DE19846271A1 - Immunoassay for enteroviral antigens useful for diagnosis, comprises binding the antigens to one or more immobilized capture antibodies directed against enterovirus group-specific determinants - Google Patents

Abstract

A method (I) for detecting enteroviral antigens, is new and comprises binding the antigens to one or more immobilized capture antibodies directed against enterovirus group-specific determinants, and detecting the bound antigens with a detector antibody. An Independent claim is also included for a kit (II) for detecting enteroviral antigens, comprising one or more antibodies directed against enterovirus group-specific determinants.

Description

The invention relates to a method for detecting entero viral antigens as well as test kits that are suitable for the implementation Enterovirus detection are particularly suitable.

Enteroviruses are a very common cause of infection especially in newborns and children. The antige niche variable genus Enterovirus, that of the Picornavi family ridae belongs to 67 known serotypes: the poliovirus Types 1 to 3 as well as the non-poliovirus enteroviruses Coxsackie virus A (23 serotypes), Coxsackievirus B (6 serotypes), ECHO- Virus (31 serotypes) and the numbered enteroviruses 68 to 71. Infection with non-poliovirus enteroviruses goes along a wide range of clinical pictures. Most often, the infections lead to a mild feverish or influenza-like disease, with severe courses it can however to aseptic meningitis, encephalitis, paralyti symptoms or acute and chronic myocarditis com men. In newborns, the most severe, occasionally fatal  ongoing septic symptoms with meningitis, myo carditis, pancreatitis and hepatitis occur.

The infection goes with an alternating pronounced cytocidal len virus multiplication, this initially in epithelium cells of the nasopharynx or gastrointestinal tract and takes place in the regional lymph nodes. Only after that can viruses spread to different organs, like the central nervous system, the skeletal muscles, the Heart or liver.

The clinical course of an enteroviral infection is often not from the courses of other viral or bacterial ones Distinguish between infections. Above all, an unnecessary to avoid unnecessary antibiotic treatment is therefore a meaningful and quick procedure needed for differential diagnosis.

The previous means of choice for the diagnosis of enteroviral Infections is the virus detection after virus isolation Cell cultures using clinical materials such as nasal and Throat swabs, rectal swabs, stool, blister contents and CSF were inoculated. Since not all enteroviruses are in one and multiply the same cell line, several must each Cell lines are inoculated in parallel. The incubation period of the Inoculated cell cultures can take up to 10 days men. The serotyping of a virus isolate is then carried out using Neutralization tests carried out. The method can only be used in specially equipped laboratories and is a very time-consuming and costly procedure for the night of enteroviruses.

Another way to diagnose enteroviral In nucleic acid detection with the help of poly merase chain reaction (PCR). This very sensitive method that on amplification specific, conserved, duck  roviral genome sequences is based, however, by their con susceptibility to tampering the risk of false positive Er results. The method requires special equipment from the performing laboratories and is associated with very high costs the.

Due to the existing situation of the very long and knockout most complex diagnosis of enteroviral infections the invention in particular the task of a fast and in addition inexpensive method, its implementation is not limited to special laboratories as well as to provide a test kit that one easy to handle detection of enteroviruses.

This task is solved by the procedure with the characteristics len of claim 1 and by the kit with the features of Claim 7. Special embodiments of this method and of this kit are in dependent claims 2 to 6 and 8 respectively to 22 described. The wording of all claims will hereby by reference to the content of this description makes.

The direct antigen detection method of claim 1 is based on the principle of the specific binding of an anti gene to immobilized antibodies (capture antibodies) Formation of an antigen-antibody complex. Come here Antibodies for use against group-specific De are directed by enteroviruses. The detection of the thus shaped antibody-antigen complex is bound performed by antibodies (detection antibodies).

