ES2374365A1 - Method and device for rapid diagnosis of hepatitis a in faecal samples - Google Patents

Abstract

Method for the diagnosis of hepatitis A, which comprises taking a representative portion from the faecal sample (1B), dispersing said sample in a specific diluent (1C), using this dispersion of the sample (1D) in a specific immunochromatographic device (1E), forming a complex between the antibodies of the device and the antigens of the sample, and visualizing this complex on the membrane of the device. This immunochromatographic device allows identification of viral proteins, fragments or particles by means of the use of a specific monoclonal antibody conjugated with stained microparticles and the immunological capture thereof in a porous membrane.

Description

Method and device for rapid diagnosis of Hepatitis A in stool samples.

Field of the Invention

The present invention fits within the area of biotechnology and specifically in the diagnosis of diseases caused by viruses. The object of the invention is a Quick and simple procedure to detect the virus Hepatitis A or its fragments in stool samples.

State of the art

Hepatitis A is a liver disease caused by the Hepatitis A Virus (HAV). Like other hepatitis (B, C, D, E) A very characteristic symptom is jaundice. The Hepatitis A infections are much more frequent in very countries towns with low hygienic-sanitary levels. It mainly affects children under 5 years of age in countries in process development where seroprevalence can reach 100%.

The hepatitis A virus is transmitted between people by the oral-fecal route. The virus is excreted abundantly in the feces of infected people and is stable in the environment for long periods of time. The Typical acquisition is by ingestion of food or water contaminated with stool (Lemon SM. Type A viral hepatitis: epidemiology, diagnosis, and prevention. Clinical Chemistry, 1997, 43 (8 (B)): 1494-1499).

The course of the disease is very variable. There are patients, especially children, who do not develop symptoms, nor Even jaundice. The intensity of symptoms between Patients who manifest them are also variable. The course of Hepatitis A (acute) can be divided into 4 phases (Hepatitis A (WHO / CDS / CSR / EDC / 2000.7) World Health Organization, 2000):

Incubation Approximately 30 days There are no symptoms, but active virus replication and great disease transmissibility.

Preictericia 4-10 days Symptoms such as: loss of appetite, fatigue, abdominal pain, nausea, vomiting, fever, diarrhea, dark urine and pale stools appear.

Jaundice Symptoms of jaundice characteristic of hepatitis occur. They usually remit the rest of the symptoms. The viremia disappears but the feces are infective one or two more weeks since the onset of jaundice.

Convalescence The recovery period of the disease is of variable duration 3-6 months.

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The expulsion of the virus in the feces as well as the viremia takes place during the incubation and pre-sterile phases and ceases upon reaching the jaundice phase. It is precisely then that antibodies begin to appear in the blood, first those of the IgM type and then IgA and IgG (Stapleton JT and Lemon SM, Hepatitis A and Hepatitis E. Infectious Diseases 5 th ed . Philadelphia, Lippincott Co , 1994; 790-797).

Currently the diagnosis is made by detecting the presence of anti-HAV antibodies in the serum blood In this way hepatitis A is distinguished from others hepatitis. More specifically, the diagnosis is based on the identification of anti-HAV IgM antibodies in the serum of the patients during the acute phase of the disease. These antibodies persist in the serum for 3 to 6 months. Anti-HAV IgG type antibodies are also available. present during the course of the disease and persist during Decades after an acute infection (Hepatitis A (WHO / CDS / CSR / EDC / 2000.7) World Health Organization, 2000).

The disease can also be diagnosed. identifying said anti-HAV antibodies in saliva samples (WO 97/02490).

The diagnosis of the disease based on the measurement of anti-HAV antibodies in serum (or saliva) has some drawbacks:

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Requires blood draw and Serum preparation

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Requires staffing specialized in conveniently equipped laboratories.

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Do not indicates if the patient has an active disease, only in some moment the immune system has been in contact with the virus.

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The vaccinated people have antibodies and therefore test positive in the test despite not having contracted the disease.

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The appearance of anti-HAV antibodies takes place approximately 4 weeks after infection (18, 21-23, 40) so there is a window period of 4 weeks in which the disease is not detectable by antibodies and the patient besides incubating the disease is excreting large amount of virus in your stool and may be the origin of a epidemic outbreak.

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There is asymptomatic carriers who are not going to be done the serum antibody test for showing no symptoms but They contribute to the spread of the disease.

