Science Review. – 2011. – volume 2 (8).

Enzyme Immunoassay Test for serological diagnosis of

A. Bulashev , S.Borovikov, M.Kuibagarov, Zh. Suranshiev, Sh. Serikova ______

Enzyme Immunoassay Test for serological diagnosis of Opisthorchiasis/ A. BULASHEV, S. BOROVIKOV, M. KUIBAGAROV, ZH. SURANSHIEV, SH. SERIKOVA.

Abstract «Sandwich» ELISA with IgG Monoclonal Antibodies (Mab) to antigens of Opistorchis felineus was used in the designing of Enzyme Immunoassay Test (EIT) for the diagnosis of Opisthorchiasis. During research helminth’s antigen suitable for serological analysis of blood is defined; algorithms of using Mab, excretory- secretory antigen of Opistorchis felineus (ES-Ag), sera samples and evaluation of test results in «sandwich» ELISA were established. Diagnostic value of developed EIT, based on the detection of antibodies to ES-Ag and circulating immune complexes (CIC) in samples of sera, was tested in comparison with foreign analogues. The high sensitivity and specificity of the elaborated EIT for serologic diagnosis of Opisthorchiasis has been established.

Keywords: felineus, Opisthorchiasis, excretory-secretory antigen (ES- Ag), circulating immune complexes (CIC), monoclonal antibodies (Mab), diagnosis, Enzyme Immunoassay Test (EIT)

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Introduction

Opisthorchiasis - widespread natural focal of and carnivorous caused by helminth Opistorchis felineus. It was first discovered in 1884 in a cat's by Sebastiano Rivolta of Italy. In 1891, Russian scientist K.N. Vinogradov found it in a human, and named the parasite a "Siberian ". In the 1930s, helminthologist Hans Vogel of Hamburg published an article describing the life cycle of Opisthorchis felineus. Contact address: Kazakhstan, Astana, S.Seifullin Kazakh Agro Technical University, tel.: +77172 38 36 57, e-mail: [email protected]

The first "intermediate hosts" of the parasite are freshwater snails Bithynia inflata (synonym: Codiella inflata), Bithynia troschelii and . The second "intermediate hosts" are freshwater fish, followed by the final , which are fish-eating such as felines and humans. A person becomes infected by eating raw, not fried and slightly salted fish. Opisthorchiasis ranges in severity from asymptomatic to severe illness. Patient outcome is dependent on early detection and treatment. Human cases of Opisthorchiasis may affect the liver, pancreas, and gall bladder. If not treated in the early stages, Opisthorchiasis may cause of the liver and increased risk of liver cancer, but may be asymptomatic in children. Two weeks after flukes enter the body, the parasites infect the biliary tract. Symptoms of infection include fever, general malaise, skin rash, and gastrointestinal disturbances. Severe anemia and liver damage may also incapacitate the infected person for 1-2 months (Fig.)

Fig. Life cycle of Opisthorchis felineus The widespread of Opisthorchiasis in some regions of the Republic of Kazakhstan (especially in the basin of the Irtysh River) creates a constant threat to public health. On the whole in Kazakhstan the incidence of the people of this invasion is one of the highest among the CIS countries [1]. Scatological study, based on the detection of helminth eggs in feces or duodenal contents is still a recognized method of diagnosis. However, helminth”s eggs are not always found in samples of patients with Opisthorchiasis.

Serological diagnosis of Opisthorchiasis by Indirect Enzyme-linked immunosorbent assay (ELISA) and Indirect Hemagglutination Test (IHAT) are possible at an early stage, ie before the release of eggs by the parasite. In chronic Opisthorchiasis these tests may be used only as helper methods due to the formation in the patient CIC. It is known that IgM class antibodies appear in blood within a week after infection, reaching maximum values in 1.5-2 weeks, and after 6-8 weeks their titres starts to decline rapidly. IgG antibodies are beginning to synthesize 2-3 weeks later than IgM class antibodies. Their concentration reaches to maximum in 2-3-month after infection and can be kept at this level up to 1 year or more. However, in the long term illness decreasing specific antibody titers below the threshold of sensitivity of serological methods is often observed due to the binding of antibodies to helminth’s antigen and the formation of the CIC.

