|Title of Invention||
"A KIT FOR DETECTION OF ANTI-TOXOPLASMA ANTIBODIES IN HUMAN AND ANIMAL SAMPLES USING DIRECT AGGLUTINATION TEST"
|Abstract||This invention relates to a kit for the detection of presence of antibodies in human and animal samples using direct agglutination test, comprising of a microtitre plate, sample diluting buffer, concentrated whole cell Toxoplasma, antigen diluting buffer (ADB), sterile distilled water, a negative control serum sample, a positive control serum sample, adhesive seals, and instruction manual.|
|Full Text||Method for detection of Toxoplasmosis Technical Field
The present invention provides a direct agglutination test method for detection of anti-toxoplasma antibodies in the samples of human and animal. The invention further provides a kit for detection and identification of toxoplasmosis.
Toxoplasmosis is an infectious disease affecting both animals and humans caused by the protozoan parasite, Toxoplasma gondii (1). Toxoplasma gondii is an obligate intracellular parasite which is classified among the Coccidia. This parasite has relatively broad host range infecting both mammals and birds. Humans and other warm-blooded animals are its hosts and the disease is prevalent world wide. The organism is ubiquitous in nature and exists in three forms: tachyzoite, cyst, and oocyst (2). Tachyzoites, found during acute infection, are the invasive form capable of invading all nucleated mammalian cells. After the acute stage of infection, tissue cysts called bradyzoites are formed within host cells and persist within the host organism for the life of the host. Cysts are important in transmission of infection, especially in humans, as the ingestion of raw or undercooked meat can result in the ingestion of bradyzoites which can infect the individual resulting in an acute infection. Oocysts represent a stage of sexual reproduction which occurs only in the intestinal lining of the cat family from which they are excreted in the feces.
Approximately one-third of human being has been exposed to this parasite. Toxoplasmic encephalitis is a major cause of morbidity and mortality in AIDS patients. Toxoplasma infection has also been shown to be a significant cause of chorioretinitis in children and adults. In the United States and in Western Europe 10 to 30% of seropositive AIDS patients die of toxoplasmosis. Due to the increased epidemic of AIDS in India, the risk of getting the toxoplasmic infection will be life-threatening (9). Because of the spread of AIDS in India the need to reacquaint physicians about problems associated with clinical toxoplasmosis is felt.
Studies on human have shown that the risk and severity of congenital toxoplasmosis is greatest when acquired during first trimester of pregnancy (5). The consequences of congenital toxoplasmosis range from spontaneous abortion, intrauterine growth retardation and premature birth, and an array of neurological symptoms, which involve ocular complications (6, 7). Infants acquiring congenital toxoplasmosis may also remain asymptomatic for several weeks after birth (8).
Toxoplasmosis in animals causes serious impact on the economic growth due to morbidity and mortality in animal husbandry and export of meat.
Acquired toxoplasmosis is generally asymptomatic (2). In immunocompromised hosts the toxoplasmic infection becomes fatal (3). In pregnant women and in animals, the infection acquires much significance as the parasite may enter into the fetal circulation through the placenta; thereby causing congenital toxoplasmosis (4). Although usually asymptomatic in immunocompetent adults, it can cause death in immunocompromised patients and in congenitally infected children. Depending on the age at the time of primary infection, the virulence of the strain of T. gondii and the immune status of the host. symptoms of disease vary from no symptoms to death. Generally, congenitally acquired toxoplasmosis is more severe than postnatally acquired infection. The severity and likelihood of infection is dependent on the trimester of pregnancy the mother becomes infected with T. gondii. Studies on human have shown that the risk and severity of congenital toxoplasmosis is greatest in infants whose mothers become infected during the first trimester than those during the later trimester of pregnancy (5). Hydrocephalus, chorioretinitis, intracerebral calcification, mental retardation, loss of hearing and death may occur. Loss of vision is the most common sequel in congenitally infected children. Infants acquiring congenital toxoplasmosis may also remain asymptomatic for several weeks after birth (8) they will develop some symptoms later in life. Although most postnatally acquired infections are asymptomatic, manifestations of toxoplasmosis include large lymph nodes (particularly of the cervical region), headaches, muscle aches and sore throat. Because these symptoms are nonspecific, postnatal toxoplasmosis is rarely diagnosed.
