RU2347225C2 - Method for evaluating phagoimmunoassay results - Google Patents

Abstract

FIELD: chemistry; biochemistry.

SUBSTANCE: invention relates to biotechnology, in particular, to immunoengineering, and may be used in digital processing of images of lysed zones on bacterial lawn, which result from antigen-antibody reaction, based on phage label principle (phagoimmunoassay or viroimmunoassay). Phagoimmunoassay results evaluation method implies analysis of lysed zones of indicator crop in plate wells with experimental and reference tests. Plate with experimental and reference tests is subsequently placed into photo or scanning unit to obtain digitised image of lysed zones. Then, each digitised image is processed by means of adaptive filter to obtain outlined individual lysed zones of various size, as well as of merged lysed zones. After that, average total area of lysed zones for experimental and reference samples is determined. When average total area of experimental lysed zones exceeds average total area of reference lysed zones by 5% minimum, phagoimmunoassay is considered positive.

EFFECT: efficient method for evaluating phagoimmunotest results.

4 tbl, 5 dwg, 1 ex

Description

The invention relates to biotechnology, in particular to immunobiotechnology, and can be used for digital processing of images of lysis zones on an indicator bacterial lawn, which are formed when the antigen-antibody reaction is based on the principle of phage label (phageimmunotest or viroimmunotest).

In the 60s of the last century, when developing new immunochemical methods, along with isotopic and enzyme labels, bacteriophages were proposed as labels [1-4], which are detected by the degree of lysis of the indicator bacterial culture.

1. The method of neutralizing the bacteriophage. The first immunological methods using bacteriophages were based on the phage neutralization reaction and were used to study the immune response [5, 6]. Sera obtained against phage have a pronounced neutralizing activity. When serum is added in different dilutions to the same amount of bacteriophage and its subsequent subculture on the microculture of bacteria sensitive to it, its biological activity decreases. It was estimated that 7-8 molecules are theoretically sufficient to neutralize one phage particle with an optimal antibody landing on the surface of phage φX174. However, when conducting specific experiments, it turned out that 1000 times more molecules of antibodies are needed [7]. The phage neutralization reaction is usually taken into account by the dilution of the antiserum, which is 50% inactivated by the bacteriophage taken in the experiment. The sensitivity of the reaction can be increased by adding a second antibody against the immunoglobulin determinants of antibodies that have joined the phage [5, 6].

2. The method of inhibition of neutralization of phage. Along with determining the number of antibodies, the method of neutralizing the bacteriophage also allowed to determine the number of haptens themselves. This was achieved by inhibiting the neutralization reaction of the phage conjugate with the addition of a specific antigen (hapten). To carry out the neutralization inhibition reaction, the amount of antibodies capable of inactivating 90-95% of the conjugate in the sample was first selected. Further, the amount of antibodies selected in this way was mixed with an unknown amount of the antigen to be determined and the optimal time was incubated together. Antigen, depending on its amount, completely or partially blocks the activity of antibodies. As a result, the neutralizing ability of antibodies will be inhibited to a varying degree, which allows you to quantify the concentration of antigen. Based on the data obtained using known amounts of antigen, a quantitative curve was constructed that reflected the dependence: the more antigen in the sample, the lower the percentage of reaction with the conjugate. Subsequently, the principles of the phage neutralization method were extended to the determination of high molecular weight antigens and antibodies against them. Using the phage hapten model, the affinity of antibodies of different classes, as well as their (Fab ') 2 and Fab' fragments, was studied.

3. Conjugates of phage with antigen. Bifunctional reagents (glutaraldehyde, bis-benzidine, carbodiimide, diisocyanates, etc.) were used to conjugate protein antigens with phage. In the synthesis of conjugates, significant difficulties arose. In the modification of phages, it was necessary to obtain intensely labeled preparations of bacteriophages with preservation of their viability. It was necessary for each protein antigen, depending on its molecular weight, to select such conditions so that the attached antigen does not block the phage sterically, but at the same time its quantity was sufficient to inactivate the phage conjugate with antibodies. The method was highly sensitive both in the neutralization reaction of the bacteriophage (10-500 pg / ml) and in the reaction of inhibition of neutralization (25-50 pg / ml), but it was too complicated [6, 8].

