KR20170015534A - Method and apparatus for diagnosing a nasopharyngeal carcinoma - Google Patents

Abstract

The present invention relates to a method and a diagnostic apparatus for diagnosing non-human cancers in a patient in which a substrate having two or more antigens immobilized thereon is cultured with a body fluid derived from the patient. In this case, the antigen is selected so that it is at least in the patient ' s body fluids in a particular area and is each bound to an antibody specific for the occurrence of an Epstein-Barr virus infection, to form an antigen-antibody conjugate. The formation of an antigen-antibody conjugate is seen with the aid of one or more markers on the substrate, which appear after the substrate has been cultured into a positive patient sample. After incubation, the surface of the substrate, in particular the area in which one or more visible markers appear during the formation of the antigen-antibody conjugate, is recorded using an optical device and this record is supplied to the evaluator. The evaluating unit generates the proposal diagnosis based on at least one characteristic of the recorded markers. The above technical solution is based on the assumption that a cell membrane based substrate, in particular a blot strip or blot piece, is used, the value of the color contrast between the markers recorded in the form of bands in each case in the form of bands and the surface of the substrate surrounding the markers is determined, It is distinguished in that a proposal diagnosis is generated based on mathematical calculation rules including contrast.

Description

METHOD AND APPARATUS FOR DIAGNOSING A NASOPHARYNGEAL CARCINOMA BACKGROUND OF THE INVENTION [0001]

The present invention relates to a method and a diagnostic apparatus for diagnosing nasopharyngeal carcinoma (NPC) in which a substrate having two or more antigens immobilized thereon is cultured in a body fluid from a patient. Where the antigens are located in body fluids from the patient at least in certain areas and are bound to an antibody specific for the occurrence of an Epstein-Barr virus infection to form an antigen-antibody complex . The formation of the antigen-antibody conjugate is visible on the substrate with the aid of marking, which is recorded by optical means. This record is ultimately sent to an evaluation unit where a proposed diagnosis is generated based on one or more characteristics of the recorded marking.

Ninh, which is often abbreviated as NPC, is cancer of the nasopharynx. Nasopharyngeal cancer belongs to the group of head and neck tumors. Epstein - Barr virus, a variety of environmental factors, diet and genetic characteristics of specific patients are the main causes of endometriosis. Although the disease is widespread worldwide, it often occurs in certain areas of southern China. Co-factors include not only genetic temperament, but also environmental influences such as eating habits.

Because of the correlation between EBV infection and the development of pharyngeal cancer, in most cases the EBV analysis system is used to diagnose the corresponding tumor disease. In this regard, a variety of assay systems are known for diagnosing EBV infection by direct detection of antigens, or otherwise serologic methods. Due to the considerable technical effort and the large amount of time spent in performing the direct detection, serology is chosen as a diagnostic method, which additionally allows for differentiation at various stages of EBV infection. Here, the complete standard is to perform corresponding serological detection through detection of specific antibodies using immunofluorescence, ELISA or immunoblot methods. Suitable assay systems include, for example, EBV-Profil 2 (DN 2790-1601-B), as a microtiter ELISA under the trade name Anti-EBV-EA (EI 2795-9601 A, G or M) in EUROIMMUN Medizinische Labordiagnostika, 2G or M), or as a Western blot with the product name Anti-EBV (DY 2790-1501 G or M).

Antibody response after EBV infection varies greatly due to the complexity of the virus and various infectious stages. For example, antibodies, particularly those with antibody class IgM, appear immediately after infection, after the acute infection has relieved, the antibodies are directed against EA (Early Antigen) and VCA (Virus Capsid Antigen) There are only those that do not exist or have very low activity. However, the anti-VCA IgG antibody formed almost at the same time or with a slight delay lasts for a lifetime. First, it is known that the highly potent reversal of VCA IgG antibody is characteristic of nasopharyngeal carcinoma. Second, the detection of anti - VCA IgG antibody provides information support in the diagnosis of NPC. Likewise, elevated anti-EA levels are characteristic of patients with nasopharyngeal cancer.

The ELISA assay systems used in many cases have attracted attention in that in particular the antigens used in VCA and EA are present as a mixture and therefore only the presence of one or more antibodies from the antibody group can be diagnosed when the patient sample is tested . On the other hand, the blot system allows the individual antigens to separate from each other on the cell membrane, thereby separating all antigen bands from each other, which ultimately provides an advantage that allows a very detailed evaluation of the band pattern for diagnosis. In this regard, it is known to use optical means, more specifically a scanner or a camera, to evaluate the band pattern of the blot system, and to evaluate the recorded image with the aid of appropriate laboratory software.