Because symptoms of toxoplasmosis are nonspecific, diagnosis is aided by serologic examination. The detection of T. gondii specific antibodies and determination of antibody titer are important tools used in the diagnosis of toxoplasmosis. Numerous serologic tests are available to detect IgG and IgM, T. gondii specific antibodies. The presence of IgG antibodies only means exposure because asymptomatic humans can develop very high (>100,000) T. gondii antibody titers, and titers may remain elevated for several years. Although an 8-fold rise in antibody titer, taken 2 weeks apart, is indicative of a recent infection, this is seldom achieved in practice because by the time patients are seen in clinic, antibody titer has usually peaked. Compared to IgG antibodies, IgM antibodies are short-lived, and they appear before IgG antibodies. However, IgM antibodies can persist for more than 12 months after exposure.
The Sabin-Feldman dye test (DT), indirect fluorescent antibody test (IFAT), indirect haemagglutination test (IHAT), latex agglutination test (LAT), direct agglutination test (DAT), and enzyme linked immunoabsorbent assay (ELISA) are some of the tests used to detect T. gondii antibodies. Although the DT is the most specific, it is rarely used now because it uses live virulent T. gondii. The IFAT is nearly as sensitive as the DT but it requires a fluorescent microscope. The IHAT, LAT, DAT and ELISA are used more commonly because kits are commercially available. The IHAT lacks sensitivity, especially during acute infection. Moreover, it lacks specificity, especially in titers of less then 1:64. The conventional ELISA requires a calorimeter reader and all ELISA kits are imported fi"om western countries enabling it to be costly and not readily available.
Due to the lack of a purified, standardized antigen or standard method for preparing the tachyzoite antigen, inter-assay variability exists resulting in different assays having different performance characteristics in terms of assay sensitivity and specificity. So, there is a need for developing a simple, accurate and indigenous diagnostic kit for the detection of toxoplasmosis in human and animals. This will help in establishing the accurate epidemiological data of toxoplasmosis infection. The kit is simple, sensitive and specific, and reagents are readily and commercially available. In the present invention the Anii-Toxoplasma gondii detection kits are developed for the first time solely in India.
Objects of the invention
An object of the invention is to develop a simple, cost effective and indigenous method for detection of Toxoplasmosis by direct agglutination test.
Another object of the present invention is to develop a method for detecting anti-toxoplasma antibodies in the samples of human and animals.
Still another object of the present invention is to develop a kit for the detection of Toxoplasmosis.
Yet another object of the invention is to provide a method for using the said kit.
Summary of the invention
The present invention provides for a simple, cost effective and indigenous method for detection of anti-toxoplasma antibodies in samples of human and animals using a direct agglutination test. Further, the invention also provides a kit for detection of toxoplasmosis.
Brief description of figure
Figure 1: Results of samples tested for detecting anti-toxoplasma antibodies
Detailed Description of the invention
In accordance with the object, the present invention provides a method for detection of anti-toxoplasma antibodies in samples of human and animals using a direct agglutination test
An embodiment of the invention, the method for detection of Toxoplasmosis is based on direct agglutination test for qualitative and semi-quantitative estimation of antibodies against toxoplasma gondii in samples of human or animals, preferably blood, serum or plasma samples.
Another embodiment of the invention provides a kit for the detection of Toxoplasmosis comprising of:
a) A Microtitre Plate,
b) Sample Diluting Buffer,
c) Concentrated Antigen,
d) Antigen Diluting Buffer (ADB),
e) Sterile Distilled Water,
f) A Negative Control Sample,
g) A Positive Control Sample, h) Adhesive seals, and.
i) Instruction manual
Still another embodiment of the invention the test is performed by obtaining test sample, diluting the test sample with a sample diluting buffer solution, adding the diluted sample solution to the wells of the microtitre plate, separately obtaining concentrated antigen, reconstituting the lyophilized antigen diluting buffer with water, adding 2 mercaptoethanol (ME) to reconstituted ADB to obtain ADB-2-ME mix, diluting concentrated antigen with ADB-2-ME mix, adding equal quantity of diluted antigen solution on to the wells containing diluted test sample solution, sealing the wells with adhesive seals and incubating at Til^C, followed by 4'*C to observe agglutination or button formation. Antibodies against Toxoplasma gondii, if present, the test sample will combine with the antigens, resulting in agglutination or if absent, button formation.