In recent years, in diagnosis of parasitic diseases highly sensitive immunological methods, including ELISA, are introducing. However, using in these tests, multi-somatic antigens of trematodes do not provide the desired results because positive reactions of people with other helminthiases are observed [2, 3, 4]. False-positive results of the analysis are also possible during investigation of sera from healthy individuals in 1% of cases, patients with non-parasitic diseases (allergosis, with the pathology of the gastrointestinal tract, hepatobiliary system, systemic diseases) - 1.5%, toxoplasmosis - 5.6%, - 7, 3% and - in 15.4%, trichinellosis - in 20.0%, fascioliasis - in 29.4% of cases [5]. These facts can be explained by the presence of cross-reacting antigens among helminths. Perhaps these common antigens are also presented in the parasite and host. This raises the need to search for antigen, specific for helminth. Such species-specific antigens can be found among the metabolic products of Opistorchis felineus.

JSC "Vector" (Russia) begans to produce diagnostic kits «Opisthorchis-IgM ELISA», «Opisthorchis-IgG ELISA» and «Opisthorchis-CIC ELISA» but there is no information about the nature of antibodies and gelminth’s antigen of these tests and efficiency of them in diagnostic practice. The aim of our research was to develop national EIT for serological express diagnostics of Opisthorchiasis based on the using Opisthorchis felineus metabolism antigens and Mab specific to them in «sandwich» ELISA.

Materials and methods

In this paper Mab to ES-Ag of Opisthorchis felineus produced by the mice hybrid cultured cells strain 4B3 were used. Mab of this strain belong to the class IgМ, have specificity to protein fraction of ES-Ag Opisthorchis felineus with molecular mass of about 28 -30 kDa. Affinity of antibodies is equal to 6x10-5M. Mab titer in ascites is 1:6400 at concentration of immunoglobulins 2-4 mg/ml.

The fish of the family Cyprinidae (lin, bream, , bream) were caught from Lake Tengiz and Irtysh River in small batches. This allowed us to more carefully examine each specimen for the presence of the parasite and to use only fresh fish for feeding laboratory animals. In order to study specimens weighing not less than 200-300 g were selected. For detection of parasites compressor method was used Metacercariae were isolated from the superficial as well as deep spinal muscles and then dogs were infected with helminth.

In order to infect 4 dogs 3 months of age were selected by excepting parasite carriers with the help of scatological investigations for the presence of trematode eggs. Animals were infected by arbitrary feeding fishes affected with metacercariae and killed at 3 months after infection. After one month following infection scatological study was periodically carried out for detection of helminth eggs inanimal’s feces.

ES-Ag of Opisthorchis felineus was prepared by the method of А.Т.Kotelkin et al. (1997) [6] with minor modifications. For this live maritas were taken from the bile ducts of experimentally infected dogs and after washing with saline, cultured in incomplete Eagle medium containing antibiotics at 37 ° C (5% CO2) for 24 hours. After that, they are transferred to mattress with fresh Eagle medium containing antibiotics and incubated for an additional 72 hours. The resulting culture medium was purified by centrifugation and filtration through membrane filters (0.45 um, Millipore) and used as ES-Ag.

Antigenicity of ES-Ag was evaluated in indirect ELISA by standard method. Samples of sera from two patients with an established diagnosis of opisthorchiasis before the start of treatment and blood sera of eight healthy subjects as controls were used to assess the antigenicity of ES-Ag. In developing the terms of EIT for the serological diagnosis of Opisthorchiasis different concentration of Mab, blocking agent - Bovine Serum Albumin (BSA), ES-Ag, dilutions of sera samples obtained from people suspected of Opisthorchiasis and healthy ones were tested. The latter were kindly submitted by infectious hospitals of Astana and Pavlodar cities. Phosphate-Buffered Saline(PBS) and Carbonate-Bicarbonate Solution (CBS) were used as the buffers for immobilization of Mab to solid phase and dilution of ES-Ag, samples of sera and Anti-Human lgG conjugate. In addition, the optimal conditions (time and temperature) required for maximum immobilization of Mab to solid-phase are determined.

Results

During the inspection of fishes for the presence of parasite we have failed to reveal larval Opisthorchis felineus in samples taken from lin, bream and carp. Metacercariae were found only in muscle tissue of ides (Table 1)

Table 1. Results of investigation of fishes

Species of Number of Number of Degree of fishes the investigated fishes, affected fishes, infestation,% pieces pieces

Lin 26 0 0

Ide 221 57 26

Bream 55 0 0

Carp 12 0 0

As is evident from Table 1, only ides were exposed to infection with the larval stage of Opisthorchis felineus. In this regard, fishes of this species were taken for further investigations. The average intensity of infection of examined specimens was equal to 8.3.

At investigations of bile ducts of each dog from 30 up to 500 adult helminths relating to the species Opisthorchis felineus were found. They were collected in Petri dishes with sterile saline, washed of blood, and then defined their activity and species under the microscope at a magnification 20 x 10.

