|Year : 2013 | Volume
| Issue : 1 | Page : 40-43
The merits of designed ELISA avidity kit in detection of Toxoplasma gondii IgG antibody in laboratory conditions
Javid Sadraie1, Ehsan Shariat Bahadory1, Vajihe Marsusi2
1 Department of Medical Parasitology, Faculty of Medical Sciences, Tarbiat Modaress University, Tehran, Iran
2 Department of Medicine, Faculty of Medical Sciences, Tehran Medical University, Tehran, Iran
|Date of Web Publication||25-Jun-2013|
Department of Medical Parasitology, Faculty of Medical Sciences, Tarbiat Modaress University, Tehran
| Abstract|| |
Background: Toxoplasmosis is a parasitic disease which may cause some laboratory symptoms in infected individuals. One of the main ways to transmit this organism is placenta to the fetus pathway. If this transmission occurs in the 3 rd month of pregnancy, the abortion, central nerve system and ocular disorder will happen. Because of this issue, the precise technique for the detection of Toxoplasma antibodies such as immunoglobulin G (IgG) and immunoglobulin M IgM are important, as they contain ELISA and ELISA avidity. Materials and Methods : i0 n this survey, the main samples are serum and amniotic fluid that were collected from 48 pregnant women infected with Toxoplasma gondii in Shariaty hospital. This survey is attempted to design ELISA avidity kit in Tarbiat Modates University. Results: The results from this survey show that, in these total pregnant women the infection by T. gondii has occurred and many of them are infected currently. Conclusions : i0 n the simple ELISA technique, the only antibody that can be detected precisely is IgM; however, using this technique the IgG antibody can also be detected. In this new technique or ELISA avidity, in addition to detection of IgG antibody against T. gondii, the month of transmission of Toxoplasma is also interpreted.
Keywords: Amniotic fluid, enzyme-linked immunosorbent assay, enzyme-linked immunosorbent assay avidity, immunoglobulin G antibody, Toxoplasma gondii
|How to cite this article:|
Sadraie J, Bahadory ES, Marsusi V. The merits of designed ELISA avidity kit in detection of Toxoplasma gondii IgG antibody in laboratory conditions. Trop Parasitol 2013;3:40-3
| Introduction|| |
Toxoplasma gondii is an obligate intracellular protozoan parasite and an important zoonotic pathogen that causes severe diseases in congenitally infected patients and in immunocompromised patients such as acquired immune deficiency syndrome victims in addition to the parasite infecting healthy persons. T. gondii infects macrophages primarily and is capable of invading and replicating within a wide variety of nucleated host cells. Acute T. gondii infection during early pregnancy in women without a history of infection may lead to fetal death in the uterus or severe neurological damage. , Transplacental infection to the fetus occurs in 12% of the cases in which the mothers acquire infection during the first trimester. The incidence of transmission increases thereafter to more than 90% when maternal infection occurs during the last weeks before delivery, which is more likely to be asymptomatic, yet may proceed to choreoretinitis later in childhood or in adolescence. One-third of mothers who acquire a primary T. gondii infection during pregnancy, transmit the infection to their fetuses. Therefore, an antenatal diagnostic method should be employed to screen this fraction. ,, and then the ELISA avidity methods help to detect the time of infecting mothers with T. gondii. This would help the mothers to make an informed decision on either treatment or therapeutic abortion. In Iran, until now, most of the decisions on infected fetuses were made based on serological findings in their mothers, which might have led to abortion of many uninfected foetuses.  The low avidity of immunoglobulin G (IgG) has been reported to be a useful marker of recent infection with Toxoplasma. Nevertheless, discrepant results on the maturation of avidity over time have been reported. The aim of this study was to investigate the maturation of IgG avidity after Toxoplasma seroconversion during pregnancy and to determine factors that could influence its evolution over time. The aim was design and preparation of ELISA avidity kit in Tarbiat Modares University and comparative surveliance with commercial ELISA avidity kit in detection of T. gondii antibody in serum and amniotic fluid samples. ,,
| Materials and Methods|| |
The study was an evaluation assessment. (1) Deepwell plates, (2) ELISA-plates, (3) Plastic seals, (4) Platereader, (5) Plate washer, (6)Phosphate buffered saline PBS pH 7.4, (7) Bovine Serum Albumine (BSA), (8) Tween-20, (9) Blockingbuffer (BB): PBS + 0.05% v/v Tween-20 + 3% w/v BSA, e.g., for 500 ml BB: 500 ml PBS +250_l Tween-20 + 15 g BSA, (10) Coatingbuffer (CB): PBS, (11) Dilutionbuffer (DB): PBS + 0.05% v/v Tween-20 + 0.5% w/v BSA e.g., 500 ml PBS + 250_l Tween-20 + 3 g BSA, (12) Sodium thiocyanate (NaSCN) Make a stock-solution of 6 M NaSCN (e.g., 97.28 g NaSCN in 200 ml PBS) and make the required concentrations out of this stock. Commonly used range: 0 M - 0,25 M - 0,5 M - 0,75 M - 1 M - 1,25 M - 1,5 M - 1,75 M - 2 M - 2,25 M - 2,5 M - 3 M. (13) MgCl2 * 6H2O: (magnesium chloride hexahydrate), (14) Diethanolamine, (15) Diethanolamine DEA-buffer: 500 ml MilliQ (MQ) water +492 _l Diethanolamine pH 9.8 + 0.15% w/v MgCl2 * 6 H2O (e.g., 0.15 g), (16) Nitro Phenyl Phosphate Hexahydrate (PNPP).
