ApplicAtion of fluorescein copolymer to improve the efficiency of counter-immunoelectrophoresis for diAgnostics of AnimAl infectious diseAses

The counter-immunoelectrophoresis (CIe) is a highly specific, simple and rapid method, which does not require expensive equipment, input materials and long-term staff training as compared with other ex press methods of viral infections diagnostic. Despite the CIe high informativeness, the results in practice can be doubtful at times. Therefore, the purpose of the study was to improve CIe method by strengthening the antigen-antibody interaction with the use of synthesized amphiphilic fluorescein-containing copolymer. It was demonstrated that the use of the fluorescein copolymer -antigen complex enhanced the interaction with serum/ plasma antibodies of rabbits immunized against hemorrhagic disease and increased protein content in the area of precipitation. This effect was more pronounced when more hydrophobic copolymer containing 5.87% fluorescein was introduced into the complex with antigen. The ability of the amphiphilic fluorescein copoly mer to enhance the antigen-antibody interaction and the ability to visualize the fluorescent precipitation zone may increase CIe

he concentration of a large number of animals in a confined space due to industrial techno logies for animal housing causes problems of rapid development and spread of various infectious diseases of both bacterial and viral origin. In case of infectious diseases it is important to conduct urgent rapid diagnostics with the aim of establishing the pathogen, and immediate development of effective measures of infection elimination and prevention.
The counter-immunoelectrophoresis (CIE), immunofluorescence (IF), solidphase hemadsorption (SPHA) and others tests are used for rapid diagnostics of viral infections. Among these methods, the CIE does not require expensive equipment and longterm staff training; it is simple and rapid to perform [1,2]. CIE is based on the precipitation reaction for the detection of specific antibodies against the pathogen. It is known that precipitating antibodies, most of which belong to class G immunoglobulins (IgG), under certain conditions of electrophoresis in an electric field move to the cathode. The antigen has a positive charge and moves to the anode. Since the doi: https://doi.org/10.15407/ubj93.01.104 antigen and antibody move in opposite directions, due to the electric field, their migration occurs towards each other. Visible precipitates appear after 30-60 minutes from the beginning of CIE [3].
This assay is used for the diagnostics of infectious diseases in veterinary and human medicine [4][5][6][7]. In particular, in the diagnosis of Aleutian mink disease of viral origin, CIE is characterized by high specificity and allows the detection of the disease on 6-15 days after infection. After experimental infection of animals, the disease is detected on 6-7 days when CIE is used [1,8]. For suckling puppies, the reaction becomes positive within the first hour after suckling the sick females' milk, which has maternal antibodies. The diagnostic efficiency of Aleutian mink disease is 98-100%. CIE assay is also highly informative for the diagnosis of coronavirus infection for cattle [9].
Sometimes, the results of CIE can be doubtful. In such cases, for antigen-antibody precipitate detection, the agar gel is immersed in 12.5% NaCl solution for 7-10 min or stained (1% Amido black 10-B or 1% tannin solution), and then immunoelectrophoregrams are washed. If the result is questionable, the study should be repeated. These additional procedures delay the diagnosis of the disease, which is undesirable because of the rapid development of viral infection.
Our previous studies [10,11] showed that one of the ways to increase the antigen-antibody interaction during immunoelectrophoresis is the appliance of an amphiphilic fluoresceincontaining copolymer. This copolymer is able to enhance protein molecule binding and facilitate visualization due to visible fluorescence. During the study of an interaction between the obtained polymers and albumin, their ability to bind up to 70% of blood proteins was established [12][13][14].
The aim of the research was to improve the specificity of the CIE reaction due to the synthesized amphiphilic fluoresceincontaining copolymer by enhancing the antigen + antibody interaction, which can be used for rapid diagnosis of infectious diseases.

