REAGENT FOR EXAMINING AGGLUTINATION OF VIRUS AND KIT FOR VIRUS EXAMINATION

Abstract: A viral agglutination test agent of the present invention is immobilized molecules having affinity for the viral envelop protein onto the surface of solid microcarriers. The agent of this invention is preferably usable for detection of HIV or hepatitis C virus.

Description: [Technical Field] This invention relates to a viral agglutination test agent and a virus test kit containing the said agent as defined in the claims, which can detect sensitively and directly the viruses in blood without using the anti-viral antibody formed in blood of patients with viral infection. The viral agglutination test agent of the invention is useful for detection of all type of viruses, DNA virus or RNA virus, especially available for detection of viruses inducing continueous infections, such as hepatitis C virus (HCV), the main pathogen for non-A non-B hepatitis, and acquired immunodeficiency syndrome (AIDS) virus (HIV). [Background Art] Among viral diseases, hepatitis C and AIDS, of which therapies have recently become a matter of serious concern, remain to be sufficiently clarified partly because the nature of the diseases had been kept unknown for a long time owing to the wide variation of the pathogenic viruses themselves. Therefore, the incidence of the secondary infection by these viruses was very high, and increase of patients could not effectively be suppressed. However the remarkable development in the gene-analytical technique in recent years has clarified the primary structure of the viruses in succession, so that the methods for detection of the viruses have been established at last and have eventually slowed down the spread of the secondary infection. It was found that in any of the above-mentioned viruses, the primary structure of the core protein is kept almost unvaried while the envelop protein is very variable. In addition, the progress in the gene technology has made it possible to mass-produce such proteins. In these circumstances, methods have been developed for detection of anti-viral antibodies formed in the serum of the patient, by using the envelop protein and also the core protein as the antigen, and these methods are now widely used in the routine examination. However, these methods are problematic partly because they cannot make any definite statement as to the presence of infection for at least a few months, a period from the occurrence of viral infection till the formation of antibody within the virus-infected patient, so that they cannot effectively prevent the secondary infection, and partly because with these methods, diagnosis is difficult owing to false-negative results frequently obtained in patients with autoimmune diseases such as collagen disease. There is another problem that the test for the antibody alone cannot always draw a correct conclusion as to the presence of infection in a child infected by the mother-to-child route because he/she has the antibody inherited from his/her mother. Additionally, RT-PCR method (reverse transcriptase-polymerase chain reaction method) to amplify its gene is used to detect RT (reverse transcriptase), which is relatively long maintained its primary structure among constitutive proteins of these virus. However, the method is more complex than the above antibody detecting method. Then, a novel method for direct detection of the virus itself has been desired to substitute the conventional diagnostic method based on the detection of antiviral antibodies in patient's serum or RT-PCR method. [Disclosure of Invention] This invention was made by taking the above-mentioned circumstances into consideration, and the object of the invention is to provide a novel agent for viral agglutination test agent which can detect directly, in a short time, and sensitively the viruses in serum without using the antibodies and which can substitute the conventional methods targeting the anti-viral antibody formed in the patient's serum. The subject matter of this invention that could achieve the above-mentioned object consists in the viral agglutination test agent prepared by immobilizing molecules having affinity for the envelop protein of viruses onto the surface of solid microcarriers and including a positive control reagent, and a negative control reagent, wherein said positive control reagent is a pseudo-viral model prepared by immobilizing a part or the whole of the viral envelop protein of the virus to be detected on the surface of a solid micro carrier. The viral agglutination test agent of the present invention is available for detecting DNA virus (Parvovirus, Papovavirus, Herpes virus, Hepatitis B virus (HBV), etc.,) and RNA