The for the method of enterovirus according to the invention Antibodies used usually meet the requirements Prerequisite for a high enjoyment specificity for the genus duck rovirus and a high cross-reactivity between the Representatives of this genus. There are monoclonal anti  body or polyclonal antibodies in question, the above preferably fixed on a solid phase, d. H. immobilized (first antibodies).

The highly specific interaction of antibodies and antigen causes, for example, after loading the solid phase which the specific antibodies are fixed with clinical trial, which indicates the presence of enteroviruses, d. H. for the presence of enteroviral antigens the enteroviral antigens should be retained the. Additional antibodies bind to these enteroviral antigens per (second antibody), which is related to the enjoyment specificity and the cross reactivity usually essentially the meet the same requirements as the first antibodies, however, against another group specific determinant of Enteroviruses are targeted. It is still possible through the use of first and / or second antibodies, which specifically against certain subgroups of Enterovi ren are directed to dif. within the group Enterovirus reference.

In preferred embodiments, the detection of the antigen retained on the solid phase by the markie tion of the second antibody, which, for. B. with horseradish Peroxidase (POD) or conjugated with biotin. In the case of Labeling with an enzyme such as POD is done di rect in the form of a color reaction. When using biotin the antibody-antigen complex is marked by the Use of POD-labeled streptavidin in turn by a Color reaction demonstrated because streptavidin has a stable Forms complex with biotin. Another preferred Colloidal gold is used as a marker for the embodiment second antibody used. Furthermore, it is with others preferred embodiments possible, the binding of the two th antibody to the antibody-antigen complex by the Detect the binding of a third, labeled antibody,  which is specifically directed against the second antibody. For example, if the second antibody is is a rabbit polyclonal antibody, it is possible to use a third antibody, the was generated specifically against antibodies from rabbits. For the color reaction caused by the labeling enzyme POD catalyzes, for example, the colorless substrate Tetramethylbenzidine (TMB) are used, which according to completed reaction to a blue color of the reaction mixture leads. By adding stop reagent, such as. B. Schwe rock acid, the color changes from blue to yellow. A photometric evaluation takes place at a wavelength of 450 nm.

The invention further comprises test kits for detecting enterovira antigens that are quick and easy to carry out enable in particular the method mentioned, and inexpensive and without any significant expenditure on equipment can be carried out. The test kits are specific interaction of the enteroviral antigen with an anti body against a group specific determinant of Enterovirus is targeted.

The antibodies used for the test kits show in the Usually a high enjoyment specificity for enteroviruses and a high one Cross reactivity between the different representatives of the Enteroviruses. According to the above description of the The method according to the invention are monoclonal, Mi different monoclonal or polyclonal Antibodies in question. All antibodies are preferably in ge cleaned form before to non-specific interactions of the Exclude enteroviral antigens with other substances.

To detect the interaction of enteroviral antigen with the (catcher) antibody comprises the test kit according to the invention preferably at least one further (detection) antibody  by. Regarding this as the second and third anti body designated detection antibody and its labeling is expressly referred to the previous description.

In a first preferred embodiment of the test kit against enteroviral group-specific determinants directed antibodies (first antibody) on a solid phase, preferably a microtiter plate, immobilized. In the latter The case for this is preferably 96-well microtiter plates used because these plates have a high sample throughput ensure and many common instruments, such as. B. Multi-channel pipettes, for use with 96-well microtiter plates are matched. These 96-well microtiter plates can be made up of twelve microtiter strips with eight Ver wells exist.

The immobilization of the antibodies on plates can be done with the help of adsorption by using suitable, pre-coated Plates are performed. Another way of Im Mobilization is the covalent linkage of the antibodies with a plate that has, for example, a hydrazide surface having. A preferred embodiment of the antibody However, immobilization on plates is in use of plates coated with protein G, since here the antibodies who fixes them via their non-antigen-binding components den and so the greatest yield of reactive, d. H. antigen binding antibodies can be obtained.

In a second preferred embodiment the test kit is against group-specific determinants of enteroviruses targeted antibodies on a membrane, preferably one Nitrocellulose membrane, immobilized.