materials and methods
Fluorescein copolymers obtained by Steglich reaction were used for the studies [13].
antigens. For immunization of rabbits were used the antigens of two strains: BG04 ≥ 640 hemagglutinating units and GBK2 of ≥ 640 hemag-glutinating units. These strains are the basis of the inactivated vaccine against hemorrhagic disease of rabbits (Lapimun GEM-2). The pathogen of rabbit hemorrhagic disease is a virus that contains RNA and possess a very high virulence. The vaccine administration protects rabbits against hemorrhagic disease for 12 months, and protective antibodies are produced on 4-14 days after vaccination [15].
animals and immunization procedure. All animals were maintained in a specific pathogenfree animal facility with water and commercial food provided ad libitum. The protocol for animal experiments was approved by the Ethical Committee of the Institute of Animal Biology of NAAS of Ukraine and the experiments were carried out in accordance with the European Convention for the Protection of Vertebrate Animals (Strasbourg, 1986). 1-month-old rabbits of the Termonic White breed (n = 9) were selected for the experiments. To obtain specific antibodies, the rabbits were immunized with the Lapimun GEM-2 vaccine, which was administered subcutaneously under shoulder-blade. Dosing was based on manufacturer's recommendation -1.0 ml per head. The experiments were conducted in two stages: for 21 days (n = 6) and, repeatedly for 28 days (n = 3). Before vaccination (0 days) and after immunization on 14, 21 and 28 days a blood samples were taken into heparin tubes from the marginal auricual vein of rabbits.
preparation of agar gel. To carry out immunoelectrophoresis, a 1% agar solution was prepared on 1:1 diluted trisglycine buffer (pH 8.8). The heated agar solution (temperature ~ 60°C) was poured by 20-25 ml onto glass plates (6×11 cm) placed on a table to prepare gel samples for horizontal electrophoresis. After the agar was solidified (after 15-20 min), two parallel rows of 10-15 μl holes were produced by the punch, with a distance of 1 cm between the rows.
Sample processing. Antigen samples were prepared as follows: 5 μl of 1.0% solutions of fluorescein copolymer in two modifications (MF37 and MF47) were added to 1 ml of vaccine against hemorrhagic rabbit disease (Lapimun GEM-2). The samples of amphiphilic fluoresceincontaining copolymer differed in the fluorescein content.
10 μl of antigen (Lapimun GEM2 vaccine) were introduced into the holes situated closer to the cathode: control one (without adding fluorescein copolymer; sample No. 1) and two experimental ones with the addition of MF37 (sample No. 2) and MF 47 (sample No. 3). Each blood sample was divided into 3 parts and 10 μl of blood plasma were introduced into the holes closer to the anode.
Counter-immunoelectrophoresis. CIE was performed in a BIO-RAD chamber (200 V; 10 mA) for 25-30 min. The movement of fluoricein and the appearance of precipitation zones were visually controlled. After immunoelectrophoresis, the agar gel plates were covered with a transparent film and the results of the studies were recorded by direct scanning of the immunoelectrophoregrams (HP Scanjet G2410, United Kingdom). Protein content in the precipitation zones was determined using TotalLab TL120 software.
Statistical analysis. Statistical calculations of results (М ± m) were performed using the Microsoft Excel 2007. The probability of differences was determined by the Student's t-test with p < 0.05 accepted as statistically significant.

