TECHNICAL FIELD

The present invention relates to a test mechanism which is suitable for usage for the detection of the presence of SARS-CoV-2 viruses which lead to Covid-19 disease and for the detection of SARS-CoV-2 antibodies which occur in persons who are vaccinated or who are patient, and which has short diagnosis duration, high reliability and low-cost characteristics, and a test method for obtaining in-vitro quantitative and/or qualitative data.

PRIOR ART

Serologic test methods are test methods used in diagnosis of the presence of various infection diseases by means of antigens and antibodies which exist in the serum or plasma of patients. The object of serology test methods is the determination of whether antigen of microorganisms (microbes) exists in the samples taken from the patient or whether antibodies exist which are generated by the body against some microorganisms (microbes) like virus, bacteria and parasites. Serologic test methods are frequently used since they are reliable test method in diagnosis of pluralities of diseases and the results are obtained in short durations. In accordance with the findings obtained as a result of serologic test, information is obtained about the diagnosis and course of the infection diseases.

Serologic test methods can be realized in various types, and some of the test methods, realized based on antibody-antigen matching, are chemiluminescence, radio immunoassay, ELFA, ELISA, agglutination and precipitation methods.

Antigen/antibody is searched in the patient sample inside a carrier surface/well coated with/comprising an antigen or antibody known essentially in serologic methods. In such tests, the formation/non-formation of antigen antibody joining is displayed as fluorescent radiation or agglutination formation/non-formation like color change/optical density measurement. In order to provide measurability, the antibody or antigen provided on the surface is marked with radioactive, fluorescent or enzymatic substances. Sometimes said tests are also worked on by bringing together an antibody/antigen and the patient serum/plasma inside a tube or well/card-test. Moreover, various latex particles/beads, synthetic particles/beams and erythrocytes are coated with known antibodies or antigens, and these tests are realized. Serologic methods can have disadvantages as follows: the result can vary depending on the person who applies the method, there is a washing step after each reaction, washing can be insufficiently realized, or erroneous results occur since the washing solution cannot be removed from the medium.

For obtaining a test method which is suitable and reliable for big mass screening and which is rapid in diagnosis of Covid-19 disease and which has low-cost, all searchers and scientists execute studies worldwide. It is apparent that the test methods having said characteristics will attract attention in the related technical field.

As a result, an improvement must be made in the related technical field for the test methods which can give rapid and reliable results for the diagnosis of Covid-19 disease and which can be applied with low costs and which are suitable for big mass screening.

BRIEF DESCRIPTION OF THE INVENTION

The present invention relates to an in-vitro test mechanism and a test method for use in diagnosis of Covid-19 diseases, for eliminating the abovementioned disadvantages and for bringing new advantages to the related technical field.

An object of the present invention is to provide an in-vitro test mechanism and method that are reliable and where result can be obtained in short durations and which are for use in diagnosis of Covid-19 disease. Thanks to this, by means of usage of serologic methods, a new test mechanism and method can be provided for the related technical field.

An object of the present invention is to provide a low-cost in-vitro test mechanism and method for use in diagnosis of Covid-19 disease. Thanks to this, together with obtaining low- cost tests, greater number of patients can be reached in much shorter durations.

An object of the present invention is to provide an in-vitro test mechanism and method where SARS CoV-2 antibody and antigen analysis can be made from the samples of the patient and for use in diagnosis of Covid-19 disease. Thanks to this, by means of diagnosis and follow-up of the Covid-19 disease, the data can be obtained which is particular for the persons who have been vaccinated.

The present invention relates to an in-vitro test method having characteristics which are suitable for big mass screening and which are reliable and which can be diagnosed in short durations and which is suitable for use in detection of antibodies which occur against the virus in the patient’s blood or the SARS-CoV-2 viruses which lead to Covid-19 disease, and the subject matter method comprises the following process steps:

• coating erythrocytes, obtained from sheep and/or humans having O Rh ⁺ blood group, and/or polymer based micro-particles with SARS CoV-2 virus antigens, wherein tannic acid, biotin, streptavidin components are used in the coating process,

• taking samples from humans, wherein said sample is serum and/or plasmas taken from human blood or upper or lower respiratory tract sample of humans,

• erythrocytes coated with antigens/antibodies and/or micro-particles and sample taken from humans are added to wells, and the mixture is obtained, wherein said well comprises gel particles which accommodates anti-human IgG antibodies or neutral gels which do not accommodate anti-human IgG,

• incubating the obtained mixture, wherein said incubation processes are realized between temperatures 35-37 ^e C and between durations 5-10 minutes,

• applying centrifuge process for duration between 10-20 minutes at 1000-3000 rpm/minute to the mixture after the incubation processes,

• checking whether red colored (if the used antigen carrier is erythrocyte, it can be red colored and the other particles can have a different color) agglutinated complex compounds are formed or not between antibodies taken from said human samples and the antigens which exist on the surface of erythrocytes obtained from sheep and/or humans having 0 rH (+) positive blood group in the centrifuged mixture, wherein red colored agglutinated compounds or different colored agglutinated compounds in accordance with the used micro or nano-dimensioned particles have big particle sizes in a manner not passing between gels and are positioned at the upper part of the test mechanism.