A corresponding system for automatic evaluation of cell membrane based assay systems is, for example, supplied and distributed by EUROIMMUN Medizinische Labordiagnostika under the product name EUROBlotCamera or EUROBlotScanner. Correspondingly recorded images can be managed and documented electronically, with the help of appropriate software supplied under the product name EUROLINESCAN, and can be obtained electronically.

Also, Patent Document 1 discloses an apparatus and method for evaluating a blot strip. The above-mentioned apparatus has attracted attention in that an image of the cultured blot strip is digitized with the help of a scanner and then transmitted to an electronic evaluation unit. The blot strip is ultimately evaluated by considering the color or brightness of the blot on this blot strip. To do this, it is desirable to be able to obtain discrete dyeing of the blots compared to the background coloring of the blot strips, by placing individual blot strips on the scanner.

In summary, therefore, it can be said that there is a different assay system available for diagnosing EBV infection. However, there is no known analysis system that can directly provide information to the doctor, more specifically, suggestion diagnosis, to the question of the extent of the risk of nasopharyngeal cancer in the patient. This is particularly because a definite diagnosis is possible only when the presence of a specific antibody in a patient sample, more specifically, its combined occurrence, and preferably even the antibody titer, can be reliably determined.

Patent Document 1: German Patent DE 100 61 352 A1

Therefore, in view of the known apparatus and methods for diagnosing EBV infection, it is an object of the present invention to suggest an assay system that can be determined with great confidence in the presence of non-human cancers in a patient. Here, the proposed analysis system is constructed based on a known method and realized relatively simply and surely. At the same time, it is very cost-effective, as well as enables the diagnosis of sensitivity and specificity of the analysis.

The above-mentioned object is achieved by the method according to claim 1 and the apparatus according to claim 8. Advantages of embodiments of the present invention are the subject of the dependent claims and are described in detail in the following description with reference to some drawings.

The present invention is based on a method for diagnosing non-human cancers in a patient in which a substrate having two or more antigens immobilized thereon is cultured in a body fluid from a patient.

Wherein the antigens are selected so that they are located at least in certain regions in body fluid from the patient and are each bound to an antibody specific for the occurrence of an Epstein-Barr virus infection to form an antigen-antibody conjugate. The formation of each antigen-antibody conjugate is visible on the substrate, with the help of one or more markings that appear after incubating the substrate with a positive patient sample. After performing the incubation, the surface of the substrate, more specifically, the area in which at least one marking is visible as it appears in the formation of the antigen-antibody conjugate, is recorded by optical means, which is ultimately transferred to the evaluation unit. Based on the one or more characteristics of the recorded markings, the evaluator generates a suggestion diagnosis. According to the present invention, the value of the color contrast between the recorded marking, which is realized in the form of a band, and the surface of the substrate surrounding this marking, is determined in each case, using a cell membrane based substrate, more specifically a blot strip or a blot piece, And the proposed diagnosis is generated based on a mathematical operation rule including this color contrast.

Therefore, the basic idea behind the present invention is to determine the contrast of two or more markings appearing on the cultured substrate, i.e. the marking in the form of bands and the difference in luminance between the substrate surface areas surrounding the band, , And may provide information about the risk of having non-human cancers for a particular patient as a result of an appropriate assessment of the determined contrast value. For evaluation and generation of the proposed diagnosis, with the help of the statistical calculation model, the contrast value of the band appearing on the substrate upon the occurrence of antigen-antibody binding is understood and evaluated. A statistical calculation model to be used is preferably a logistic regression model, more specifically, a binomial logistic or a polynomial logistic regression model, and the contrast values recorded and quantified for individual bands are inserted as parameters into the calculation model. Based on the results provided by the computation model using the contrast value, a proposal diagnosis is generated in an evaluation unit, which ultimately determines whether the tested patient sample is indicative of whether the patient has nasopharyngeal cancer To the doctor.

To obtain a quantified value for the contrast of the marking, it is desirable to examine each recorded band, analyze it according to gray shades, and specify the corresponding value for the contrast. Here, in a particular embodiment, the value 0 (zero) is assigned when the minimum contrast value of the band, i.e. the band, is not prominent in terms of color for the substrate surface surrounding it, and the value 255 (255) The maximum value, i.e., the maximum contrast. Therefore, it is preferable that the contrast of the recorded marking is graded within the range of 0? L? 255.

