DE102008028295A1 - Immunological on-site-rapid test kit for detecting surface protein of pathogen, i.e. Borrelia burgdorferi, after the stinging or biting of the arthropods - Google Patents

Abstract

Immunological on-site-rapid test kit for detecting pathogens in arthropods, is claimed, where the surface protein of the pathogen is detected after the stinging or biting of the arthropods.

Description

The The present invention relates to an on-site rapid immunological test kit for direct detection of Borrelia burgdorferi sensu lato in arthropods.

It There are a variety of arthropods, human or human, worldwide animal hosts affected by sting or bite. These arthropods are carriers of a variety of pathogens, z. As viruses, bacteria, protozoa or worms. The best known of arthropods, especially ticks, transmitted pathogens is Borrelia burgdorferi, which causes Lyme disease.

The Lyme disease or Lyme disease is an infectious disease, which is caused by the bacterium Borrelia burgdorferi from the group of Spirochaetes is triggered. The Borrelia are pulling soon after infection from the bloodstream into the Tissue back. It can be any organ, the nervous system, that Joints and the tissue are attacked. That is why we speak this disease also from a multisystemic disease. The Disease occurs in humans and all other mammals, as well as birds.

Of the Disease progression of Lyme borreliosis is characterized by stages. As a result of the tick bite occurs after an incubation period of several days to a few weeks initially to a local infection (Stage I, erythema migrans), later on to an exciter generalization (Stage II; nervous system, heart or joint symptoms) and after months or years to organ manifestation (stage III, late stage; Lyme arthritis or acrodermatitis chronica atrophicans). The individual disease stages can but also be skipped so that z. B. the typical Wanderer (Erythema migrans) is missing. A spontaneous healing is rare Cases possible.

All in all is both clinical and laboratory diagnostics of Lyme disease difficult and in some cases of illness with big ones Uncertainty. Because the direct detection of the pathogen in materials Patients are considered to be unreliable, are usually serological Procedure used as aid to the diagnosis. However, in early stages of the disease become common not found increased antibody titers, though an infection has taken place, so here's the serology sometimes not a reliable criterion. This is partly true even of neuroborreliosis, the one threatening clinical picture and their demarcation of neurological diseases of other cause of great Meaning is.

at Lyme disease is of the utmost importance to recognize the disease as early as possible and to start with a treatment. Only then will the best chances of recovery exist for the patient.

One The first step is to get the arthropod directly and so fast as possible to examine the pathogens. So can provide a first indication of contagion risk become.

Information on the risk of infection after tick bite has been found sparse and usually without reference to the positivity or negativity of the tick. American research suggests that 1-3% of tick bites in endemic areas lead to infection with Borrelia burgdorferi. Come here Magid et al., N. Engl. J. Med. 327, (1992), 534-541 in a statistical cost-benefit analysis, concluded that in areas with very high levels of infection of the ticks with Borrelia burgdorferi, prophylactic antibiotics should be administered after each tick bite.

Direct detection of Lyme disease pathogens in the tick by means of a PCR laboratory procedure ( United States Patent 6087097 . United States Patent 6051382 ) is already known and commercialized. Disadvantage is that the tick must be sent by post to the respective laboratory. Important time passes in which a treatment could already be carried out. The waiting time for the result is about 2-3 days.

The It is an object of the present invention to provide a test kit with which it is possible, directly after arthropod sting to examine the arthropod on the spot for Borrelia. So useful time can be saved to make a first estimate to get the infection risk. The / the stingy can with this Indicate immediately go to the doctor to discuss appropriate therapy and / or initiate. Using the on-site quick test kit can the doctor, supporting to the signs of disease, his Make a diagnosis and quickly initiate appropriate therapy.

According to the invention this by an on-site rapid immunological test kit according to claim 1 reached. Advantageous embodiments emerge from the subclaims.

The immunological on-site rapid test kit according to the invention Based on the pathogens in the tick surface proteins possess specific to Borrelia burgdorferi sensu lato are and it must be proven.

The term "arthropod" includes the terms insects, especially ticks and mosquitoes, Spiders and crabs. Furthermore, the term "pathogens" includes viruses, bacteria, protozoa and worms.

Of the Detection of surface proteins of the pathogen in arthropods using an immunological on-site rapid test kit. For this, the arthropod is removed from the host, in a special Buffer solution prepared and placed in a test cassette. There is the presence of specific surface proteins and thus detected the presence of Borrelia in the tick.

The Invention is that with this on-site immunological rapid test kit the arthropod removed, on the spot and after the bite on pathogens can be examined. The result is within 10 minutes in front.

The Advantages over the previous tests are that of Test applied anytime and anywhere without any aids can be. Submitting the tick is no longer necessary and no waiting times of 2-3 days are more necessary.

in the Subsequently, the immunological according to the invention On-site rapid test kit described for the detection of ticks the Borrelia burgdorferi, the causative agent of Lyme disease in itself wear. This pathogen is located in the midgut of the ticks, but not or rarely in saliva this before. In a first phase after Tick bite is introduced into the puncture wound tick saliva, which anesthetizing and tissue-liquefying properties has. In a second phase, blood and tissue components become sucked. Regurgitation of midgut content often occurs during this phase. which pathogen, in particular Borrelia, may contain, in the puncture wound.

Borrelia have numerous opera surface proteins known as OspA are designated OspF, especially OspA, OspB and OspC play an important role in the pathogenesis. OspA is expressed by Borrelia in the intestine of the tick, but after onset Blood meal, migration into the salivary gland and blood contact down regulated by the Säuwirirtes and then it finds one Expression of OspC instead. (Ohnishi 2001).