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To solve all these problems we have developed the present invention consisting of a rapid test for detection of viral particles or fragments of the virus Hepatitis A in faecal samples. Present the following advantages:

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It is not blood collection and serum preparation necessary, just A sample of stool.

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I dont know need complex laboratory equipment or personnel specialized.

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Indica presence of virus and therefore active disease.

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Be Can perform on vaccinated people.

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He Window period is reduced from 4 weeks to just a few days (see figure one).

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He virus can be identified in the stool of patients asymptomatic

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Is Fast (10 minutes) and easy to carry out.

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The identification of viral particles or HAV fragments can be carried out by ELISA techniques or PCR The Mediagnost HAV-Antigen EIA (Reutlingen, Germany) is an ELISA immunoassay for the identification of HAV in various types of sample with cell cultures and feces. The detection of HAV by identification of RNA sequences by using a transcriptase Reverse (RT) and Polymerase Chain Reaction (PCR) (Patent WO 2008145196). However, this type of evidence presents some inconveniences that have made its use very limited:

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Time: ELISA requires between 2 and 3 hours and PCR about 8-10 hours.

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Both of them they need complex laboratory equipment available only in specialized laboratories and expert staff in said techniques

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He ELISA has a limited sensitivity since it is necessary dilute the sample a lot.

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They present a high number of false positive. In the case of ELISA, all positive results must be be reevaluated after being incubated with a solution neutralizing ensuring its blocking effect.

brief description

The Hepatitis A diagnostic method of The present invention consists of: taking a portion representative of the stool sample, its dispersion in a diluent specific, the application of this dispersion of the sample in a specific immunochromatographic device, the formation of a complex between the antibodies of the device and the antigens of the sample and visualization of this complex in the membrane of the device.

The immunochromatographic device used is single use only. No instrumentation is required to carry out the method or interpret the result. As shown They use stool.

Sample preparation time is about two minutes and the test development time is 5 to 10 minutes, which means 10 to 20 times less than rehearsals EIA (ELISA), much more complex to carry out and need laboratory instrumentation

Because of its simplicity the test can be taken to conducted in the doctor's office even in nurseries or places where an epidemic outbreak is suspected.

The test can be performed before the symptoms of the disease appear in suspicious individuals or likely to have been infected by HAV for being near a focus of the disease or by belonging to risk groups.

The test can also be performed as soon as the first symptoms appear: especially diarrhea, but also loss of appetite, fatigue, abdominal pain, nausea, vomiting, urine dark or pale stools (no color).

The test works best before they appear Hepatitis symptoms, typically jaundice or also high blood bilirubin levels and high aminotransferase activity in the blood serum.

The Hepatitis A diagnostic method of invention is useful during the incubation period of the disease in which the concentration of the virus in feces can be high.

Since the virus can be detected in stool before the onset of symptoms, the method is especially useful for disease prevention and management of epidemic outbreaks to know promptly and in advance at the onset of symptoms the origin and magnitude of the focus epidemic.

Similarly, the proof of the invention is suitable for establishing early surveillance procedures for occurrence of the disease especially in high areas virus prevalence

Description of the figures

Figure 1: Scheme of the steps of the method of diagnosis. The specific device or vial for taking faecal samples (Figure 1A) is a plastic vial of about 3-4 mL capacity, which has previously been dispensed 1 ml of diluent for dispersion, with a cap thread that has a rod whose end, designed for that purpose, allows the collection of a portion of stool (Figure 1B) solid or semi-solid and its introduction in the vial. After this, it closes and it stir vigorously (Figure 1C) until complete dispersion of the sample. Finally, the upper part of the device is broken (Figure 1D) and a few drops are added on the application site of the immunochromatographic device sample (Figure 1E).

Figure 2: Device views immunochromatographic

Figure 2A: Top view of the device immunochromatographic strip format. That strip includes the following elements: an absorbent material (1), porous membrane (2) with a test line (3) and a control line (4), a place of Sample application (5) and a place for the conjugate antibody-marker (6).

Figure 2B: View of the device section immunochromatographic strip format in which they are also distinguished of the elements described in Figure 2A, the plastic support (7).

Figure 2C: Projected view of the device immunochromatographic, in which the elements are distinguished described in Figures 2A and 2B.