This membrane is particularly part of a test strip fens (Dip Stick), which enables a very fast execution of the Allows testing for enteroviral antigens. Furthermore, the  Use of test strips has the crucial advantage that no other instruments, such as B. a photometer, benö be done.

With the test kit, especially when running as a test strip, a functional check is preferably pre to safely rule out false negative results can. An additional antibody is preferably used for this fixed on the test strip that binds the antibody, which is directed against the enteroviral antigen, as from the the following description of the figures becomes clear.

The described features and other features of the invention result from the following description of preferred th embodiments in connection with the subclaims and the drawings. Here, the individual characteristics individually or in combination with each other be realized.

The drawings show:

Fig. 1 Schematic representation of phase in a hard fixed antibodies and the interactions with enterovirus antigen and other antibodies

Fig. 2 shows a schematic representation of the structure of an enterovirus test strip.

Solid phase 1 , to which first antibody 2 is fixed, is shown in the lower part of FIG. 1. This (scavenger) antibody is directed against group-specific determinants of enteroviruses 3 . The various possibilities for fixing the antibodies 2 to the solid phase 1 are explained in more detail in the example below. The ente roviral antigens 3 retained by the interaction with the fixed antibody 2 are additionally bound by a further, second antibody 4 . If this antibody 4 , as shown in the left part of the picture, is labeled with an enzyme 5 , such as peroxidase, the complex formed of fixed antibody 2 , antigen 3 and second antibody 4 can be detected by a color reaction which is caused by the Enzyme 5 is catalyzed. Another way of proof is shown in the right part of the picture. The second antibody 4 is conjugated to biotin 6 . This biotin 6 can form a stable complex with streptavidin 7 . If the streptavidin 7 is conjugated with an enzyme 8 , for example Peroxi dase, the complex formed can be detected by an enzymatic color reaction. Furthermore, the detection can take place in that the second antibody 4 is bound by a third antibody which is specifically directed against the second antibody 4 and which bears a label, for example peroxidase.

Fig. 2 shows schematically the structure of a preferred embodiment of the test kit. It is a test strip (dip stick), which is constructed using common components. The carrier material of the test strip 11 be made of an approximately 9 cm long and 0.5 cm wide plastic strip, which is provided with absorbent membranes. In each case about 2 cm from the two ends 12 and 13 of the Strei fens absorbent components 15 and 15 a are arranged over its width, each having a length of about 1.5 cm. A section 14 is provided above the lower suction component 15 , which carries a mouse antibody 16 which is specifically directed against enteroviral antigens and has a marking made of colloidal gold. The area between the section 14 and the upper suction component 15 a has a nitrocellulose membrane on which two bands 17 and 18 of different antibodies 19 , 20 of about 0.1 cm width are applied at a distance of about 2 cm. The antibody 19 of band 17 is specifically directed against enterovinal antigens and corresponds to the first antibody with reference to the preceding description. The anti-body 20 of the band 18 is used to check the function of the test strip and is specifically directed against the antibodies 16 from the mouse.

The operation of the test strip is explained below: After the lower end 12 of the test strip has been immersed in a clinical sample 21 , the soluble components are drawn up along the test strip in the direction of the absorbent component 15 as a result of the capillary forces. In this way, the enteroviral antigens from the clinical sample 21 come into contact with antibodies 16 of the section 14 , so that a stable antigen-antibody complex can form if necessary. The complex formed from enteroviral antigens from the clinical sample 21 and the specific, labeled antibodies 16 reaches the band 17 of the test strip as a result of the capillary forces and is held in place by the antibodies 19 immobilized there. In this case, the band 17 turns purple. Above this band 17 is the band 18 with the immobilized antibody 20 which is directed against antibodies from the mouse. This antibody 20 binds the specific antibody 16 and also leads to a violet discoloration of the band 18 . This latter discoloration is independent of whether enteroviral antigen has been bound and thus serves as a functional check.

