous for the screening of clone supernatants in the context of, hybridoma technique, since only a small number of samples can be evaluated simultaneously. Further disadvantages are the high cell number required for the incubation and the relatively large expenditure for the fixation of the antigen. Reuse of the carrier is also not possible. Also known is a glass substrate for 12 samples with glued glass rings for the subdivision of the reaction sites, wherein a heat fixation or acetone fixation of the material to be examined is carried out (DD-PS 145 133). Again, the relatively small number of samples to be evaluated simultaneously is disadvantageous. This carrier is also in its ready for use form also not reusable. Object of the invention The aim of the invention is to develop an easily manageable method for carrying out immunofluorescence optical examinations, which makes it possible to manage with a low cell number in order to reduce the costs for the cell production (fewer experimental animals). In addition, the time and material-technical effort that is necessary for the attachment of the cells to the test plate to be lowered. A further object of the invention is to provide a reusable carrier for carrying out the method which makes it possible to evaluate a substantially larger number of samples simultaneously. Explanation of the essence of the invention The invention has for its object to perform immunofluorescence optical examinations so that a small cell count is sufficient and the procedure so elaborate steps as the drying and lyophilization of the samples to attach the cells to the test plate, superfluous and still allows a good readability of the samples , Another object is to provide a reusable carrier for carrying out the method which makes it possible to evaluate a larger number of samples simultaneously. According to the invention the object is achieved in that 5000 to 20,000 cells are pipetted into the wells of the carrier and centrifuged at 150 g for 15 minutes. Surprisingly, it has been found that the centrifugal force causes a sufficiently firm adhesion of the cells to the bottom of the carrier, so that the cells are not rinsed off in subsequent washing processes. The time-consuming drying and subsequent lyophilization is thus completely eliminated. The carrier used in the invention consists of a 1 mm thick glass plate (120 mm by 85 mm), to which an opaque, 5 mm thick plastic plate with 12 times 8 holes, each having a diameter of 5 mm, was adhered. The carrier has two supports each on the longitudinal and transverse sides of the surface, as well as two more in the middle of the plate (see Fig. 1). Per carrier (96 samples) about 1.5 million cells are needed. This is much less than with conventional methods, which is associated with a significant reduction in the cost of cell production (fewer animals). After attachment of the cells by centrifugation, they are incubated with the antiserum to be determined and the fluorescein isothiocyanate-labeled antibody.