Yet another embodiment of the invention provides storage of kit at a temperature ranging between 2°C and 8 C.
The terms test sample, sample diluting buffer, concentrated antigen, antigen diluting buffer, a negative control, and a positive control, referred herein in the application is described below:
1. TEST SAMPLE
Blood, serum or plasma obtained from human beings or animals
2. SAMPLE DILUTING BUFFER
Phosphate buffered saline for dilution of test samples
3. CONCENTRATED ANTIGEN
Tachyzoites of Toxoplasma gondii fixed in formaldehyde along with preservatives and protein stabilizers
4. ANTIGEN DILUTING BUFFER
Tube containing lyophilized preservatives and protein stabilizers, to be reconstituted in sterile double distilled water and used for assay.
5. NEGATIVE CONTROL SAMPLE:
Human serum sample found negative on testing for anti-toxoplasma antibodies along with preservative
6. POSITIVE CONTROL SAMPLE:
Human serum sample found positive on testing for anti-toxoplasma antibodies along with preservative
The invention is illustrated by the following examples and should not be construed to limit the scope of the present invention. The present invention is described in term of its specific embodiments and any modifications and equivalents to a person skilled in the art should be included within the scope of the present invention.
Collection of sample: Blood, plasma or serum samples are collected carefully as it may contain infectious agents infecting animal species or human. The serum or plasma may be stored between 2-8 C for not longer than 72 hrs after collection. Sample containing visible particulate matter are clarified by centrifugation before the test. Care is taken not to
eat and inactivate the samples, as it may lead to false positive or sometimes false negative results.
Method of using the kit: All the reagents are allowed to attain room temperature (20-25 °C) before performing the test. The lyophilized Antigen Diluting Buffer (ADB) is reconstituted in 10 ml of sterile distilled water and to require aliquot of ADB, 2-mercaptoethanol (2-ME) is added to get the final concentration of 1.4%. The required dilution of the concentrated antigen is prepared using ADB-2-ME mix. For obtaining 1:25 (antigen: ADB-2-ME mix), mix 10 µl of concentrated antigen with 240 µl of ADB-2-ME mix. 1:50 dilutions of control and test specimens are prepared by adding 10 µl of test sample to 490 µl of sample dilution buffer. 50 µl of diluted antigen is dispensed to each well containing diluted sample in the micro titer plate. The plate is gently swirled avoiding any spillage. The plate is covered with an adhesive sealer and incubated at 37 "C for 16-18 hrs. The plate is further incubated at 4 0C for 30 min. The plate is examined for visualization of agglutination or button formation.
Interpretation of results A positive result for the detection of Toxoplasmosis is indicated by the development of clearly visible clumps of the antigen-antibody agglutination complex at the bottom of the well. A negative result is indicated when the parasites are settled down at the bottom of the well forming a button like spot. An improper agglutination or button formation implies the repetition of the test. It is also recommended to carry out the quantitative estimation to determine the titer of a positive sample.
Quantitative estimation: Test samples which showed agglutination is subjected to double dilution with sample dilution buffer. Each dilution is tested with antigen (working dilution) using the method as given in Example 3. The antibody titer value is arrived at as a reciprocal of the highest dilution of test sample.