Below is a possible implementation of the fiction procedure with the provision of the various described for required components.

1. Manufacture of reagents and test kits for various Enterovirus direct detection method 1.1 Provision of antibodies against enteroviruses

As described above, are suitable for the invention Process and the kit according to the invention both monoclonal as well as polyclonal antibodies. Suitable monoclonal type Antibodies are also commercially available, for example from from Chemikon, Temecula, USA. You can get cleaned up Form or as a raw product (mouse ascites). When using the preferred mouse for cost reasons Ascites is a purification, for example as follows described under 1.3. Suitable poly clonal antibodies are, for example, by the Immu rabbits (1.2) followed by purification (1.3) won.

1.2 Production of a polyclonal antiserum against Entero viruses

A mixture of different strains of enterovirus was used to immunize rabbits. The viruses were propagated in Vero cells. The cultures were inoculated with a virus dose of 3 × 10 ⁵ TCID ₅₀ (tissue culture infective dose 50%) per 175 cm ² cell culture bottle. After a 100% cytopathic effect appeared, the viruses were harvested. Infected cells were pelleted and taken up in 5 ml TE buffer (0.01 M Tris, 1 mM EDTA, 2% Nonidet P-40, pH 7.4). The cell suspension was disrupted in an ice bath using a dounce homogenizer and then centrifuged twice at 4500 rpm. The pellet was discarded and the supernatant processed further. The medium supernatant from the cell culture flasks was concentrated to approximately 80 ml using ultra filtration and pelleted at 4500 rpm for 10 minutes. The pellet was discarded and the supernatant was pelleted in an ultracentrifuge for 50 minutes at 30,000 rpm in a 45 TI rotor. The pellet was taken up in 3 ml of TE buffer, resuspended and combined with the virus supernatant from the digested cells.

The concentrated virus was placed on a 30% sucrose cushion worn and in the 45 TI rotor for 60 minutes at 35,000 rpm centrifuged. The pellet was buffered in PBS (phosphate Saline) added. The purified virus was Irradiation inactivated and lyophilized. The protein determ The micro BCA-Pro tein assays performed by Pierce, Rockford, USA.

Three rabbits were sub 4 times at 2 week intervals cutaneous (100 µg protein / injection) and the titer certainly.

1.3 Antibody purification

5 ml rabbit serum or possibly 1 ml mouse ascites became 1: 5 diluted in PBS and fil through a 0.2 µm Millipore membrane trated. HiTrap Protein A columns (Pharmacia, Uppsala, Sweden) were washed with PBS (3 × column volume). The The antibodies were eluted with 0.1 M citric acid pH 3. The 0.5 ml fractions were collected in tubes and the pH with 50 µl carbonate buffer (pH 11) to a neutral value set. The protein content was in all fractions determined (Micro BCA protein assay, Pierce). Fractions with high protein content was pooled.

1.4 Coating of solid phases for antigen detection

Antibodies were purified according to 1.3 for the coating used.

1.4.1 Polysorb sheets (e.g. Nunc, Roskilde, Denmark)

100 µl antibody solution (5 µg / ml) in carbonate buffer (0.05 M, pH 9.6) were pipetted into each well and over Incubated at 4 ° C overnight. The plates were washed with PBS  blocked with 1% BSA (bovine serum albumin), the The supernatant was then removed and dried.

1.4.2 Covalent binding on a plate with a hydrazide surface (e.g. company Corning Costar, Bodenheim, Germany)

Periodate activation: The antibodies (1 µg / ml) were diluted in 10 ml 10 mM sodium acetate buffer pH 4.0. 32 mg of NaJO _{4 were} added to this solution and incubated for 30 minutes at room temperature.

Coating: The solution with periodate-activated antibodies (100 µl / well) was at room temperature for 1 hour incubated. The plates were washed with PBS, with 1% BSA blocked, the supernatant removed and dried.

