PROCESS FOR THE DIAGNOSIS OF INFECTIONS CAUSED BY HTLV III VIRUS

Abstract: Process for the diagnosis of viral infections (caused in particular by the HTLV III virus and others) capable of detecting the presence in blood of IgM antibodies (in addition to other classes) that characterize the initial stages of infections in general, and those leading to AIDS in particular. The antibodies, after being separated from serum by the respective antigens, are hot eluted and then fixed again by means of antigens. Their presence can be then evidenced by adding the conjugate.

Description: Process for the diagnosis of infections caused by HTLV III virus. The present invention provides an improved process for the laboratory diagnosis of infections caused by the HTLV III virus in particular, process that has a great¬ er specificity compared to the known methods, and that allows to disclose the presence of antibodies of the IgM class, which are typical of the first stages of the in ection. The serological diagnosis methods presently used are based on the search for specific antibodies for a certain kind of infection, through the examination of blood samples. It is well known that antibodies are immunoglobulins that is to say proteins so structured as to perfectly fit to the antigens against which they act. Each kind of antibodies (that is specific for a determi¬ nate antigen) may consist of immunoglobulins that, though having the same specificity, have different im- - 2 - munochemical properties, on the basis of which they are divided into five classes, distinguished by the abbre¬ viations: IgG, IgA, IgD, IgE, IgM. There are thus antibodies of different classes that have the same specificity; accordingly, antibodies of the IgM and IgG classes may coexist, acting both against the HTLV III virus. t is also known that each class of antibodies, if injected into animals, causes the formation of anti- antibodies to the same class. For instance, if human IgG immunoglobulins are injected into an animal, they will produce anti-antibodies act¬ ing on the human IgG, no matter which specificity they have, and only against them. As hereinabove said, the serological diagnosis of the infections may consist too in the search for the rele¬ vant antibodies present in the subject's blood. One of the presently used methods provides preparing a compound containing known antigens, and fixing it to a solid medium, e.g. a small polystyrene ball. Then a blood sample taken from the subject under examin¬ ation is reacted with the small ball coated with anti¬ gens. During this stage, if the specific antibodies for a determinate infection are present in blood, they are caught by the antigens present on the small ball and fix onto them. At this point the small ball is reacted with the conjugate anti-antibody (that is with a reactant consi¬ sting either of anti-antibodies - or anti-immunoglobu- lins - usually obtained from immunized goat, and conju¬ 0 gated with special enzymes such as peroxidase, phospha- tase etc.); the subsequent addition of the relevant medium brings about a colouring that shows the presence of antibodies, and therefore the infection state. * 5 This method of serological diagnosis, based on the determination of the specific antibody by catching it by means of its own antigen and revealing the presence thereof by means of an anti-antibody has, in comparison with other methods, a very good sensitivity, but an 0 insufficient specificity. More specifically, the greatest non specificity occurs in the search for the IgM antibodies which, in the case of the HTLV III virus, characterize the first stages of ^ the infection. At the present time, in the case of the AIDS virus, the HTLV III, the diagnosis methods base on the search for the relevant IgG class antibody. The methodology of one of the nowadays most widespread techniques is set out hereunder. 0 In such a case, the purified antigens are fixed onto the surface of a polystyrene ball having 6 mm diameter, introduced in a steam heated vessel. The serum under examination, after dilution up to 400 times in order to avoid non specific reactions, is introduced in the vessel where it reacts with the ball coat. The temperature of the water bath is kept, at this stage, at 40°. At the end of the incubation, the small balls are washed by means of suction- and force pump apparatus of known type, after which the specific IgG class antibody is added, after having been previously conjugated with peroxidase . At the end of the second incubation another washing is effected, and the medium is added, upon which acts - if present - the anti-antibody conjugate. The presence of the latter - if any - is indicated by a colouring that evidences the infective state. Although the serum under examination is very diluted, by this method false positivities may result all the same, partly due to the non specific absorption of some IgG immunoglobulins. In addition, by this kind of diagnosis only the pre¬ sence of this class of immunoglobulins can be revealed, as non specific positivities are often likely to be obtained by the IgM immunoglobulins. Since IgM immunoglobulins only are formed in the start¬ ing period (of some months' duration) of HTLV III virus infections, the diagnostic methods known at present do not allow to detect the presence of such infection during this first stage. Therefore, the need for a diagnostic process is strong¬ ly felt, that allows to determine with certainty this kind of infection as early as in the first stage, both to prevent contagion (as in the case of blood donors), and to take up treatment more timely. To this end, the present invention provides an improved process for the laboratory diagnosis of HTLV III virus infections, comprising the following steps: a) separation of the antibodies attacking HTLV III par¬ ticularly, by combining them with the relevant anti¬ gens in the solid phase; b) subsequent hot elution of same; c ) second incubation of the thus purified antibodies on a second small ball coated with antigens of the same type, or partly of the same type; d) third incubation with anti-antibodies of the chosen class, and subsequent detection. The present invention will be now described in detail by means of the following EXAMPLE 200 _uJ/ (or more) of the serum under exam are put into a vessel containing also the ball coated on the surface with the purified antigens of HTLV III. Contrary to what happens by the known methods, in this 0 case the serum is undiluted, and the incubation takes place first in a stirrer at 180 r.p.m. for 45' and then in water bath for 15' , at a temperature of 40°. At the end of the incubation, the ball is washed with *^ the usual washings, and there are added 250 _u of diluent consisting of phosphate buffer with a certain amount of goat serum. The temperature of the water bath is brought to a value 0 between 50° and 58° for 30*. The optimum temperature resulted to be 56° for 30'. At such a temperature, the specific immunoglobulins of the various classes separa¬ te from their respective antigens, and there is obtain- ed in the buffer liquid an eluate of specific anti- bodies of the various classes, with few non specific antibodies. At the end of the elution period, the liquid is quickly sucked in, so as to avoid a decrease in temperature inside the vessel, in which case the antibodies would be again absorbed by the antigens present on the ball. The liquid so collected with the purified antibodies is then put into a second vessel containing a second ball, similar to the first one, coated with the same antigens. A third incubation is then started, always at 40°, one hour long, in the course of which only the immunoglobu¬ lins specifically active against HTLV III fix on the ball. At the end of this step, the balls are washed and 200 ;ul of conjugate are introduced into the vessel. Then a further incubation at 40° takes place, for 2 h 10' , after which the ball is washed and put into a test-tube. Then 300 tl of 0PD are added, and the whole is again incubated for 30' at room temperature. One ml of IN sulphuric acid is then added. By determining the optical density of the thus obtained sample it is possible to obtain the desired result. By this process the presence of antibodies of the classes IgG and IgM may be detected, which makes it possible to diagnose with certainty the HTLV III virus infection at the very beginning of the disease. The detection system consisting here of peroxidase may be based on other enzymes such as phosphatase or A galactosidase or the like; alternatively, the anti-anti¬ body may be conjugated with a radioisotope employed as detector. It is pointed out further that the method consisting in eluting the antibodies by heat at 56° is already known in the field of immunohematology to detach from the erythrocytes the antibodies by which they are attacked, antibodies that are then purified in the eluate to assess the specificity and to determine against which hematic system or group they act. In the case of the invention, instead, the process is applied reversely, as the antibodies under examination, caught by known antigens, are eluted with a view to evidencing the same with the maximum specificity, after making them react again with other purified antigens, of the same kind as the previous ones. A characteristic feature of the invention process is therefore that of bringing about the elution of the antigens of the antibodies caught by means of the same relevant antigens, and the subsequent fixation of the thus purified antibodies to a second layer of antigens, before the addition of the respective conjugate. An expert in the art may provide, if necessary, modi¬ fications and variations that should all fall, however, within the ambit of the present invention.