Comparative Standardization of DAT for Toxoplasma with multiple dilutions: Fifty samples are used for the standardization of the DAT for the detection of anti-toxoplasma antibodies in the serum samples with multiple dilutions. These serum samples are found positive for anti-toxoplasma antibodies by DAT. The serum samples were serially diluted
starting from 1:100 to 1:25600 at an arithmetic progression. The Standardization cut-off was kept at 1:50. The results are indicated in table below:
DAT-JPD: HS-Ag antigen obtained from J.P.Dubey (USA) used as reference standard
SS-DAT: Antigen obtained from the inventor's lab
Comparative DAT profile of serum samples using HS-Ag of JPD and SS (New)
A total of 423 serum samples from the patients coming to AIIMS for TORCH test are included in the comparative study of their DAT profile for the detection of anti-toxoplasma antibodies using the antigens obtained from Dr. J. P. Dubey (USA), antigens prepared previously and the newly prepared antigen in the inventor's laboratory. Out of the 423 samples, 113 (26.71 %) were found positive for anti-toxoplasma antibodies and 310 (73.28 %) were found negative for anti-toxoplasma antibodies using ELISA (Organon). Whereas 148 (34.98 %) samples were found positive using the HS-Ag from Dr. J. P. Dubey and the HS-Ag developed previously in our laboratory. In comparison 159 (37.58 %) samples were found positive using the HS-Ag newly developed in our laboratory. Therefore it has been found that there is 93 % correlation between the HS-Ag of JPD and SS (New). It has been observed that there may be 7 % false positive results in DAT using HS-Ag of SS (New).
Comparison of DAT & ELISA: A total of 423 serum samples were taken for the comparative ELISA and DAT. Out of 113 serum samples found positive using ELISA (Organon), all the 113 samples were again found positive using DAT (JPD), DAT (SS, previously prepared) and DAT (SS, newly prepared). Interestingly out of 310 samples found negative using ELISA, 35 (11.3 %) samples were found positive using DAT of JPD and SS (previously prepared) whereas 46 (14.84 %) samples were found positive using DAT of SS (newly developed). Thus the DAT has got an overall 21 % more sensitivity than ELISA.
Main Advantages of the present Invention
The following are the advantageous features associated with the present invention:
1. The kit is unique being the only kit which can be used for detection of Toxoplasmosis in both human and animal samples,
2. The kit of the present invention comprises of all essential items namely; antigen, buffers, other reagents, positive control sample, negative control sample and instruction manual in a suitable pack,
3. The kit can be used for qualitative as well as quantitative estimation;
4. The tachyzoite antigen is prepared from the indigenously maintained RH strain of Toxoplasma gondii adapted to mice model and which can also be adapted and maintained in various cell lines. The tachyzoites fixed in formalin are non-infectious and harmless to the user.
The parasites (antigen) are stainable with vital dyes,
The test is simple and does not require any expensive/sophisticated instruments for analysis.
8. The kits have been evaluated on human and animals samples and validated with 100
% sensitivity and 93% specificity.
9. The kit is indigenously developed providing cost effectiveness and easy
1. Dubey, J.P. and Beattie, C.P. (1988). Toxoplasmosis of animals and man. Florida : Boca Raton : CRC press Inc. p 220.
2. Remington, J.S.; McLeod, R. and Desmonts, G. (1995). Toxoplasmosis. In: Remington, J.S., Klein, O.D. (eds). Infectious diseases of the fetus and new-bom infant. 4th Ed. Philadelphia : W.B. Saunders Co. 140.
3. Luft, B.J.; Hafiier, M.D.; Korzun, A.H. et. al. (1993). Toxoplasmic encephalitis in patients with acquired immunodeficiency syndrome. N. Engl. J. Med. 329 : 995.
4. Desmonts, G and Couvreur, J. (1974). Congenital toxoplasmosis. A prospective study of 378 pregnancies. N. Engl. J. Med. 290 : 1110.
5. Daffos, F.; Forestier, F; Capella-Pavlovsky, M. et. al. (1988). Prenatal management of 746 pregnancies at risk for congenital toxoplasmosis. N. Engl. J. Med. 318:271.
6. Frenkel, J.K. (1974). Pathology and Pathogenesis of Congenital Toxoplasmosis. Bull. N. Y. Acad. Sci. 50 : 182.
7. Desmonts, G. and Couvreur, J. (1974). Toxoplasmosis in Pregnancy and its Transmission to the Fetus. Bull. N. Y. Acad. Sci. 50 : 146.