In a possible embodiment of the present invention, as the antigen provider, there are erythrocytes obtained from sheep and/or from humans, having O Rh ⁺ blood group, coated with SARS CoV-2 antigens, and besides, at least one micro-particle(s) is/are used which is/are coated with SARS CoV-2 antigens selected from the material group of latex, polystyrene.

In a possible embodiment of the present invention, as said gel, it comprises at least one or some or all selected from the material group of crosswise bonded agaroz, sephadex, sephacryl, silica gel, poly-acrylamide, poly-dextran, sitren-divinyl benzene polymers. In a possible embodiment of the present invention, the particle size of the gels is between 10-200 mcm.

The present invention relates to an in-vitro test mechanism having characteristics which are suitable for big mass screening and which are reliable and which can be diagnosed in short durations and which is suitable for use in detection of antibodies which occur against the virus in the patient’s blood or the SARS-CoV-2 viruses which lead to Covid-19 disease, within this context, it comprises the following items in its body:

• sheep erythrocyte(s) and/or human erythrocyte(s) having O Rh ⁺ blood group coated with chemicals like tannic acid, biotin and streptavidin as the antibody provider, and/or other various micro-particle(s),

• serum and/or plasma samples obtained from humans or animals (for instance horses) as the antibody provider which can enter into interaction with said antigens,

• gels having particle sizes between 10-200 mcm and which realize separation processes.

BRIEF DESCRIPTION OF THE FIGURES

In Figure 1 , a representative view of the negative antigen/antibody test result, obtained from persons who do not have suffered from Covid-19 disease and who have not been vaccinated in the subject matter test mechanism, is given.

In Figure 2, a representative view of the weak positive antigen test result in the samples taken from the person who suffers from Covid-19 disease and the weak positive antibody result in the serum of the person who has undergone the disease or who has been vaccinated in the subject matter test mechanism is given.

In Figure 3, a representative view of the positive antigen test result in the samples taken from the person who suffers from Covid-19 disease and the positive antibody result in the serum of the person who has undergone the disease or who has been vaccinated in the subject matter test mechanism is given.

DETAILED DESCRIPTION OF THE INVENTION

In this detailed description, the subject matter relates to a test mechanism which is suitable for usage for the detection of the presence of SARS-CoV-2 viruses which lead to Covid-19 disease and for the detection of SARS-CoV-2 antibodies which occur in persons who have been vaccinated or who are patient, and which has short diagnosis duration, high reliability and low-cost characteristics, and a test method for obtaining in-vitro quantitative and/or qualitative data, and is explained with references to examples without forming any restrictive effect only in order to make the subject more understandable.

The subject matter in-vitro test method is essentially a serologic method, and has an operation principle based on the interaction between the erythrocyte, coated with antigen/antibody, and other similar components and the antigens/antibodies which exist in patient samples.

In the subject matter test mechanism, as the antigen provider, specific polyclonal SARS CoV-2 antibodies, obtained from humans or animals who have undergone the disease or who have been vaccinated by means of SARS CoV-2 virus spike and nucleo-capsitis antigens, are used. Said antigens are coated to sheep erythrocytes and/or human erythrocytes having blood group of O Rh ⁺ or latex particles. The innovative characteristic of the present invention is that the chemicals like tannic acid and/or biotin and streptavidin are used for coating erythrocytes with SARS CoV-2 virus antigens/antibodies.

In the invention, as the antigen/antibody carrier, sheep erythrocytes and/or human erythrocytes having blood group of O Rh ⁺ are used, and besides, latex or similar micro particles can also be used which are coated with the mentioned SARS CoV-2 virus antigens/antibodies. Here, as the mentioned micro-particles, latexes, nano-particles and polymeric products like polystyrene can be used.

In the invention, as the antibody provider, there are samples taken from human or animal (for instance horses) sample. Here, the antibodies to be tested are antibodies which occur against the SARS CoV-2 virus. Here, the mentioned antibodies have IgG and IgM structure. Antibodies can be tested as total IgG+lgM or separately.