Preferably, a blot strip and / or a blot strip having two or more antigens immobilized thereon are suitable for evaluation performed in accordance with the present invention. It is particularly advantageous to use five or more antigens immobilized on a Bull substrate, preferably the antigens VCA GP 125, VCA P 19, EBNA-1, P 22 and EA-D.

Certain assays are preferably accomplished by providing a blot strip and / or a blot strip for use in each case with the antigen, whereby the patient sample is tested for the presence of two or more different antibody grade antibodies. In a highly unusual development of the present invention, the patient samples provided for testing are tested for the presence of antibodies of antibody grade IgA, IgG and IgM using appropriate enzyme conjugates.

The method practiced in accordance with the present invention is substantially based on the fact that the proposed diagnosis is established with the help of a mathematical operation model, more specifically a binomial logistic regression model. Such a model forms the ratio from the convergence probability and the converse probability with the help of the appropriate exponential function. In the particular development of the present invention, the contrast value of the individual bands is used as a parameter of the binomial logistic regression model and forms at least some of the exponent of the exponential function used. Thus, the ultimately performed operation establishes the probability of being able to judge the presence of non-cancerous cancer in the test patient. Here, the tested patient sample is in each case assigned to one of two groups, a positive patient sample or a group of negative patient samples. To ensure proper distribution of the patient sample, a suitable probability value is defined as a limit.

The appropriate contrast value of the band provides an optical recording section for recording the color contrast of the cultivating blot pieces (EUROAssay) with respect to their surroundings so that the calculation model used in the evaluation section can be used as a parameter. Such a recording unit is a camera or a scanner. When the corresponding substrate is cultured in a positive patient sample, this leads to the formation of a so-called antigen-antibody conjugate, thereby forming a black band as a marking. The intensity of the band staining depends on the antibody titer of the patient sample, and thus the contrast between the band and the substrate area surrounding the band may vary. With the aid of the optical recording section, the band of the incubated substrate is recorded, and the color difference or contrast for the surroundings is determined.

An image recorded with the aid of a camera or a scanner is compared with a gray level image having a staining value of 0 to 255 in the evaluation part in relation to its staining. Therefore, based on the determined color intensity of the band or the contrast between the band and the substrate area surrounding the band, the proposed diagnosis is ultimately created. To this end, a determined contrast value is inserted into a mathematical operation model, more specifically a logistic regression model, and based on the determined result a determination is made whether the patient sample is a positive or a negative sample.

The method described above and the apparatus used for the method are particularly notable because it means that the calibration of the automatic recording means only takes place before the start of the measurement of the instrument and thereafter needs to take place at relatively large intervals over time. On the other hand, no calibration is required at the beginning of each measurement.

Without limiting the general inventive concept, the present invention will be described in more detail with reference to the drawings on the basis of an exemplary embodiment.

Figure 1 shows EUROLine EBV-Profil 2 cultivated from EUROIMMUN medizinische Labordiagnostika.
Figure 2 shows the cultured Anti-EBV Western blot from EUROIMMUN medizinische Labordiagnostika.

To date, the EUROLine assay set from EUROIMMUN shown in Fig. 1 has shown that for five EBV antigens VCA GP 125, VCA P 19, EBNA-1, P 22 and EA-D in serum or plasma, It was first used for qualitative in vitro determination of globulin class IgG. Except for the antigen VCA GP 125 negative in affinity chromatographic purification form, the antigens are recombinant antigens. By using the method according to the present invention, with the help of the above-described analysis, more specifically, it is also possible to provide quantitative information on the probability that nasopharyngeal cancer is present in the test patient.

The assay strip coated with the antigen described above is incubated with the serum of the patient diluted in the first incubation step. In the case of positive samples, specific antibodies are bound to each antigen. In the second step, the assay strip is incubated with an enzyme conjugate, an enzyme labeled anti-human IgG, IgM or IgA conjugate that catalyzes the subsequent color reaction. This makes it possible to detect antibodies of the immunoglobulin class IgG, IgM or IgA in the patient sample, depending on the enzyme conjugate to be used.

If the result is an antigen-antibody conjugate, each band appears as a marking at the intended location of the assay strip.

Where IgG and IgM antibodies are bound to VCA while no IgG antibody is bound to EBNA-1, a primary or new EBV infection can be estimated. If IgG antibodies are bound to EBNA-1 and VCA but no IgM antibody binding to VCA, elapsed EBV infection can be estimated. On the other hand, if there is a binding of IgG antibody to P22 and VCA but the IgG antibody is not bound to EBNA-1, it is possible to estimate an EBV infection in which the EBNA-1 antibody is defective.