The results of the study of antigenicity of ES-Ag have shown that it comes into specific reaction with the antibody-containing serum of two patients with opisthorchiasis. For example, if the optical density of the liquid in the wells with blood sera of sick people were 2.468 and 0.977, but the figures in the wells with the negative controls were much lower - 0, 157. The antigenicity of the isolated preparation in relation to serum antibodies was determined in five replicates.

For working out of EIT protocol for detecting ES-Ag Mab, specific to mentioned antigen, were immobilized to solid-phase of 96-well polystyrene microtiter plate. Mab were immobilized into the wells in PBS, pH 7,2-7.4 or in CBS, pH 9.6, in the following concentrations: 15 mkg /ml, 10 mkg /ml, 5 mkg /ml and 1 mkg /ml. Immobilization of Mab to solid phase was carried out at 37 ° C for 1 hour or leaving overnight at 4°C. The excess of Mab was washed away by PBS or CBS. ES-Ag was introduced into the wells for 1 hour in solutions of the corresponding buffers. Samples of blood serum of healthy individuals and patients with Opisthorchiasis in the titers of 1:50, 1:100, 1:200, 1:400 in PBS with the addition of Tween-20 (PBS-Tw) was used. After incubation of plates at 37 ° C for 1.5 h washing procedure was repeated by the respective buffers, and anti- human lgG conjugate was applied to the wells for 1 hour. After the final wash step, reaction was developed by adding an enzymatic substrate - tetramethylbenzidine for 10-15 minutes at room temperature to produce a visible signal, which indicates the quantity of antigen in the sample. Cleavage of the substrate was stopped by adding solution of 0.5M sulfuric acid into the wells. The EIT results were detected by spectrophotometer with a vertical flow of light (ASYS Expert 96, Austria) at a wavelength of 450 nm.

To determine the optimal parameters of EIT for detecting CIC the same algorithm was defined with the only difference that in this version immune complexes in blood serum fixed to the solid phase by Mab were acting as antigen. In both cases, the optimal conditions for EIT when using ES-Ag and CIC of Opisthorchis felineus were defined in five replicates.

Below are the final results by definition of conditions that allow to get more precise data by proposed EIT (Table 2). Table 2. Optimal parameters of detecting ES-Ag and CIC in EIT

Parameters

Reagents concentration buffers exposure primary Mab 10 mkg / ml CBS , рН 9,5 12 hours at 4 ° C

BSA 0,01 mkg / ml PBS, рН 7,4 1 hour at 37 ° C

ES-Ag 5 mkg / ml PBS, рН 7,4 1 hour at 37 ° C

Serum blood 1:50 PBS -Тw, рН 7,4 1 hour at 37 ° C

Serum blood of 1:100 PBS -Тw, рН 7,4 1 hour at 37 ° C finding CIC

Anti-Human lgG. 1:5000 PBS -Тw, рН 7,4 1 hour at 37 ° C conjugate

EIT based on investigation of human blood sera for antibodies to ES-Ag of Opisthorchis felineus and specific CIC was validated in comparison with commercial tests of JSC "Vector-Best," (Novosibirsk). Sera from 141 people who suspected of infecting with Opisthorchiasis were subjected to serological analysis (Table 3).

Table 3 - Results of comparative studies of sera samples by ELISA for the diagnosis of Opisthorchiasis

Total of Opisthorch- Opisthorch Opisthorch samples IgМ IgG CIC EIT (IgG) EIT (CIC) ELISA ELISA ELISA

Number of positive samples

141 0 4 (2,83%) 4 (2,83%) 5 (3,54%) 4 (2,83%) (100%)

As the Table 3 shows, Test-set «Opisthorch-IgM ELISA» in neither case did not show positive result. «Opisthorchis-IgG ELISA» revealed the presence of the antibodies to the ES-Ag in blood sera of 4, or 2.83%, of patients. It should be noted that the «sandwich» ELISA confirmed all positive results of «Opisthorchis- IgG ELISA» and additionally revealed one more person with specific antibodies.

It is known that in the later stages of Opisthorchiasis active centers of antibodies occupied by parasite antigens. That is why such antibodies are not detected in serological tests. Such immune complexes can be determined by other antibodies specific to free epitopes of antigen. Therefore, in the development of EIT for the diagnosis of Opisthorchiasis Mab were also used as specific ligands for binding to the solid-phase CIC, consisting from antibody and antigen molecules. It is interesting to underline that in either case antibodies to both substrates - serum IgG and CIC were not simultaneously detected (Table 4).