4. Immunosorbent variant of fagoimmunotest. Later, an immunosorbent variant of the fagoimmunotest was developed both for the determination of antibodies and antigens [2, 9]. The immunosorbent synthesized on different bases, due to its large capacity, allows to extract the test substance in small quantities from a complex mixture, concentrating it on its surface. The immunosorbent principle was used in the phage immunoassay to determine antibodies to phage by extracting phage-antibody complexes. It was shown that during the sorption of phage loaded with rabbit antibodies on the sorbent associated with antibodies against rabbit immunoglobulin, antiphage antibodies in the amount of picogram fractions can be detected [9]. A similar technique was used by Pestka et al. [10] to detect anti-hapten antibodies using phages conjugated with haptens. In this variant of the method, the sensitivity was several orders of magnitude higher than that of the phage neutralization method.

An immunosorbent variant of the fagoimmunotest was developed to determine diphtheria toxin [11]. In this case, purified antibodies were used to obtain the conjugate. Antibodies were pre-activated with glutaraldehyde and, after being released from its excess, were attached to the bacteriophage. Using the obtained conjugate, it was possible to determine the antigen at a concentration of 50 pkg / ml.

However, the above variant of the fagoimmunotest provided a method for producing a conjugate. Using glutaraldehyde, antibodies were fixed randomly on the bacteriophage, thereby creating steric barriers to interaction with the antigen, which reduced the sensitivity of the method. In addition, glutaraldehyde inactivates the biological activity of the bacteriophage.

Further Skvortsov V.T. [7] proposed a more effective variant of the immunosorbent phagoimmunotest for direct determination of antigen. This variant of the fagoimmunotest differed in the method for producing the conjugate. The bacteriophage ϕX174 was conjugated to Fab'-fragments of antibodies obtained by digesting antibodies with pepsin. For the synthesis of conjugates, the reagent N-acetyl-DL-homocysteine-γ-thiolactone was used. This made it possible to preliminarily introduce an active SH group into the bacteriophage capsid, which, as a result of activation, selectively reacted with the free thio group of Fab'-antibody fragments located at the opposite end from the active center. Thus, the active antibody center in the conjugate was optimally oriented for subsequent interaction with the antigen.

This principle has also been applied to the synthesis of high-capacity immunosorbent with orientedly attached Fab'-fragments of antibodies to obtain antigens. For the synthesis of conjugates, a radical containing a free sulfhydride group was attached to the bacteriophage, which was then converted to mixed disulfide using Elman's reagent (5,5-dithiobis (2-nitrobenzoic acid)). Fab'-fragments of antibodies added to the bacteriophage activated in this way were attached via a thiol disulfide exchange reaction. Using this variant of the immunosorbent phagoimmunotest, up to 3.0 pg / ml of antigen could be determined. For direct determination, the antigen in different dilutions was added to the samples of the immunosorbent and, after incubation, the sorbent was washed with buffered saline. Samples were transferred to clean tubes, conjugate was added and incubated. After incubation, the sorbent was washed from the non-conjugated conjugate, dithiothreitol was added, thereby destroying the disulfide bond between the bacteriophage and Fab'-fragments of antibodies, which led to the restoration of the biological activity of the phage. The amount of conjugate added was determined by reseeding the samples (after restoration of the biological activity of the bacteriophage) on a Petri dish with an indicator culture of microbes on a solid nutrient medium. Depending on the concentration of antigen in the sample on a plate with indicator culture, a certain number of lysis zones (plaques) appeared.

Despite the high sensitivity of the immunosorbent phagoimmunotest, the method was used only in laboratory studies, since the analysis required a large amount of work and laboratory glassware.

5. Screening version of the fagoimmunotest. The method of immunosorbent phagoimmunotest proved to be possible to simplify. A screening variant of the fagoimmunotest was developed using the conjugate of phage φX174 and Fab'-fragments of antibodies oriented to it. The immunosorbent analysis step was carried out in 96-well polystyrene plates, which greatly simplified the formulation of the method and increased the number of analyzed samples. The analysis scheme at the initial stages did not differ from the ELISA using the sandwich technique. This variant of the phageoimmunotest described by the authors of [12] was developed on a model experiment to determine rabbit IgG using polyclonal Fab'-fragments of antibodies and was not inferior in sensitivity to ELISA variants similar to the formulation scheme.

The above-described variant of the fagoimmunotest has an indisputable advantage in comparison with the previously proposed methods using bacteriophages as a label and, in terms of simplicity of analysis, came close to the widely used ELISA method. The advantages of this method (high sensitivity, the ability to test a large number of samples, a relatively short analysis time of 6 hours, and the use of a more accessible label - bacteriophages) served as the basis for the development and improvement of the method in terms of routine use in the clinic as a quick and effective method determination of α2 and γ interferons. As a result, an original screening variant of the phageoimmunotest for the analysis of interferon in biosubstrates was developed, including the synthesis of conjugates based on the bacteriophage φХП4 with covalently attached Fab'-fragments of monoclonal antibodies against α2- and γ-interferons [13]. The use of the phagoimmunotest for the analysis of interferons made it possible to detect α2 and γ interferons in solutions and in biological fluids at a concentration of up to 1.0-1.5 IU / ml for 5-6 hours. A comparative analysis of the determination of interferons using a highly sensitive biological test and phage immunoassay did not reveal a significant significant difference between these indicators (P <0.05) [13].