In each case, the culture is performed by filling the culture with 1.5 ml of the diluted universal buffer prepared for use according to the number of patient samples to be tested. Use tweezers to remove the required number of analytical strips from the packaging and place each directly into the buffer filled with buffer. Next, a first incubation is carried out on the rocker to culture the assay strips at room temperature for 15 minutes. Thereafter, the liquid is completely removed from the bath.

Next, 1.5 ml of the diluted patient sample is filled into each incubation tank filled with the assay strips and incubated on the rocker for 30 minutes at room temperature (+ 18 ° C to + 25 ° C). Thereafter, the liquid is completely removed from each bath and, in each case, cleaned three times for 5 minutes on a rocker with 1.5 ml of a universal buffer prepared for dilution for use. Next, in each case, a 1.5 ml enzyme conjugate prepared for dilution for use, in this case an alkaline phosphatase-labeled anti-human IgG or IgA, is pipetted into the culture tank and incubated on the rocker for 30 minutes at room temperature Respectively. After orienting the conjugate, the liquid is completely removed from the bath again and, in each case, washed three times for 5 minutes on a rocker with 1.5 ml of the universal buffer prepared for dilution for use. For subsequent substrate incubation, in each case, 1.5 ml of the substrate solution is pipetted into the incubator and incubated on the rocker for 10 minutes at room temperature. Then, the liquid is completely removed from each bath and each analytical strip is rinsed with distilled or ionized water for three minutes for one minute.

For evaluation, an analytical strip is mounted on the evaluation record, and after air drying, the band pattern of the analytical strip is recorded with the aid of a scanner or camera. The recorded image is transferred to the evaluation unit in an electronic form. Within the evaluation section, the contrast of the individual bands to the surface of the analyte strip in the vicinity of the band is determined. The contrast or color intensity of the recorded band is determined by the evaluator with the help of gray gradation grading and classification accordingly. To this end, a gray scale of 0 to 255 is provided, whereby a corresponding contrast value is assigned to each band. In this way, quantification of the intensity of the individual bands in the analytical evaluation occurs first.

Therefore, the contrast values of the obtained various bands are inserted as parameters into the logistic regression model, and ultimately the probability values are determined with the help of the model. If the probability value is above a predetermined limit, the test patient sample is classified as a positive sample and a corresponding proposal diagnosis is generated. In this case, it is expected that the patient who has taken the sample has nasopharyngeal cancer. On the other hand, if a probability value below a predetermined limit is determined, it can be estimated that there is no tumor corresponding to the test patient.

In principle, it may be considered to use one of the binomial or polynomial logistic regression models and to insert the recorded contrast value of the individual bands into the corresponding model as a parameter. Depending on the computation model used in each case, various numbers of contrast values of various antigen bands are used. In addition, consider whether antibodies of the antibody class IgG, IgM or IgA can be detected in the patient's serum, or whether the contrast values of the individual bands (e.g. IgG-P22 and IgA-EBNA) are inserted into the regression model as parameters. A particularly advantageous computational model when using the method according to the present invention is a binomial logistic regression model with various inclusions (all parameters) and exclusion (retractions) according to Wald, Which is sent back in succession) is also a multiple logistic regression model. In order to determine the probability of occurrence of non-human cancer, various contrast values of two or more, and generally more, recorded bands are inserted as parameters into the corresponding regression model. Here, therefore, each recording band showing a color difference to the periphery represents the formation of antigen and antibody conjugates present in the patient's serum. By analyzing the patient sample equally for the presence of antibodies of different antibody classes, i. E. Grades IgA, IgG and IgM, highly accurate prognosis diagnosis can be generated.

With the help of a logistic regression model that is stored in an evaluation unit and forms a ratio between the probability of occurrence and the opposite probability, the test patient sample is divided into two groups: a positive sample group and a negative sample group. Based on this distinction, the evaluator generates a prognosis diagnostic that ultimately provides information to the patient who tested the serum whether there is a risk of non-human cancer. Considering the color contrast or color intensity of individual bands, the evaluation of the band patterns revealed on the cultured analysis strips can be carried out using a relatively simple method of producing a very accurate diagnosis of the presence of nasopharyngeal cancer using known statistical methods to provide.