Of the immunological on-site rapid test is based on the immunological "sandwich assay "for the detection of surface proteins of Group Borrelia sensu lato. Anti-Borrelia antibodies are on the test line and rabbit anti-chicken antibodies on the Control line fixed. Located on one side of the membrane a stained nonwoven which contains a mouse anti-Borrelia antibody-gold conjugate is soaked.

at The Borrelia antigen is used to carry out the test the tick using the extraction reagents (sample vessel with Buffer solution), a comminution aid and the pipette put to the test. Here, the Borrelia antigen reacts with the colored gold conjugate particles into a complex.

The Shredding aid includes the terms plastic rod, pestle, stir bar, Disposable stir bar, disposable pestle.

at the sample vessel is a term such as Eppendorf tube, cryotube, reaction tube, Cup and Vial.

By the effect of capillary forces runs the sample mixture on the membrane and binds to the test line, which turns red.

is Borrelia antigen is present, forms in the area "T" one red line through formation of the "sandwich" complex Solid phase anti Borrelia antibody / Borrelia antigen / anti Borrelia antibodies-gold conjugate. Absence from Borrelia no line appears in the test field.

Independently reacted by the presence of Borrelia antigens in the sample the sample mixture always passes when passing the control line "C" with the immobilized rabbit anti-chicken antibody and forms a red line.

The Presence of this line serves as confirmation that the correct sample volume was applied and the solution properly over the membrane is.

Included in the invention the on-site immunological rapid test kit of the above method all materials and reagents which for the detection of surface proteins of Pathogens in arthropods are necessary. This includes a sample tube with buffer solution, a test strip with test cassette, pipette, a crushing aid, as well as a Manual.

in the The following is the detailed description of the test procedure and test evaluation shown.

Test procedure

• 1. Tick is in the sample vessel with shredded on the enclosed plastic rod (see drawing 1)
• 2. The sample is aspirated using the pipette (see drawing 2).
• 3. 3 drops of the sample solution is in the round Sample opening of the test cassette given (see drawing 3)

test evaluation

Negative:

In the control region "C" appears after 2-3 minutes a red colored line. In the test region "T" is no red line visible after 10 minutes. Borrelia are not detectable.

Positive:

in the Results window are 2 red lines ("T" and "C") after 10 minutes to recognize. This result shows that Borrelia is in the sample available.

Appear within 10 minutes neither in the control region nor in the Test region a line, the test is invalid. The investigation must be repeated with a new test.

Of the Test is read out at the latest after 10 minutes and with compared to the results below. Later read out Results are no longer usable. (see drawing 4).

100 Collected ticks were first transferred individually to Eppendorf tubes and numbered. Subsequently, each sample was overcoated with PBS and homogenized by means of a disposable pestle. Part of the homogenate was used to carry out the tick test Lyme disease transferred to the associated sample tube with buffer solution. Of the Rest of the sample was frozen for reference analysis and thawed as needed.

Of the immunological on-site rapid test kit was performed according to instructions and the result (pos./neg.) noted.

The associated frozen sample was then sorted by genus Diagnostic PCR was investigated, with one for the Borrelia SP. Specific band as positive and a missing band as negative was designed. Previous counted Borrelia cultures served as controls and for the purpose of establishing the identity with selected samples. In case of discrepancies was as a further reference method the dark field microscopy was used

46 According to the reference method, ticks were laboratory PCR positive, of which the on-site rapid test kit identified 44 samples as positive. 49 out of 52 ticks can be treated with the on-site immunological rapid test kit be identified as being really negative.

These Data show that the immunological on-site rapid test kit a fast-acting alternative to the PCR detection method represents.

QUOTES INCLUDE IN THE DESCRIPTION

This list The documents listed by the applicant have been automated generated and is solely for better information recorded by the reader. The list is not part of the German Patent or utility model application. The DPMA takes over no liability for any errors or omissions.

Cited patent literature

• - US 6087097 [0009]
• - US 6051382 [0009]

Cited non-patent literature

• Magid et al., N. Engl. J. Med. 327, (1992), 534-541 [0008]

Claims (10)

Immunological on-site rapid test kit for the detection of pathogens in arthropods, characterized in that after the sting or bite of the arthropod in the latter, the surface proteins of a pathogen is detected. Test kit according to claim 1, characterized that the detection of the surface proteins of the pathogen carried out by immunological rapid test method Test kit according to claims 1-2, characterized that on the spot and right after bite or sting of an arthropod, the same with the help of the immunological on-site rapid test kit can be examined directly for pathogens. Test kit according to claims 1-3, characterized that the immunological on-site rapid test kit from a test cassette, a sample vessel with buffer solution and a crushing aid exists Test kit according to claims 1-4, characterized that the sample vessel with a special buffer solution filled to prepare the sample (arthropod) serves. Test kit according to claims 1-5, characterized that the enclosed shredding aid (drawing 5) for crushing, d. H. Preparing the arthropod in the sample vessel is used. Test kit according to claims 1-6, characterized that the pathogen is Borrelia burgdorferi sensu lato acts. Test kit according to claims 1-7, characterized that in the immunological rapid test method specific surface proteins of the pathogen Borrelia burgdorferi sensu lato become Test kit according to claims 1-8, characterized that as a sample the crushed arthropod in the special Buffer solution used. Test kit according to claims 1-9, characterized that the sample to be examined is in contact with the test cassette and thus the antibodies for detection of surface proteins the pathogen is brought.