Detailed description

To carry out the test, a portion the stool sample in the diluent in a proportion Approximately 1/10. If the sample is liquid 100 microL of sample is mix with 1 mL of diluent. If the sample is solid they separate about 100 mg of sample with the help of a spatula and disperse in 1 mL of diluent.

It is very convenient to use a specific device or vial for stool sampling. This device is a plastic vial of about 3-4 mL of capacity, in which 1 ml of diluent has been previously dispensed for dispersion, with a screw cap that has a stem whose end, designed for this purpose, allows the collection of a portion of solid or semi-solid stool and its introduction into the vial. In US 5543115 a document can be seen more detailed description of a similar device.

Once the sample has been introduced into the vial, Close with the screw cap and stir vigorously. After this I know break the upper part of the cap and by the created tightness are added 3-4 drops of stool dispersion over the area of Application of the immunochromatographic device sample.

After the sample application is Wait 10 minutes and interpret the result. The test is consider negative if only one line appears (for example, blue) in the results window and positive if two lines appear (for example, one red and one blue).

Immunochromatographic devices for the detection of both antigens and antibodies have been described above (for example, in EP 1248112). For the detection of the HAV virus or its fragments, monoclonal or polyclonal antibodies or a combination of both can be used. In a preferred embodiment, a single anti-HAV monoclonal antibody is used. This monoclonal antibody (HA1) has been developed using semi-purified extracts of in vitro cultures of the Hepatitis A virus. The obtained monoclonal antibody has a high affinity and specificity against a portion of the structural protein of the VP1 virus.

In a preferred embodiment, this antibody monoclonal (HA1) is used both in the membrane and in the conjugate. The conjugate is prepared with colored microparticles and is deposited in one of the device materials immunochromatographic downstream of the place of application of the It shows and acts as a mobile phase. The same antibody is immobilized. on the porous membrane and acts as a fixed phase, in which it occurs the immunological capture of viral particles or fragments of virus. The membrane and the material with the conjugate are mounted together with other absorbent or adhesive materials on a support plastic that is cut in strip form.

All the elements described to carry out the test (the immunochromatographic device, the vial for taking of faecal samples, the sample dispersion diluent, ...) together with the instructions for use can be included in a set or kit for easy testing.

Examples of realization Example 1 Preparation and purification of monoclonal antibodies anti-HAV

The monoclonal antibody HA1 was obtained from the hybridoma of the same name. The hybridoma producing the monoclonal antibody HA1 was obtained using known protocols according to the method of cell fusion and fusion of the obtained clones that was initially described by Köhler and Milstein in 1975 (Köhler G, Milstein C. Continuous cultures of fused cells secreting antibody of predefined specificity Nature, 1975 Aug 7; 256 (5517): 495-7).

BALB / c type mice were immunized with the proportional dose the Havrix vaccine (GlaxoSmithKline). The lymphocytes of immunized mice were fused with cells Myelomics of the SP20 line and the hybridomas obtained were selected by ELISA techniques. The wells of a plate 96-well microtiter were upholstered with polyclonal antibody rabbit anti-HAV in 100 mM pH carbonate buffer 9 to 37 degrees for 2 h. To these wells the viral extract semi-purified as described below. After washing it add the different hybridoma culture samples and the presence of anti-HAV antibody is revealed using an anti-mIgG conjugate with peroxide (Sigma) and the corresponding substrate.

The hybridomas were selected that greater affinity and what better specificity they presented. Among these are they selected the most producers and the ones with the best benefits offered in the tests of thermal stress and reaction speed against the antigen.

The semi-purified HAV extract was obtained at from a culture of MRC-5 cells in medium minimum essential (Eagle) supplemented with 10% fetal bovine serum, who were inoculated with the Hepatitis A virus. It was incubated for several days at 37 degrees with 5% CO2. They were treated with proteases and the virus was partially purified by centrifugation

The selected hybridoma HA1 was cultured in RPMI-HT medium for several days at 37 degrees with 5% of CO2. From the culture medium, the antibody by protein A affinity chromatography according to column manufacturer's instructions (GE Healthcare) after which was dialyzed in PBS pH 7.4.