Figure 2 shows a Western blot assay strip for the primary purpose of qualitative in vivo determination of human antibodies, mainly immunoglobulin grade IgG, against EBV antigen from serum or plasma. By using the method according to the present invention, with the aid of the above-described analysis, more specifically, quantitative information on the probability of having non-human cancer in a test patient can be provided.

The blot strips are coated with an electrophoretic separation antigen extract of Epstein-Barr virus. As already described in connection with FIG. 1, first, the blot strips are incubated with the diluted patient sample. In the case of positive samples, the specific antibody binds to the corresponding antigen. To mark these antibodies, in a second step, cultures are performed with a correspondingly conjugated enzyme conjugate, enzyme-labeled anti-human IgG IgA or IgM, which catalyzes a subsequent color reaction.

The western blot strip shown in Fig. 2 is cultured through blocking, sample culture, washing, conjugation culture, washing, substrate culture and quiescence. The band pattern formed in incubation of the blot strip with the patient sample, which in each case is at least partially positive, is again automatically evaluated with the aid of an optical recording medium, more particularly a scanner or camera and evaluation unit.

According to the present invention, the color contrast or color intensity of individual bands, that is, the luminance difference between the band and the background, is again confirmed in the evaluation section by gray gradation gradation from 0 to 255 values occurring in the evaluation section. The contrast value of the individual bands is considered as a parameter of the binomial logistic regression model stored in the evaluation unit, together with the contrast value forming at least some of the exponential exponents. Based on the color contrast of the determined individual bands, therefore, the probability value, which is a value considering the generation of the proposed diagnosis, is determined with the help of the regression model. The proposal diagnosis is based on a comparison between a determined probability value and a predetermined limit so that individual patient samples are divided into two groups, one group having positive patient samples and one group having negative patient samples do. For a patient whose patient sample is identified as benign, it is highly probable that the patient has benign tumor.

Claims (11)

Diagnosis of nasopharyngeal carcinoma in a patient;
A substrate having two or more antigens immobilized on a substrate is cultured in body fluids from a patient and said antigens are located in body fluids from a patient at least in certain areas and are capable of producing an Eppstein- Antibody conjugate, wherein the formation of the antigen-antibody conjugate is in each case visible on a substrate based on one or more markings, wherein the two or more markings are visualized by optical means And transmitted to an evaluation unit where a proposed diagnosis is generated based on one or more characteristics of the marking recorded and recorded,
The value of the color contrast between the marking realized in the form of bands in each case and the background surrounding the band is determined, Is generated based on a mathematical computation model that at least some of the determined color contrast is used as a parameter. The method according to claim 1,
Characterized in that a blot strip or bloat strip having two or more, more specifically five or more, antigens immobilized thereon is used. 3. The method according to claim 1 or 2,
Wherein said antigen (s) used is VCA gp125, VCA p19, EBNA-1, p22 and / or EA-D. 4. The method according to any one of claims 1 to 3,
Wherein the patient sample is tested for the presence of antibodies of two or more different antibody classes. 5. The method according to any one of claims 1 to 4,
Wherein said patient sample is tested for the presence of antibodies of antibody class IgG, IgM and IgA. 6. The method according to any one of claims 1 to 5,
Wherein the mathematical operation model used is a logistic regression model. 7. The method according to any one of claims 1 to 6,
Wherein the mathematical operation model used is a binomial logistic regression model. 8. The method according to any one of claims 1 to 7,
Wherein the at least one contrast value of the marking realized in the form of a band is used in a mathematical operation model as a paramater. Diagnosis of nasopharyngeal carcinoma in a patient;
Wherein the antigens bind to an antibody specific for the occurrence of an Epstein-Barr virus infection and that are located in body fluids from a patient at least in certain areas, Antibody conjugate, wherein the marking begins to be visible upon formation of the antigen-antibody conjugate and is transmitted to an evaluator that produces a record of one or more markings and generates a proposal diagnosis based on one or more characteristics of the recorded markings The optical system comprising:
Wherein the substrate is realized on the basis of a cell membrane, more specifically a blot strip or a blot piece, wherein the evaluating unit determines the value of the color contrast between the marking realized in the form of bands and the background surrounding the band, And the proposed diagnosis is generated using a mathematical operation model in consideration of the determined color contrast. 10. The method of claim 9,
Wherein two or more antigens selected from the group consisting of VCA gp125, VCA p19, EBNA-1 and EA-D are immobilized on the platelets or the platelets. 11. The method according to claim 9 or 10,
Wherein the optical means is realized as a scanner and / or a camera.

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