Table 4 - Indicators of of the optical density of samples blood sera by ELISA

Оrdinal Opisthorch- Opisthorch- Opisthorch- № IgМ IgG CIC EIT (CIC) EIT (IgG) ELISA ELISA ELISA

1 0.105 0.163 0.017 0.044 0.370

±0,003 ±0,005 ±0,005 ±0,001 ±0,01

2 0.012 0.030 0.339 0.367 0.166

±0,004 ±0,001 ±0,01 ±0,01

3 0.021 0.084 0.279 0.299 0.119

±0,007 ±0,003 ±0,009 ±0,009 ±0,003

4 0.014 0.088 0.139 0.246 0.186

±0,004 ±0,003 ±0,004 ±0,009 ±0,006

5 0.011 0.012 0.362 0.299 0.129

±0,003 ±0,004 ±0,012 ±0,009 ±0,003

6 0.148 1.249 0.031 0.121 1.269

±0,006 ±0,04 ±0,001 ±0,004 ±0,04

7 0.046 0.011 0.113 0.031 0.103 ±0,004 ±0,003 ±0,003 ±0,001 ±0,003

8 0.079 0.059 0.014 0.092 0.123

±0,002 ±0,002 ±0,004 ±0,003 ±0,004

9 0.013 0.007 0.037 0.177 0.159

±0,004 ±0 ±0,001 ±0,005 ±0,005

10 0.020 0.025 0.009 0.102 0.096

±0,006 ±0,008 ±0 ±0,003 ±0,003

11 0.034 0.013 0.033 0.143 0.114

±0,001 ±0,004 ±0,001 ±0,004 ±0,003

12 0.011 0.003 0.029 0.140 0.171

±0,003 ±0 ±0,006 ±0,004 ±0,003

13 0.047 0.018 0.024 0.054 0.195

±0,001 ±0,006 ±0,008 ±0,001 ±0,006

14 0.030 0.977 0.022 0.153 1.241

±0,001 ±0,003 ±0,007 ±0,003 ±0,04

15 0.025 0.016 0.022 0.008 0.102

±0,008 ±0,005 ±0,007 ±0 ±0,003

16 0.009 0.004 0.041 0.026 0.198

±0 ±0 ±0,001 ±0,008 ±0,006

17 0.022 0.018 0.015 0.032 0.076

±0,006 ±0,006 ±0,005 ±0,001 ±0,002

18 0.015 0.008 0.052 0.057 0.100

±0,006 ±0 ±0,002 ±0,001 ±0,003

19 0.017 0.007 0.018 0.034 0.057

±0,005 ±0,001 ±0,006 ±0,001 ±0,001 20 0.020 2.468 0.027 0.102 1.294

±0,006 ±0,08 ±0,009 ±0,003 ±0,04

21 0.009 1.248 0.047 0.022 0.803

±0,001 ±0,04 ±0,001 ±0,006 ±0,03

Analysis of the table 4 data testifies that the average optical density of the sera showing positive results in Тest «Opisthorchis-IgG ELISA» were an average of 1.485±0.020, whereas these values in our EIT were slightly lower – 0.995±0.030. Moreover, the negative sera had a low background of optical density in the investigation by foreign analogue (0.03 ±0.002) as compared with proposed EIT (0.130±0.001).

Average value of the optical density of positive and negative samples when testing sera for the presence of CIC by commercial test Opisthorch-CIC-ELISA were 0.279 ± 0.003 and 0.032 ± 0.006, respectively. Similar indicators amounted to 0.302 ± 0.001 and 0.008 ± 0.001, respectively, were also obtained in the case of using of EIT. Thus, EIT gives comparable evidence for a sufficiently degree of its specificity in diagnosis of Opisthorchiasis.

Discussion

Mab, produced by the mice hybrid cultured cells strain 4B3 can be used in EIT as ligand for binding to the solid phase not only ES-Ag Opisthorchis felineus, but the CIC in the blood of people with Opisthorchiasis.

Availability and specificity of the antibody-ligand making this test more attractive for diagnostic practice. However, the weakness of EIT is the difficulty in obtaining ES-Ag (catching fish, definition of affected individuals, infection of dogs with their subsequent killing and extracting mature parasite from organs, culturing helminth, stockpiling and purification of specific antigen), and in monitoring the invasive process. Thus, in our experiments, we have never managed to find helminth eggs in the feces of infected dogs, although, as it shown by the results of the autopsy, all of them were infected with the parasite. In this regard, there is a need in replacing ES-Ag more accessible antigen.

From this viewpoint, the research aimed at obtaining anti-idiotypic antibodies will have great practical importance. Such antibodies can be used as original antigen. The advantage of this approach is the availability of standard source of antigen in the form of Mab producer-strain, elimination of the need of antigen purification and subsequent stockpiling it in large quantities.

References

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