In general, antigenic and antibody-based diagnostics based on the principle of phage tag (phage immunotest or viroimmunotest) have been developed for:

- determination of the concentration of interferons α and γ;

- determination of the concentration of γ-globulins;

- detection of chlamydial LPS;

- identification of growth factor of nerve cells;

- detection of diphtheria toxin.

Obtaining a diagnostic result is based on a visual assessment by the operator of images of lysis zones. Below is a typical technique for the formulation of the phageoimmunotest (viroimmunotest) on the example of the diagnosis of chlamydia using "ChlamyViroskrin." The test system is a set of reagents for the detection of lipopolysaccharide antigen (LPS) of chlamydia viroimmunotest. For the reaction according to the known method, 0.05 ml of a solution of chlamydial monoclonal antibodies to LPS is added to each well of a 96-well plate. The plates are covered and incubated at 37 ° C. for 1 hour or at room temperature for 18 hours. Then, the contents of the wells are shaken out and washed with buffered saline with Tween-20 (PBS). 0.025 ml of the positive control are added to wells C6 and D6, 0.025 ml of the negative control to wells C7 and D7, and 0.025 ml of the studied sera to the remaining wells, each serum being examined in two wells. Then, 0.025 ml of conjugate solution (phage anti-LPS chlamydia) is added to all wells. The tablet is covered with a lid and incubated for 2 hours or overnight at 37 ° C. During incubation of the plate, nutrient agar is prepared. After incubation, the contents of the plate are shaken out and washed with PBS, after which 0.05 ml of reducing reagent is added to each well, the plate is closed with a lid and placed in a thermostat at 37 ° C for 30 minutes. During incubation of the tablet, indicator lawns are prepared. To do this, 2 polystyrene plates are treated with alcohol and placed on a horizontal table. In a vial with an indicator culture of E. coli, add 1 ml of PBS, mix and transfer the resulting suspension into a test tube with molten agar at a temperature of 42-48 ° C, the tube is immediately removed from the water bath, the contents are quickly mixed, rotating the tube between the palms of the hands, and immediately poured onto the plate, evenly distributing the agar over the entire area of the plate (using the same tube). Two indicator lawns are prepared in this way. Agar is allowed to freeze. Plates with indicator lawns should be prepared 5-15 minutes before the end of the incubation of the tablet, so that the agar could freeze, but did not have time to dry. Samples from each well of the plate are orientedly transferred onto the agar using a 96-channel applicator, the hollow tips of which are located respectively in the wells of the plate. The applicator is inserted into the tablet and by gently tapping on the side walls of the tablet to achieve uniform liquid suction in the tips. The applicator is carefully placed on the agar plate and slightly “stirred” without tearing off the surface of the agar, and then removed, lifting vertically, so as not to smear the “prints” - drops transferred onto the surface of the agar. The applicator is reinserted into the plate and samples are transferred to a second agar plate. The applicator is used repeatedly. After each analysis, the applicator is thoroughly washed with water and detergent, the detergent is washed off, rinsed with distilled water and treated with alcohol. The plates are covered with an unused plate or tablet lid pretreated with alcohol, fastened with pieces of adhesive tape or patch and incubated at 37 ° C for 3 hours. If during this time the drops are not absorbed into the agar, the plate is left for 5-10 minutes at room temperature without a lid, after which they evaluate the results. The results of the reaction are taken into account visually by the presence of transparent zones of bacterial lysis at the site of application of the samples. The reaction is considered positive if a continuous lysis zone or separate plaques (at least 6) are formed at the site of application of the sample. The number of plaques at the site of application of the negative control sample should not exceed 3.

However, in this method of recording the results of the reaction of the phageoimmunotest, the result can not always be expressed in an exact digital measurement. First of all, this concerns the situation in which there is a merger of the lysis zones on the indicator lawn.

The technical result of the invention is to increase the accuracy of image analysis of the reaction of phageoimmunotest in tablets, plates and Petri dishes, eliminating the subjectivity of image evaluation and introducing quantitative indicators for images of plaque-forming units and assessing the total area of lysis zones.