In this study, we injected the toxoplasma tachyzoites to brain of BALB/c mice (5 mice) and within 1 week we aspirated peritoanal fluid and centrifuged this fluid. The upper level of this centrifuged fluid could be used for extracting excretory - secretory antigens to design ELISA avidity kit. ,,
The coating buffer was PBS with pH 7.4. We coated plates with this buffer for 24 h in 37°C in incubator. Then we used blocking buffer to end this design. The blocking buffer was PBS + 0.05% v/v Tween-20 + 3% w/v BSA, e.g., for 500 ml BB: 500 ml PBS + 250_l Tween-20 + 15 g BSA. The time of blocking stage was 1-2 h in room temperature. 
- Coat ELISA-plates with the desired coating. Coating diluted in CB, 100_l/well. Incubate o/n at 4°C, covered with a plastic seal or a lid.
- Prepare samples and standards in deepwell plates. Dilute samples in DB, make 100_l/well at a concentration of 1 absorbance unit AU. Incubate o/n at 4°C.
- Remove coating from plates by inverting the plates with a vigorous wrist action.
- Block the plates with 200_l/well BB, incubate 1 h at room temperature RT.
- Wash with plate washer.
- Add 100_l/well of sample at 1 AU. Add 100_l/well DB for the standard.
- Add 100_l of standard and dilute it 2-fold over 11 wells. Leave the Blank blank. Incubate 1 h at RT.
- Wash with plate washer.
- Add 100_l/well NaSCN in different concentrations to the wells with samples. For the wells with standard or blank just add DB. Incubate for 15 min at RT.
- Wash with plate washer.
- Add 100_l/well conjugated antibody diluted in DB. Incubate for 1 h at RT.
- Wash with plate washer.
- Add 100_l/well PnPP (1 mg/ml) in DEA-buffer. Incubate for 30 min at RT.
- Read optical density OD at 450 nm on the plate reader.
The main part of this design was the Bradford protein assay:
Bradford protein assay
- Prepare a series of protein standards using BSA diluted with 0.15 M NaCl to final concentrations of 0 (blank = NaCl only), 250, 500, 750, and 1500 μg BSA/mL. Also prepare serial dilutions of the unknown sample to be measured.
- Add 100 μL of each of the above to a separate test tube (or spectrophotometer tube if using a Spec 20).
- Add 5.0 μL of Coomassie Blue to each tube and mix by vortex, or inversion.
- Adjust the spectrophotometer to a wavelength of 595 nm, and blank using the tube which contains 0 BSA.
- Wait for 5 min and read each of the standards and each of the samples at 595 nm wavelength.
- Plot the absorbance of the standards versus their concentration. Compute the extinction coefficient and calculate the concentrations of the unknown samples.
- We added 100 λ of serum or amniotic fluid to ELISA avidity wells.
- Incubated for 30 min in 37°C.
- We washed plates for 3 times with PBS (ELISA WASHER).
- We added 100 λ enzyme conjugate to these plates.
- Incubated for 30 min in 37°C.
- We added 100 λ substrate Tetra Methyl Benzidine (TMB) .
- After 15 min we added stop solution and read at 450 nm ELISA reader.