virus [Paramyxovirus, Togavirus, Retrovirus (human immunodeficiency virus (HIV), Human T-lymphotropic virus (HTLV-1), etc.,), Hepatitis C virus (HCV), etc.,], especially available for detecting HIV and HCV. Molecules having affinity for the viral envelop protein used in this invention are desirably proteins [monoclonal antibodies (anti-gp 120 monoclonal antibody, anti-HCV monoclonal antibody, anti-HBV monoclonal antibody, anti-Herpes Simplex Virus I monoclonal antibody, anti-Herpes Simplex Virus II monoclonal antibody. anti-Varicella Zoster Virus II monoclonal antibody, anti-Cytomegalovirus monoclonal antibody, anti-Epstein-Barr Virus monoclonal antibody, anti-Measles Virus monoclonal antibody, and anti-HTLV-1 monoclonal antibody, etc.), polyclonal antibody, CD4, etc.] such as antibodies, or peptides (anti-gp 120 polypeptide, etc). The subject matter of the virus test kit of this invention consists in that the kit contains the above viral agglutination test agent and the positive control reagent. The kit contains the negative control reagent as well. The positive control reagent is desirably the one prepared by immobilizing a part of or the whole of the viral envelop protein onto the surface of solid microcarriers, while the negative control reagent contains desirably a part of or the whole of the viral envelop protein. [Best Mode for Carrying Out the Invention] As the result of our researches to obtain a novel test agent which can directly detect the virus in serum and which can substitute the conventional antibody test methods, we found that a virus can directly be detected, in a short time, and sensitively by using agglutination of carriers labeled with molecules having affinity for a domain of the viral envelop protein present in the patient's blood, and we completed this invention. Therefore this invention includes ones, as far as molecules having affinity for the viral envelop protein are available, and carriers on which the said molecules are immobilized are available. As mentioned above, the viral agglutination test agent of the invention is characterized in that molecules having affinity for the viral envelop protein are immobilized onto the surface of solid microcarriers. Hereinafter the solid microcarriers without the above-mentioned molecules having affinity for the protein are sometimes called, for convenience, unlabeled carriers, while the carries with the molecules having affinity for the viral envelop protein (the carriers of this invention) are sometimes called molecules-labeled carries. The molecules having affinity for the viral envelop protein mean those which can recognize the envelop protein and be bound electrochemically or chemically to the said protein; such molecules are exemplified by soluble proteins such as polyclonal/monoclonal antibodies (anti-gp120 monoclonal antibody, anti-HCV monoclonal antibody, anti-HBV monoclonal antibody, anti-Herpes Simplex Virus I monoclonal antibody, anti-Herpes Simplex Virus II monoclonal antibody, anti-Varicella Zoster Virus II monoclonal antibody, anti-Cytomegalovirus monoclonal antibody, anti-Epstein-Barr Virus monoclonal antibody, anti-Measles Virus monoclonal antibody, and anti-HTLV-1 monoclonal antibody, etc.), CD4 (known as the binding sites for lymphocytes in HIV), etc. and polypeptides (anti-gp 120 polypeptide, etc.). For the viruses, as defined in this invention, to which antibodies could hardly be prepared because of the wide variability, Tam et al. proposed a method for efficient preparation of antibodies which uses only the peptide in a specified domain as the antigen, and this method was applicable also to this invention (James P. Tam. In Synthetic Peptide: Approaches to Biological Problems, ed. J. P. Tam and E. T. Kaiser, Multiple antigen peptide systems: A model design for synthetic peptide vaccine and immunoassay, pages 3-18, 1989, Alan R. Liss, Inc.). The unlabeled carriers used in this invention are not specified as far as they are globular carries used in usual agglutination methods such as latex, gelatin, polystyrene, colloidal gold, etc. The size of these carriers may be selected from the range of 0.3 nm to 20 µm in diameter, and an optimal size can be selected according to the evaluation method of agglutination to be used. For example, for macroscopic evaluation of agglutination, it is desirable to employ carriers of 0.2 to 3 µm in diameter with which macroscopic judgment is easier, and for spectroscopic evaluation of agglutination, carriers of 0.2 µm or less in diameter may be used. The color of carriers is not restricted and is diameter may be used. The color of carriers is not restricted and is desirably selected according to the method of evaluation. These unlabeled