The technical result is achieved by the fact that in the method for evaluating the results of the phageoimmunotest, which involves the analysis of the lysis zones of the microbial indicator culture in the wells of the tablet with experimental and control tests, according to the invention, the tablet with experimental and control tests is sequentially placed in a photographic or scanning unit to obtain a digitalized image of the lysis zones , then each digitized image is processed using an adaptive filter to obtain contoured separate lysis zones of various Dimensions as well as the fused zones lysis, then determine the average total area of the zones of lysis for test and control samples, and if it exceeds the average of the total area of lysis zones in an experiment on the magnitude of the average of the total area of the lysis zone in the control by at least 5% fagoimmunotest considered positive.

In addition, the evaluation of the results of the fagoimmunotest is carried out automatically.

The essence of the method. In Petri dishes, culture plates and plates containing various media, initial processing of the images is carried out with the reaction of the phageoimmunotest. Initial processing includes: digitizing the image of the entire tablet (plate or Petri dish) using a scanner or camera, which provides images of the reactions of the phage immunotest using a slide module or camera, fixing the basic coordinates on the digitized image of the tablet (plate or Petri dish), cutting images of the tablet (plate or Petri dish) into separate images and their subsequent storage. The resulting individual images are recycled. To obtain images from tablets, plates or Petri dishes, specially selected scanning or photographing modes are used with changing brightness and contrast parameters, as well as color. Processing digitized images using an adaptive filter allows you to get a clear outline of plaque-forming units (PFU) of various sizes, as well as merged PFU (see figure 1). The contouring of PFU, both single and merged, allows us to differentiate the lysis zone from the indicator lawn and as a result to determine the total lysis area included in the selected zones (see figure 2). The total allocation of lysis areas allows a quantitative assessment of the total number of phages in this test sample, i.e. objectively and automatically take into account the results of the phagoimmunotest.

The invention is illustrated by drawings.

Figure 1 shows an example of a 96-well plate with the formulation of the phagoimmunotest.

Figure 2 shows the allocation of lysis zones of E. coli With phage φX174 by contouring PFU.

Figure 3 shows an example of determining the area of digitized and contoured E. coli lysis zones with a phage φXP4 using a specialized computer program, indicating individual lysis zones and groups of merged lysis zones.

Figure 4 shows an example of evaluating the result of a fagoimmunotest based on a certain area of the experimental and control wells having different numbers of lysis zones.

Example.

After staging the reactions in the wells of the plate (in a Petri dish or on a plate), as described above, in accordance with the instructions for use of the diagnosticum, the plate with the tests is placed in a specially modified scanner or photographic unit, which enables digitalization of images. Using the developed specialized computer program, scanning is performed and, in the case of setting the reaction on the plate, the digitized image is cut into separate “holes”. Next, the program processes each individual digitized image using an adaptive filter, resulting in contoured separate lysis zones of various sizes, as well as merged lysis zones. The program recognizes the types of lysis zones and distributes them into groups, then it calculates the average total area of the lysis zones in a particular image. Further, based on the experimental design, the correspondence of the obtained values of the area of lysis zones for control and experimental samples is determined. Evaluation of the results of the phageoimmunoassay is carried out by determining the ratio of the average total area of lysis zones in experimental samples to the average total area of lysis zones in control samples. Exceeding the average total lysis zone in the experiment over that in the control is considered a positive result; in this case, a specific threshold figure is determined separately for each version of the test system, however, it should differ from the control by at least 5%.

The results of the analysis are shown in tables 1-5.

Table 2.
Automated Recording of the Results of the Fagoimmunotest: Total Area of Lysis Areas LPS, ng / ml The total area of lysis zones in the dilution of the reaction. mixtures Ref. 1/2 1/4 5 770 1110 1080 700 710 780 470 790 760 570 150 220 0.156 430 460 830 810 500 570 620 660 540 180 440 300 0 40 0 0 0 70 0 40 10 0 0 7 6

Table 3.
Automated Recording of the Results of the Fagoimmunotest: Area of Lysis Areas by Group LPS, ng / ml The area of the lysis zones (in groups) when diluting the reaction. mixtures Ref. 1/2 1/4 5 620 880 880 520 640 520 310 540 600 340 0 0 0.156 250 140 670 700 150 200 560 250 380 120 300 180 0 0 0 0 0 0 0 0 0 0 0 0 0