8. Barker, K.F. and HoUiman, R.E. (1992). Laboratory techniques in the investigation of toxoplasmosis. Genitourin. Med. 68 : 55.
9. Singh, S. (1997). Human toxoplasmosis in India before and after AIDS epidemic. Ann. Natl. Acad. Med. Sci. (India). 33(1): 15.
10. Desmonts, G. and Remington, J.S. (1980). Direct agglutination test for diagnosis or toxoplasma infection: Method for increasing sensitivity and specificity. J. Clin. Microbiol. 11(6): 562.
11. A Procedural Guide to the Performance of the Serology of Toxoplasmosis. Centers for Disease Control, Atlanta, 1976.
1. A kit for detection of anti-toxoplasma antibodies in human and
animal samples using direct agglutination test, comprising of
a) A Microtitre.Plate,
b) Sample Diluting Buffer,
c) Concentrated Antigen,
d) Antigen Diluting Buffer
e) Sterile Distilled Water,
f) A Negative Control Sample,
g) A Positive Control Sample, h) Adhesive seals,
i) Instruction manual,
2. A kit, as claimed in Claim 1, wherein human and animal samples is selected from the group consisting of blood, serum and plasma samples.
3. A kit, as claimed in Claim 1, wherein the sample diluting buffer is a phosphate buffer saline solution.
4. A kit, as claimed in Claim 1, wherein the concentrated antigen is whole parasite (tachyzoite) from the indigenously maintained RH strain of Toxoplasma gondii adapted to mice model or in various cell lines, fixed in formaldehyde with preservatives and stabilizers.
5. A kit, as claimed in Claim 1, wherein the Antigen Diluting Buffer is obtained by lyophilizing with preservatives and protein and reconstituted with sterile distilled water.
6. A kit, as claimed in Claim 1, wherein the negative control sample is a sample with preservative found negative on testing for anti-toxoplasma antibodies.
7. A kit, as claimed in Claim 1, wherein the positive control sample is a sample with preservative found positive on testing for anti-toxoplasma antibodies.
8. A kit, as claimed in Claims 6 and 7, wherein the preservative used is sodium azide.
9. A kit, as claimed in Claim 1, provides qualitative and quantitative
estimation of anti-toxoplasma antibodies in human and animal
10. A kit, as claimed in Claim 1, wherein the instruction manual
comprises instructions given herein below:
a) obtaining concentrated antigen,
b) lyophilizing antigen diluting buffer solution with preservatives and protein, reconstituting the lyophilized buffer with sterile water.
c) Adding 2-mercaptoethnaol (2-ME) to the reconstituted buffer of step (b) to obtain antigen diluting buffer-2-mercaptoethanol mix,
d) Adding the mix of step (c) to concentrated antigen of step (a) to obtain dilute antigen solution,
e) Obtaining the test sample,
f) Diluting the test sample of step (e) with sample diluting buffer solution,
g) Adding the diluted sample solution of step (f) on to the wells of
microtutre plate, h) Adding equal quantity of diluted antigen solution of step (d) on
to the wells of step (g) with swirling, i) sealing the wells of step (h) with adhesive seals, incubating at
37°C for 16-18 hrs followed by 40°C for about 30 min, and j) observing agglutination or button formation against positive
and negative control.
11. A kit, as claimed in Claim 1, wherein the kit is applicable for detection of anti-toxoplasma antibodies in human and animal samples using direct agglutination test.
|Indian Patent Application Number||1611/DEL/2003|
|PG Journal Number||11/2011|
|Date of Filing||26-Dec-2003|
|Name of Patentee||ALL INDIA INSTITUTE OF MEDICAL SCIENCES,DIV OF CLINICAL MICROBIOLOGY DEPARTMENT OF LABORATORY MEDICINE|
|Applicant Address||CLINICAL MICROBIOLOGY, DEPT. OF LABORATORY MEDICINE, ANARI NAGAR, NEW DELHI-110 0291, INDIA.|
|PCT International Classification Number||G01N 33/00|
|PCT International Application Number||N/A|
|PCT International Filing date|