carriers may be dispersed in advance in a solution such as purified water, buffer, or serum, or lyophylized unlabeled carriers may be dissolved in a suitable solvent (e.g. purified water, buffer, etc.) before use. The following is illustrated the method for detection of virus using the kit containing the above-mentioned viral agglutination test agent of this invention. The virus test kit of this invention comprises, in addition to the above-mentioned viral agglutination test agent, a positive control reagent, and a negative control reagent. The positive control reagent is a pseudo-virus model prepared by immobilizing a part of or the whole of the viral envelop protein onto the surface of the unlabeled carriers. Namely, the use of the pseudo-virus model is recommended from the viewpoint of the safety of handling during the test and the quality assurance of the positive control. The negative control reagent may be selected among the serum from a normal subject having no target virus, buffer, salt-containing aqueous solution, a reagent containing the viral envelop protein, etc. From the viewpoint of quality assurance of the negative control in the kit of this invention, it is again recommended on a similar basis to use a reagent containing a part of or the whole of the viral envelop protein; in the concrete, the reagent may be dispersed in advance in a solvent such as purified water, buffer, or serum, or a lyophilized reagent may be dissolved before use in a suitable solvent (e.g. purified water, buffer, etc.). As an example of the kit for virus test of the invention, the kit for HIV test is illustrated as to how to use it. [HIV test kit] Components: (1) Anti-gp120-molecule-labeled latex reagent, 2 ml (2) positive control [pseudo-HIV; gp120-labeled colloidal gold], 500µl (3) negative control [gp120], 500 µl (4) agglutination plate, 20 plates (5) diluted test sample, 50 ml How to use: 1. Before use, allow to stand the molecule-labeled latex reagent at room temperature until the normal temperature is reached. Before addition, mix carefully to avoid bubbles forming until a homogeneous suspension is obtained. 2. Add 20 µl of the molecule-labeled latex reagent into each well of the agglutination plate. 3. Add 20 µl each of the test sample, or the positive control/negative control, and mix well with the tip of a micropipet. 4. Rolling the agglutination plate for 1 to 2 minutes in hands. 5. Confirm that the agglutination is evidently recognizable with the positive control reagent, while no agglutination is obtained with the negative control reagent. The virus agglutination test agent of the present invention is available for all type of viruses, DNA virus or RNA virus, especially available for detecting virus inducing continueous infections, such as DNA virus [Parvoviridae {Parvovirus (Kilham virus, etc.,)}, Papovaviridae {Papillomavirus (Shope pappilloma virus, etc.,), Polyomavirus (polyoma virus, etc.,),}, Herpesviridae {Herpes simplex virus I, Herpes simplex virus II, Varicella zostervirus, Cytomegalovirus, Epstein-Barr virus, etc.,}, Hepadnaviridae (HBV, etc.,) etc.,] , RNA virus [Paramyxoviridae {Pneumovirus (Respiratory syncytial virus, etc.,), Paramyxovirus (Sendai virus, etc.,), Morbillivirus (Measles virus, etc.,), etc.,}, Togaviridae {Rubivirus (Rubella virus, etc.,), Flavivirus (Japanese encephalitis virus, etc.,), etc.,}, Retroviridae (HIV, HTLV-1 etc.,), Arenaviridae {Arenavirus (Lymphocytic choriomeningitis virus, etc.,) etc.,}, HCV, etc]. In the following, the invention is illustrated in more detail with the Preparations and Examples. Preparation 1 To a suspension of activated latex beads (Polyscience Co., particle diameter: 0.2 µm), was added an equivalent amount of anti-gp120 monoclonal antibody (1 mg/ml of borate buffer, pH 8.3), and the mixture was allowed to react at 37°C for 1 hour. After completion of the reaction, was added bovine serum albumin (10 mg/ml) so that its concentration became 0.1%, and blocking of the unreacted active groups was performed at room temperature for 30 minutes. Unreacted substances were removed by repeated centrifugation, and the buffer was substituted by the phosphate buffer (pH 7.4), to prepare HIV test agent. The positive control was prepared by labeling colloidal gold of 0.3 µm in diameter (NanoProbe Co.) by the reaction with the recombinant gp120 expressed in vaculovirus. The negative control was prepared as a solution of the said recombinant gp120 in the phosphate buffer (pH 7.4). Preparation 2 To the avidin-labeled latex beads (Molecular Probe Co.) suspension, was added an equivalent amount of biotinylated anti-NS(HCV) monoclonal antibody, and the mixture was allowed to react at 37°C for 1 hour, to