Claims: CLAIMS 1. A process for the diagnosis of the HTLV III virus infections, characterized by providing the following steps: a) selecting the serum antibodies by catching them by means of antigens, by incubation; b) separating the thus purified antibodies by hot elu¬ tion; c) second incubation with antigens of the same kind as the previous ones; d) third incubation with anti-antibodies of the chosen class, and subsequent detection by addition of the respective conjugate. 2. A process according to claim 1, characterized in that the first incubation takes place partly by stirrer and partly by heating. 3. A process according to claim 1, characterized by providing the separation of the purified antibodies from the respective antigens, by elution at a temperatu¬ re ranging from 50° to 58°, and the subsequent fixation to a second layer of antigens, analogous to the pre- vious ones, by incubation. 4. A process for the diagnosis of HTLV III infections, characterized by providing the following steps: a) separating the specific antibodies from a not neces- sary diluted serum by incubation; b) adding the diluent and eluting further at a tempera¬ ture of about 56°; c) separating the eluate keeping it at the same tempera¬ ture; d) selecting the antibodies further by- means of anti¬ gens analogous to the previous ones, by incubation; e) adding the conjugated (or marked with radioisotope) antibody, and incubating further; f) adding substances suitable to produce a colouring in the presence of the antibodies, or _ζ ~ " counter reading if the employed antibody is marked with radioisotope. 5. A process according to claim 4, wherein the elution takes place at 56°. 6. A process according to claim 4, characterized in that the first incubation takes place partly by stirrer and partly by heating. 7. A process for the diagnosis of viral infection syndromes according to one or more of the previous claims .

Inventors: BEDARIDA GUGLIELMO, BEDARIDA, GUGLIELMO
Applicants: TECHNOGENETICS SPA, TECHNOGENETICS S.P.A.
Application year: 1986
Main classification: G01N33
IPC codes: C12Q1, G01N33