In the test mechanism of the invention, a suitable medium is provided for realization of the processes in a stable manner and in short durations, and this medium is a well medium in neutral gel structure which comprises or which does not comprise Anti-Human IgG.

The subject matter test mechanism comprises gels with specific particle sizes in order to be able to test the sample taken from the patient. Here, the gel is selected from the material group of crosswise bonded agaroz, sephadex, sephacryl, silica gel, poly-acrylamide, poly- dextran, sitren-divinyl benzene polymers. The particle size of said gels is at a value between 10-200 mcm. The gel materials used in the present invention are inert materials which do not enter into reaction and related to antigens and antibodies.

The present invention comprises the following process steps for the diagnosis of the presence of SARS CoV-2 virus antigen/antibody, which leads to Covid-19 disease, in the samples taken from the humans:

• coating latex micro-particles or nano-particles or erythrocytes, obtained from sheep and/or humans having O Rh ⁺ blood group, with SARS CoV-2 virus antigens,

• taking samples from humans, wherein said sample is serum and/or plasmas taken from human blood or upper or lower respiratory tract sample of humans,

• erythrocytes coated with antigens/antibodies and the sample taken from humans are added to wells in order to be tested, and the mixture is obtained, wherein said well comprises gel particles which accommodates anti-human IgG antibodies or neutral gels which do not accommodate anti-human IgG,

• incubating the prepared well, wherein said incubation processes are realized at a value between temperatures of 35-37 ^e C and durations between 5-10 minutes,

• applying centrifuge processes to the mixture at values between 1000-3000 rpm/minutes and 10-20 minutes after the incubation processes,

• examining the results afterwards.

As a result of the processes mentioned in the invention, observing a line and/or layers having red color or a different color (if the used antigen/antibody carrier is erythrocyte, it can be red colored and the other particles can have a different color) on the gel particles shows that the test result is positive.

, have sizes in a manner allowing passing between gels, they advance until the lo In the subject matter test mechanism, SARS CoV-2 antigen/antibody which exists on the surface of micro-particles and erythrocytes and the probable antigen or IgG+lgM antibodies in the samples taken from the human enter into interaction and they form agglutinated compounds in red color, or in other colors if other particles are used. Said agglutinated compounds must have a size such that they cannot pass between gels and therefore, they cannot pass between gels, and as shown in Figure 3, they remain at the upper part of the test mechanism as a red colored structure or a different colored structure. If there is no antibody in the samples taken from humans and which can enter into interaction with SARS CoV-2 antigen, said red colored agglutinated compounds do not occur, and afterwards, since the micro-particles and/or erythrocytes, coated with antigen/antibody which cannot enter into interaction partially with antibodies/antigens wer part of the test mechanism as shown in Figure 1. If there is weak positive antigen test result in the samples taken from the patient of Covid-19 disease and if there is weak positive antibody in the serum of the person who has undergone the disease or who has been vaccinated, and if said person is tested by means of the subject matter test mechanism, this means that there is small amount of antibody formation/small amount of antigen in the sample, and therefore, it enters into interaction with small amount of antigen/antibody. Therefore, the view in Figure 2 is obtained which is a view between Figure 1 and Figure 3.

The subject matter test mechanism is a Covid-19 disease diagnosis and follow-up method which is simply applicable and which has high reliability. The test results obtained here are based on qualitative observations, and if preferred, the test results can be easily automized and can be turned into quantitative data thanks to the suitable programs in the computer. The subject matter test mechanism can be easily used in small and big automized systems used in blood banking. These devices realize result evaluation by means of high resolution cameras in an independent manner from human errors. Since these devices can be integrated to such systems, they can be easily provided to be used in big-scaled public screening.

Any kind of test component used in the subject matter test mechanism can be obtained substantially easily and with low-costs. The coating process of erythrocytes and micro particles used in the present invention are substantially easily applicable and low-cost processes. Thanks to this, test mechanisms can be obtained with lower-costs when compared with the present test kits or mechanisms used in the art.

In the subject matter test mechanism, test durations are substantially short and the process which has the longest duration among the process steps is the incubation and centrifuge step and these process steps long for at most 30 minutes. When compared with the present test kits and mechanisms, it is apparent that the subject matter test mechanism gives reliable results in shorter durations.

The protection scope of the present invention is set forth in the annexed claims and cannot be restricted to the illustrative disclosures given above, under the detailed description. It is because a person skilled in the relevant art can obviously produce similar embodiments under the light of the foregoing disclosures, without departing from the main principles of the present invention.