prepare HCV test agent. The positive and negative controls were colloidal gold preparations labeled with the recombinant NS and with human albumin, respectively. NS means non structure protein. Preparation 3 Equivalent amounts of aldehyde-group-modified polystyrene beads (Interfacial Dynamics Co., particle diameter: 0.2 µm) and the anti-gp120 monoclonal antibody (1 mg/ml of purified water) were mixed, to which triethylamine (10 mg/ml of purified water) was added to the concentration of 0.1%, and the mixture was allowed to react at room temperature for 1 hour, to prepare HIV test agent. Preparation of the positive control/the negative control was performed as described in the Preparation 1. Preparation 4 Maleimidized colloidal gold (Molecular Probe Co., particle diameter: 3 nm) was allowed to react with the SH-group-introduced Fab' antibody prepared by using the anti-NS(HCV) monoclonal antibody, to prepare HCV test agent. Preparation of the positive /negative control was performed as described in Preparation 2. Preparation 5 Amino-group-modified latex beads (Interfacial Dynamics Co., particle diameter: 0.2 µm) were treated with glutaraldehyde, to which an equivalent amount of anti-gp120 monoclonal antibody (1 mg/ml of borate buffer, pH 8.3) was added, and the mixture was allowed to react. After completion of the reaction, the unreacted active groups were blocked with triethylamine, to prepare HIV test agent. Preparation of the positive control/negative control was performed as described in Preparation 1. Preparation 6 To a suspension of activated latex beads (Polysciences Co., particle diameter: 0.2 µm), was added an equivalent amount of the recombinant CD4 (100 ug/ml of borate buffer, pH 8.3) expressed in vaculovirus, and the mixture was allowed to react at 37°C for 1 hour, to prepare HIV test agent. Preparation of the positive control/negative control was performed as described in Preparation 1. Preparation 7 To a suspension of colloidal gold (Polysciences Co., particle diameter: 0.03 µm), was added an equivalent amount of an anti-gp120 polypeptide solution, and the mixture was allowed to react at 37°C for 1 hour, to prepare HIV test agent. Preparation of the positive control/negative control was performed as described in Preparation 1. Preparation 8 To a suspension of activated latex beads (Polysciences Co., particle diameter: 0.2 µm), was added an equivalent amount of the NS (HCV) monoclonal antibody solution, and the mixture was allowed to react at 37°C for 1 hour, to prepare HCV test agent. Preparation of the positive control/negative control was performed as described in Preparation 2. Preparation 9 To a suspension of activated latex beads (Polysciences Co., particle diameter: 0.2 µm) was added an equivalent amount of the HBV monoclonal antibody, and the mixture was allowed to react at 37°C for 1 hour, to prepare HBV test agent. The positive control and negative control were colloidal gold of 0.3 µm in diameter (NanoProbe Co.) preparations labeled with HBS antigen and human serum albumin, respectively. Preparation 10 To a suspension of activated latex beads (Polysciences co., particle diameter: 0.2 µm), was added an equivalent amount of the anti-Herpes Simplex Virus I monoclonal antibody solution, and the mixture was allowed to react at 37°C for 1 hour, to prepare Herpes Simplex Virus I. The positive control was prepared by labeling colloidal gold of 0.3 µm in diameter (NanoProbe Co.) by the reaction with partial purified Herpes Simplex Virus I cultured in Velo cell. The negative control was performed as described in Preparation 9. Preparation 11 To a suspension of activated latex beads (Polysciences Co., particle diameter: 0.2 µm) was added an equivalent amount of the anti-Herpes Simplex Virus II monoclonal antibody, and the mixture was allowed to react at 37°C for 1 hour, to prepare Herpes Simplex Virus II test agent. The positive control was prepared by labeling colloidal gold of 0.3 µm in diameter (NanoProbe Co.) by the reaction with partial purified Herpes Simplex Virus II cultured in Vero cell. The negative control was performed as described in Preparation 9. Preparation 12 To a suspension of activated latex beads (Polysciences Co., particle diameter: 0.2 µm), was added an equivalent amount of the anti-Vaicella Zoster Virus monoclonal antibody, and the mixture was allowed to react at 37°C for 1 hour, to prepare Vaicella Zoster Virus test agent. The positive control was prepared by labeling colloidal gold of 0.3 µm in diameter (NanoProbe Co.) by the reaction with partial purified Vaicella Zoster Virus cultured in human fibroblast cell. The negative control was performed as described in Preparation 9. Preparation 13 To a suspension of activated