results and discussion
The appearance of new strains of viruses predetermines both the improvement of existing and the search for new rapid diagnostic tests for infectious diseases. To improve the effectiveness of CEI for rapid diagnostics of viral diseases, we combined a precipitation reaction (precipitation of a certain antigen from a colloidal solution under the influence of serum specific antibodies with the formation of the antigen + antibody complex and sedimentation) with fluorescence using new fluorescein copolyesters. A generalized scheme of the elemental unit structure of fluoresceincontaining copolyesters is shown in Fig. 1.
Polymeric products based on lipophilic 2-(octadecanoylamino) pentanedioic acid, hydrophilic polyoxyethylene diol with a molecular weight of 1000 or 1500, dipropylene glycol and fluorescein were obtained according to the procedure and their characteristics are shown in Table 1.
The synthesized copolyesters exhibit amphiphilic properties and are capable of forming self-stabilized dispersed media in water [17]. Moreover, they contain a dye (fluorescein) capable of fluorescence at pH 7 and higher pH. Due to this, the new copolyesters are used as fluorogenic markers to obtain images from the biological objects environment [18].
After immunoelectrophoresis, the precipitation zones between the antigen (inactivated vaccine viruses) and serum antibodies were formed on the gel plates. The zones were formed in response to the introduced vaccine and characterize the reaction-response of the immune system of the rabbit organism (Fig. 2).
Precipitation zones and, respectively, the formation of antibodies on the 14 th day of research are clearly observed. At the same time, before the vaccination, the protein content of the antigen + antibody precipitation zones (both without and with the use of the fluorescein copolymer in the antigen composition) was in the range of 31.3-36.1% (Fig. 3, Table 2).
On the 14 th day after vaccination, the antigenantibody complex content in the precipitation zone changed: it was by 15.1-23.6% (p < 0.01-0.001) higher in CIE with complex between fluorescein copolymer and the antigen than in CIE without polymer. The established differences were maintained during the study of the content of complexes between antigen and antibody after 21 days. The difference between the use of a fluorescein copolymer in the complex with the antigen, compared to pure antigen, was smaller (5.8-8.7%), but remained statistically significant (p < 0.05-0.01).
It should be noted that for some animals the antigen-antibody precipitation zones were detected before vaccination. Apparently, their presence was caused by passive immunity provided by immunized female rabbits. Thus, the presence of antibodies against hemorrhagic disease agents in the blood of young rabbits (up to 1 month) can indirectly charac-   terize the immunity state of female rabbits from which they are originated. Repeated studies of the antigen-antibody complex content in the fluorescein copolymer complex confirm that fluorescein copolymer addition to the antigen makes the appearance of precipitation zones more intense (Figs. 4, 5).
Particularly, before vaccination, the concentration of the antigen-antibody complex in precipitation zones (without and with the use of the fluorescein copolymer in the antigen composition) was in the range of 31.3-36.2% (Table 3).
After 14 days of vaccination the highest protein content in the precipitation zone (41.1 ± 4.86%) was observed for the samples using the fluorescein copolymer MF37; the lower value (32.5 ± 4.15%) was observed using the polymer MF47 and the lowest one (6.1-14.7%, p < 0.05) was determined for the samples without polymers. The established relation between the samples with and without fluorescein copolymer was also preserved after 21 and 28 days of imminization (9.2-16.7%, p < 0.05) and 1.7-2.7%, respectively.

. The dynamics of complex formation between antigen (with fluorescein copolymer) and antibody after 28 days from the immunization of young rabbits against hemorrhagic disease, (M ± m)
Note. Data were compared to the control. The difference is judged to be statistically significant when: *p < 0.05; **p < 0.01; ***p < 0.001

Experimental conditions, days
The ratio between the content of antigen-antibody complexes in the samples, % Control (without polymer), n = 6 Polymer МF37, n = 3 Polymer МF47, n = 3 0 (before vaccination) 13 The efficacy of using fluorescein copolymer to improve the specificity of the antigenantibody binding reaction during CEI was confirmed by changes in the formation of antigen-antibody complexes for 28 days (Table 4).
Hence, the percentage content of antigen-antibody complexes increased from 11.9-13.1% (before vaccination) to 25.2-30.9% (p < 0.05-0.001) on 28 th day after vaccination. Moreover, in each period of study, the difference between the values of protein content in the precipitation zone without and with the use of the fluorescein copolymer was within the error of the arithmetic mean (0.3-4.9%). The experimental results indicate the formation of antibodies in the animal body in response to the introduced pathogen and the suitability of the fluorescein copolymer for diagnostic studies. At the same time, a strong correlation (η = 0.983) between the increase of protein content in the precipitate zone and the day of study characterizes the advantage of the fluorescein copolymer MF37 for diagnostic study. This copolymer enhances the antigen + antibody interaction for detection of specific antibodies (or pathogene(s) if specific antibodies are present) during the ascertainment of the strength of the immune response and infectious disease diagnostic by CEI.