1.4.3 Reacti-Bind ™ Protein G coated plates (company Pierce, USA)

Antibody solution (100 µl, 1 µg / ml) in SuperBlock / Tween Buffer (Pierce) was pipetted into wells and 1 hour incubated at room temperature. After that, the plates washed three times with PBS.

1.4.4 Membranes for immunochromatography

Commercially available strips were used for the test Coating used (e.g. company Advanced Microdevices MDI, Ambala Cantt, India).

Anti-enterovirus anti were applied to the nitrocellulose membrane body (5 µg / ml) with the help of a pen gene.

Approx. Goats were anti- Mouse IgG antibody (e.g. Sigma-Aldrich, Deisenhofen,  Germany, 5 µg / ml) applied. The nitrocellulose membrane was blocked with 1% BSA in 0.01 M Tris / HCl buffer pH 7.6 and then air dried.

1.5 Labeling of the antibodies 1.5.1 Biotinylation

1 ml of purified antibodies (2 mg / ml) were raised against 50 mM Sodium bicarbonate buffer pH 8.5 in Slide-A-Lyser ™ dialysis cassettes (Pierce) dialyzed. Sulfo-NHS Biotin (74 µl, Pierce) was added to the antibody solution and on ice incubated. Unbound biotin was removed by dialysis distant.

1.5.2 Labeling with peroxidase using the periodate method

2 mg of horseradish peroxidase was distilled in 500 ml Water dissolved. After adding 100 µl 0.1 M sodium periodate the solution was shaken for 20 minutes at room temperature and dialy against 1mM sodium acetate buffer pH 4.4 overnight siert. 0.5 ml antibody (2 mg / ml) against 10 mM Natri Umbikarbonat buffer dialysed. The pH of the peroxidase Solution was added by adding 10 µl of 0.2 M sodium carbonate buffer pH 9.5 adjusted to 9.0. The two solutions were found mixes and shake at room temperature for 2 hours. After that 50 ul sodium borohydride solution (4 mg / ml) were added and shaken for a further 2 hours at room temperature.

1.5.3. Gold marking

100 ml of the antibody solution was dialyzed against 2 mM Borax pH 9.0. The antibodies were then pelleted at 100,000 x g for 1 hour. The marking was carried out with 5 nm colloidal gold (British BioCell International, Cardiff, Great Britain). 50 ml of gold sol were adjusted to pH 9.0 with 100 mM K ₂ CO ₃ and shaken for 5 minutes at room temperature. Then 5 ml of 10% BSA in 2 mM Borax pH 9.0 was added. The gold-conjugated antibodies were separated from the non-conjugated antibodies and the free gold particles by centrifugation at 45,000 × g for 1 hour. The pellet was resuspended in 1 ml Tris / HCl buffer (pH 8.2 with 1% BSA and 1% NaN ₃ ) and applied to a 10-30% glycerol gradient, which was also in Tris / HCl buffer (pH 8, 2 with 1% BSA and 1% NaN ₃ ). After centrifugation (125,000 × g, 1 h), the fractions with gold-labeled IgG with Tris / HCl buffer (pH 8.2 with 1% BSA and 1% NaN ₃ ) became the optical density 3.0 at 530 nm diluted and frozen with 20% glycerine.

2. Implementation of a qualitative invention Enterovirus detection on microtiter plates

In a preferred embodiment of the method for Detection of enteroviral antigens are 96-well microtiter plates used on which the purified antibodies are fixed.