latex beads (Polysciences Co., particle diameter: 0.2 µm) was added an equivalent amount of the anti-Cytomegalovirus monoclonal antibody, and the mixture was allowed to react at 37°C for 1 hour, to prepare Cytomegalovirus test agent. The positive control was prepared by labeling colloidal gold of 0.3 µm in a diameter (NanoProbe Co.) by the reaction with partial purified Cytomegalovirus cultured in human fibroblast cell. The negative control was performed as described in Preparation 9. Preparation 14 To a suspension of activated latex beads (Polysciences Co., particle diameter: 0.2 µm), was added an equivalent amount of the anti-Epstein-Barr virus monoclonal antibody, and the mixture was allowed to react at 37°C for 1 hour, to prepare Epstein-Barr virus test agent. The positive control was prepared by labelling colloidal gold of 0.3 µm in diameter (NanoProbe Co.) by the reaction with partial purified Epstein-Barr virus from infectioned cells. The negative control was performed as described in Preparation 9. Preparation 15 To a suspension of activated latex beads (Polysciences Co., particle diameter: 0.2 µm), was added an equivalent amount of the anti-Rubella Virus monoclonal antibody, and the mixture was allowed to react at 37°C for 1 hour, to prepare Rubella Virus test agent. The positive control was prepared by labeling colloidal gold of 0.3 µm in diameter (NanoProbe Co.) by the reaction with partial purified Rubella Virus from infectioned cells cultured in Vero cell. The negative control was performed as described in Preparation 9. Preparation 16 To a suspension of activated latex beads (Polysciences Co., particle diameter: 0.2 µm), was added an equivalent amount of the anti-HTLV-1 monoclonal antibody, and the mixture was allowed to react at 37°C for 1 hour, to prepare HTLV-1 test agent. The positive control was prepared by labeling colloidal gold of 0.3 µm in diameter (NanoProbe Co.) by the reaction with partial purified HTLV-1 from infectioned cells. The negative control was performed as described in Preparation 9. Example 1 The sensitivity of the HIV test agent prepared as described in Preparation 7 was examined by using the HIV stock culture (HTLV-IIIB). The supernatant of this stock culture showed a negative result with the known HIV antibody test agent (CellodiaHIV, Fuji Rebio Co.). Before the examination, it was confirmed that the positive control described in Preparation 7 showed a positive result and the negative control showed a negative result, and also that the controls did not react at all to LAV-2, a HIV-2 type virus. Then the supernatant of the stock culture was diluted serially, and the resultant dilutions were allowed to react with the test agent of Preparation 7. The results are summarized in Table 1. The following tables 1-7 show that "+"/"-" means positive or negative, respectively. HIV (dilution) 10 1 10 2 10 3 10 4 10 5 10 6 gp120/culture supernatant (negative control) gp120/colloidal gold (positive control) 100ug protein/ml + + + + + + - HTLV-IIIB (HIV-1) stock culture supernatant (10 4-5 TCID 50 /ml) + + + + + - - LAV-2 (HIV-2) stock culture supernatant (10 4-5 TCID 50 /ml) - - - - - - - As shown in Table 1, it was found that HIV test agent had good sensitivity of detection even at the dilution of 10 5 fold. The limit of dilution of the HTLV-IIIB stock culture (10 4-5 TCID 50 /ml) was 10 5 fold, and this dilution fold was the limit at which the virus was supposed to be detected. Example 2 The HIV test agent of Preparation 7 was used to examine the sensitivity of the test agent with the 10-fold serial dilutions of HTLV-IIIB stock culture supernatant (10 4-5 TCID 50 /ml) in inactivated human serum. The results are summarized in Table 2. dilution in inactivated human serum 10 1 10 2 10 3 10 4 10 5 10 6 HTLV-IIIB (10 4-5 TCID 50 /ml) + + + + - - As shown in Table 2, it was found that HIV can be detected with the test agent of this invention without any influence on the sensitivity even when HIV was diluted 10,000 folds in human serum alone. Example 3 The HCV test agent of Preparation 8 was used to examine the sensitivity of the test agent with serum from various patients shown in Table 3. The results are summarized in Table 3. disease hepatitis C hepatitis B AIDS SLE progressive sclerema sarcoidosis HCV (Preparation 8) + - - - - - As shown in Table 3, it was found that only hepatitis C can be specifically detected with the HCV test agent of this invention. Example 4 The HCV test agent of Preparation 9 was used to examine the sensibility of the test agent with serum from various patients shown in Table 4. The results are summarized in Table 4. disease hepatitis B hepatitis C SLE sarcoidosis HCV (Preparation 9) + - - - As shown