In ELISA avidity, we added 2 buffers to wells prior to adding enzyme tracer (conjugate), consist of urea buffer to on well and phosphate buffer to another well of same sample.
| Results|| |
The formula of ELISA avidity (semi quantitative):
The normal range is <0.8 but the results >1.1 are positive for congenital toxoplasmosis.
The formula of quantitative ELISA avidity:
The results beyond 60% were in high avidity ranges. The results between 40% and 60% were in intermediate avidity ranges and the results lower than 40% were in low avidity ranges.
The analysis calculated with SPSS version 18.0 program.
In amniotic fluid the mean of ELISA avidity titer elevated that associated with number of abortion. For example in patients with 4 th abortion, the mean of commercial ELISA avidity titer was 67% and in designed ELISA avidity, the mean of ELISA avidity titer was 71% (quantitative avidity). But in patients with 1 st abortion, the mean of commercial ELISA avidity titer was 45% and in designed ELISA avidity, the mean of ELISA avidity titer was 48% (quantitative avidity in amniotic fluid) [Table 1]and [Table 2].
|Table 1: The mean of titer semi quantitative ELISA avidity in toxoplasmosis mothers (amniotic)|
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|Table 2: The mean of titer quantitative ELISA avidity in toxoplasmosis mothers (amniotic)|
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In serum samples the mean of ELISA avidity titer elevated that associated with number of abortion. For example, in patients with 4 th abortion the mean of commercial ELISA avidity titer was 72% and in designed ELISA avidity, the mean of ELISA avidity titer was 78% (quantitative avidity). But in patients with 1 st abortion the mean of commercial ELISA avidity titer was 65% and in designed ELISA avidity, the mean of ELISA avidity titer was 68% (quantitative avidity in serum samples)[Table 3] and [Table 4].
|Table 3: The mean of titer semi quantitative ELISA avidity in toxoplasmosis mothers (serum)|
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|Table 4: The mean of titer quantitative ELISA avidity in toxoplasmosis mothers (serum)|
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| Discussion|| |
Among the T. gondii antigens characterized so far, Excretory/secretory antigens ESA is peculiar since it is expressed both during the acute and chronic phase of parasitemia. ,, Thus, in principle, ESA could play a role in the persistent stimulation of cell-mediated immunity in chronically infected healthy subjects. Excretory/secretory antigens may be the best form of antigens for stimulation of the cell-mediated immune response and a good candidate as a vaccine for toxoplasmosis prevention. However, only few studies have been focused on the fractionated forms of ESA. Excretory/secretory antigen was the main antigen to design ELISA avidity kit in the laboratory. , In this survey we showed that if the kit prepared in the laboratory with fresh Excretory/secretory antigens ES toxoplasma antigens, the mean results could be in high level, that was because of (1) fresh antigens and (2) the use of RH strain toxoplasma antigens. The ESA confers a significant protection against a lethal challenge with the 76K strain cysts in both mice, by direct immunization, and nude rats, by the passive transfer of immune sera or T cells.  The ESA can stimulate a better cell-mediated immune response as compared to soluble or cysts antigen. Therefore, this antigen is a good candidate for research into immunizing agents against T. gondii infection. These studies have been performed on total-ESA of T. gondii. One important question that needs to be answered is, what fraction(s) of total-ESA is/are responsible for the cellular immune response? Therefore, in the present study, we analyzed the immune responses against ESA-fractions of T. gondii in BALB/c mice.
Prevention of congenital toxoplasmosis in pregnant women has been based mainly on serological tests for anti-Toxoplasma antibodies. Many serological tests, including the haemagglutination test, latex agglutination test (LAT), ELISA, and indirect fluorescence antibody test, have been utilized in the detection of antibodies against T. gondii. There have been several reports regarding the screening of anti-T. gondii antibodies among Koreans. Ryu et al, (1996) demonstrated 4.3% and 0.94% of positive rates, using ELISA and LAT kits among pregnant women who visited medical institutes in Yangpyong-gun and Kwangju-gun of Kyonggi-do. However, infection should be diagnosed at the early acute stage, when treatment is more effective. Therefore, the ELISA avidity methods were useful in detection of congenital toxoplasmosis and the results were reliable, if we use designed ELISA avidity kit from toxoplasma ES antigens. ,,
| Acknowledgments|| |
This study was financially supported by Tarbiat Modares University. The authors wish to thank DR. V. Marsusi from Medical University of Tehran. The authors declare that they have no conflicts of interest.
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[Table 1], [Table 2], [Table 3], [Table 4]