Clinical samples were diluted in dilution buffer (VP). Throat and rectal smears were taken in about 1.5 ml of buffer. Liquors were diluted 1: 2. Stool samples were processed with VP to 30% suspensions and heavy components centrifuged at 1000 × g. The supernatants were used for the investigation. Materials from patients without enterovirus infection were used as negative controls. Negative patient material, which had been contaminated with purified enteroviruses from cell culture supernatants, served as a positive control. 100 μl of positive and negative controls and 100 μl of the samples to be tested were pipetted into the plates coated with antibody and incubated at 37 ° C. for 1 hour. After washing three times with PBS, the incubation was carried out with diluted labeled antibody (1: 500 in VP, 100 μl / well, 1 hour at 37 ° C.). After washing again using peroxidase-labeled antibodies, substrate (TMB, tetramethylbenzidine) was added. When using biotinylated antibodies, an incubation (0.5 hours, 37 ° C.) with 100 μl peroxidase-labeled streptavidin (eg Sigma-Aldrich) was carried out, followed by the addition of substrate (TMB). After 10 minutes, the reaction was stopped with 1 MH ₂ SO ₄ and the absorption was measured at 450 nm. The results were considered positive if the absorption of the tested samples reached at least twice the absorption of the negative control.

3. Dip stick rapid test according to the invention

The clinical samples were prepared as above described under 2. The bottom end of the test stick was immersed in a sample to be tested. In the positive case two purple lines appear on the stick, in the negative case only one (function check).

Claims (22)

1. A method for the detection of enteroviral antigens, wherein the Antigen from at least a first immobilized against at least one group-specific determinant Antibodies targeted by enteroviruses under training of an antibody-antigen complex bound and under Binding of additional antibodies is detected. 2. The method according to claim 1, characterized in that the immobilized antibody is monoclonal. 3. The method according to claim 1, characterized in that the immobilized antibody is a mixture of different represents monoclonal antibody. 4. The method according to claim 1, characterized in that the immobilized antibody of polyclonal origin is. 5. The method according to any one of the preceding claims characterized in that the detection by Markie tion of a binding to the antibody-antigen complex second antibody.   6. The method according to any one of claims 1 to 4, characterized ge indicates that the detection by marking a third antibody occurs, which against one of the Antigen-antibody complex binding antibody gerich is. 7. Kit for the detection of enteroviral antigens, comprising at least one antibody ( 2 , 19 ) which is directed against at least one group-specific determinant of enteroviruses. 8. Kit according to claim 7, characterized in that the antibody is immobilized on a solid phase ( 1 ). 9. Kit according to claim 7 or 8, characterized in that the antibody is monoclonal. 10. Kit according to claim 7 or 8, characterized in that the antibody is a mixture of different monoclona len antibodies. 11. Kit according to claim 7 or 8, characterized in that the antibody is of polyclonal origin. 12. Kit according to one of claims 7 to 11, comprising a second antibody ( 4 , 16 ) directed against at least one determinant, preferably group-specific determinant of enteroviruses, which is labeled. 13. Kit according to one of claims 7 to 11, comprising a further, against at least one, preferably groups specific determinant of enteroviruses second antibody and a third antibody, wel directed and labeled against the second antibody is.   14. Kit according to claim 13, characterized in that the second antibody is also marked. 15. Kit according to one of claims 12 to 14, characterized records that the or the labeled antibody with Biotin, labeled with enzyme or with colloidal gold are. 16. Kit according to one of claims 8 to 15, characterized records that the solid phase is a microtiter plate. 17. Kit according to claim 16, characterized in that it the microtiter plate is an adsorbing one Plate acts. 18. Kit according to claim 16, characterized in that it the microtiter plate is a covalently binding one and / or a plate coated with protein A / G delt. 19. Kit according to one of claims 8 to 15, characterized records that the solid phase is a membrane, in particular for immunochromatography. 20. Kit according to claim 19, characterized in that the membrane is part of a test strip (dip stick) ( 11 ). 21. Kit according to one of claims 7 to 20, characterized in that a function check ( 18 ) is provided. 22. Kit according to claim 21, characterized in that the function control ( 18 ) comprises at least one immobilized antibody ( 20 ) which detects other antibodies ( 16 ) which are directed against at least one determinant, preferably group-specific determinant of enteroviruses.