in Table 4, it was found that only hepatitis B can be specifically detected with the HBV test agent of this invention. Example 5 The various virus test agent shown in Table 5 was used to examine the sensitivity of the test agent with serum from various positive control shown in Table 5. The results are summarized in Table 5. As shown in table 5, it was found that only objective virus can be specifically detected with the test agent of this invention. Example 6 The various virus test agents shown in Table 6 were used to examine the sensibility of the test agent with serum from various positive control shown in Table 6. The results are summarized in Table 6. Positive control HIV HIV Rubella virus HTLV-1 HIV (Preparation 1) + - - - HCV (Preparation 8) - + - - Rubella virus (Preparation 15) - - + - HTLV-1 (Preparation 16) - - - + As shown in Table 6, it was found that only objective virus can be specifically detected with the test agent of this invention. Example 7. The various virus test agent shown in Table 7 was used to examine the sensitivity of the test agent with serum from various patients shown in Table 7. The results are summarized in Table 7. Patients' serum HIV HBV HCV SLE sarcoidosis HIV (Preparation 7) + - - - HBV (Preparation 8) - + - - - HCV (Preparation 9) - - + - - As shown in Table 7, it was found that only objective virus can be specifically detected with the test agent of this invention. [Industrial Applicability] This invention has been constituted as described above, and shows following several advantages over the conventional antibody test methods. (1) Because this invention directly detects viruses without using any.antibody, the time lag (about 2 to 3 months) required for formation of an antibody in the patient's serum in the conventional antibody test methods is no more needed, so that the target virus can be detected immediately after viral infection. (2) Viruses can sensitively be detected even in children infected through the mother-to-child route and in patients with autoimmune diseases such as collagen disease, where viruses were hardly detected with the conventional antibody test methods. (3) Even an infectious viruses, which could not be detected with the conventional antibody test methods, can sensitively be detected. (4) The conventional antibody test methods required the patient's serum containing the target virus as the positive control, whereas this invention uses solid microcarriers with the viral envelop protein immobilized on their surface. This invention is highly recommendable also from the viewpoint of assuring the safety on the examiner's side.

Claims: A method for detecting directly a virus in a patient's serum, comprising contacting a patient's sample with a viral agglutination test agent prepared by immobilizing molecules having affinity for the envelop protein of the virus onto the surface of a solid microcarrier, and confirming that the agglutination or no agglutination is obtained using a positive control reagent and a negative control reagent, wherein said positive control reagent is a pseudo-virus model prepared by immobilizing a part of or the whole of the viral envelop protein of the virus to be detected onto the surface of a solid microcarrier. The method of claim 1, wherein said negative control reagent is a solution containing a part or the whole of the viral envelop protein of the virus to be detected. The method of claim 1 or 2, wherein the solid microcarrier is selected from the group consisting of gelatine, latex beads, polystyrene beads, and colloidal gold. The method of claim 1 to 3, wherein the virus is a DNA virus or a RNA virus. The method of claim 4, wherein the virus is selected from the group consisting of Parvovirus, Papovavirus, Herpes virus, Hepatitis B virus, Paramyxovirus, Togavirus, Retrovirus, and Hepatitis C virus. The method of claim 1 to 5, wherein said molecules are selected from the group consisting of polyclonal antibodies, monoclonal antibodies, CD4, and polypeptides. The method of claim 6, wherein said molecules are monoclonal antibodies. A virus test kit for detecting directly a virus according to the method of any one of claims 1 to 7, containing a viral agglutination test agent as defined in claim 1, 3, 6 or 7, a positive control reagent as defined in claims 1 or 3, and a negative control agent as defined in claims 1, 2 or 3.

Inventors: FUJII TAKERU, HAMAMOTO HIDETOSHI, YOKOYAMA HIDEAKIRA, FUJII, TAKERU, HAMAMOTO, HIDETOSHI, YOKOYAMA, HIDEAKIRA, HAMAMOTO, Hidetoshi Yamaichikan 101, YOKOYAMA, Hideakira Park Court Josanjima 102
Applicants: TEIKOKU SEIYAKU KK, TEIKOKU SEIYAKU KABUSHIKI KAISHA
Application year: 1996
Main classification: G01N33
IPC codes: G01N33, C12Q1