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Example 2 Conjugate Preparation

A conjugate of the HA1 antibody was prepared with polystyrene microparticles. Colored particles with carboxyl groups on their surface of 300 nm in nominal diameter were used. 1 mL of 10% particles is washed by centrifugation and resuspended in MES buffer (2- (N - morpholino) ethanesulfonic acid) 10 mM pH 6 and was added EDC (1-ethyl-3- (3-dimethylaminopropyl) - carbodiimide) to a concentration of 5 mM. Incubate 1 h at 37 degrees and remove excess reagent by centrifugation. The activated particles are resuspended in 10 mM MES of pH 6 and monoclonal antibody is added to a surface concentration of 2 mg / m2 after which they are incubated 4 h and washed in 0.1% Tween 20. The particles thus obtained are diluted in a solution containing 10% sucrose, 2% bovine albumin, 1% PEG-6000 and 2% Tween-20 in TRIS buffer (tris (hydroxymethyl) aminomethane) of pH 8, to a concentration of 0 , 05%.

This solution is deposited at a rate of 15 uL / cm in a winding material of 29mm non-woven polyester fibers of width that dries during 24 h in a camera to 30 degrees and 20% relative humidity

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Example 3 Preparation of the test and control lines

HA1 antibody dialyzed in phosphate buffer Saline (PBS) is brought to a concentration of 1 mg / mL and is linearly deposited on a laminated 25 nitrocellulose membrane mm wide and pore size between 10 and 30 micrometers at the rate of 1 microL / cm after which it dries for 24 hours in a chamber at 30 degrees and 20% relative humidity.

At the same time it is deposited and in them conditions deposit rabbit polyclonal antibody anti-mIgG that will serve as a control line.

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Example 4 Immunochromatographic test assembly

The material with the conjugate, the membrane and the Absorbent material is mounted as indicated in Figures 2 and 3 on a plastic support with an adhesive sheet and the strips are cut transversely to its assembly to a width of 4 mm. Optionally the strip can be placed inside a plastic device as a housing that protects and facilitates Its use.

A solution for the dispersion of stool samples consisting of an aqueous solution of 300 mM sodium chloride, 1% Triton X-100 and nonspecific 100 microgr / mL mouse IgG antibodies in a buffer 200 mM TRIS at a pH of 9. This preparation is dispensed in vials for sampling at a rate of 1 mL / vial.

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Example 5 HAV detection

Serial dilutions 1/2 of HAV will be prepared in the sample dispersion diluent described in section previous. 100 uL of these dilutions are applied to the devices immunochromatographic of the invention and the assay is allowed to progress for 10 minutes at room temperature (25 ° C) after which proceed to interpret the result by visual appreciation of appearance of the test line. The same operation is performed but diluting the HAV in a pool of stool samples dispersed approx. 1/10 in the same buffer. The results are shown in the table one.

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TABLE 1

one

Claims (8)

1. Method of diagnosis of Hepatitis A by detecting viral particles or virus fragments of Hepatitis A using specific antibodies comprising the following stages:

to.

the taking a portion of stool sample,

b.

the dispersion of a portion of stool sample in a diluent,

C.

the application of this dispersion of the stool sample in a immunochromatographic device,

d.

the formation of an antibody-fragment complex viral,

and.

the visualization of the complex in the membrane of the device immunochromatographic

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2. A method of diagnosing hepatitis A according to claim 1 wherein the used specific antibodies are polyclonal or monoclonal. 3. A method of diagnosing hepatitis A according to claim 2 wherein a single monoclonal antibody is used. 4. A method of diagnosing hepatitis A according to claim 3 characterized in that the detected antigen is a structural protein of hepatitis A. 5. Method of diagnosis of Hepatitis A according to claim 1 characterized in that the sampling and its dispersion is carried out with the help of a specific vial for faecal samples. 6. An immunochromatographic device for the diagnosis of hepatitis A in the form of a strip with a sample application site, a porous membrane in which the result is visualized and an absorbent characterized in that:

to.

be have immobilized specific antibodies against the virus Hepatitis A in the porous membrane test line,

b.

It has been impregnated a conjugate prepared with antibodies against the virus Hepatitis A and a marker particle downstream of the site Sample application,

C.

be have used materials whose characteristics and pore size they allow the flow of particulate matter from feces.

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7. An immunochromatographic device according to claim 6 characterized in that the strip can be inside a plastic housing that protects it. 8. A Hepatitis A diagnostic kit that comprises the immunochromatographic device of claim 7 and a diluent for dispersion of the faecal sample that can go or no, in a specific vial for stool sampling.