Table 4.
Statistical processing data for automatic recording of the results of the phageoimmunotest for immunodetection of F.tularensis LPS, ng / ml Total average area ± Stand. off The average area in groups ± Stand. off Ref. 1/2 ¼ Ref. 1/2 1/4 5 915 ± 210 688 ± 149 425 ± 289 725 ± 184 503 ± 139 235 ± 291 0.156 555 ± 186 588 ± 69 365 ± 158 440 ± 287 290 ± 185 245 ± 117 0 10 ± 20 30 ± 32 3 ± 4 0 0 0

Table 5.
Evaluation of the fagoimmunotest for immunodetection of F.tularensis on a 96-well plate according to the formula P = (Experience / Control) · 100%; threshold value of 105%. LPS, ng / ml Ref. 1/2 1/4 Value Output Value Output Value Output 5 9150 Pol., P <0.01 2293 Pol., P <0.01 14167 Pol., P <0.01 0.156 5550 Pol., P <0.01 1960 Pol., P <0.01 12167 Pol., P <0.01

Calculation Parameters:

1. The main calculation parameter, which determines the value of the phageoimmunotest reaction and the difference between experiment and control, as well as between individual test samples, is the total area of lysis zones during dilution of the reaction mixture (see Table 2 and Table 4). The lysis area by groups is an additional additional parameter that allows us to estimate the magnitude of the reaction at the moment when individual lysis zones begin to merge: the more the lysis zones merged and their area, the stronger the reaction.

2. Statistical processing of the data obtained in the above experiment illustrates the change in the intensity of the reaction of the phageoimmunotest with a change in antigen concentration (LPS F.tularensis), and also indicates a significant difference between the experimental and control samples.

References

1. Skvortsov V.T., Suslov A.P. Viroimmunotest for mass determination of nanogram amounts of antigen. // Bull. exp biol. honey. - 1988. - No. 12. - S.704-706.

2. Haimovich J., Sela M. Protein-bacteriophage conjugates: Application in detection of antibodies and antigens. // Science. - 1969. - V.164. - P.1279-1280.

3. Haimovich J., Sela M. Inactivation of poly-DL-alanyl poly-DL-alaT4 with antisera spesific toward poly-DL-alamine. // J. Immunol. - 1966. - V.97, No. 3. - P.338-343.

4. Macela O. Assay of anti-hapten antibody with the aid of hapten-coupled bacteriophage. // Immunology. - 1966. - V.10, No. 1-2. - P.81-86.

5. Haimovich J., Hurwitz E., Novic N., Sela M. Preparation of protein-bacteriophage conjugates and their use in detection of anti-protein antibodies. // Biochem. Biophis Acta. - 1970. - V.207. - P.115-124.

6. Haimovich J., Hurwitz E., Novic N., et al. Use of protein-bacteriophage conjugates in quantitation of proteins. // Biochem. Biophis Acta. - 1970. - V.207. - P.125-129.

7. Skvortsov V.T. Determination of ultramicroquantities of antibodies and antigens using viroimmunotest. In: Immunosorbents and their use in biology. - M .: - 1986. - S. 49-60.

8. Andreev J.M., Mamas S., Dray F. Viroimmunoassay of prostaglandin F2athe picogramm level. // Prostaglandins. - 1974. - V.6. - P.15-22.

9. Skvortsov V.T., Bondarenko V.M. Determination of ultramicroquantities of antibodies to a bacteriophage based on the specific extraction of phage-antibody complexes using immunosorbents. // Q. virusol. - 1971. - No. 4. - S. 603-605.

10. Pestca S., Rosenfeld H., Harris R. Communication to the editor viroimmunoassays: detection of virus-antigen, antibody complexes with an immunoadsorbent. // Immunochemistry. - 1974. - V.I 1. - P.123-218.

11. Skvortsov V.T., Vertiev Yu.V. Using Viroimmunotest for the quantification of diphtheria toxin. // Lab. a business. - 1981. - No. 4. - S. 437-439.

12. Skvortsov V.T., Suslov A.P. Viroimmunotest for mass determination of nanogram amounts of antigen. // Bull. exp biol. honey. - 1988. - No. 13. - S.704-706.

13. Skvortsov V.T., Bologa V.F., Denisov L.A., Malinovskaya VV, Vetchinin S.S., Grechko G.K., Khlebnikov V.S., Miller G.G., Suslov A.P. Screening viroimmunotest for determination of human α2- and γ-interferon in biological fluids. // Immunology. - 1995. - No. 2. - S.26-28.

Claims (1)

A method for evaluating the results of the phageoimmunotest, which includes analyzing the lysis zones of the microbial indicator culture in the wells of the tablet with experimental and control tests, characterized in that the tablet with experimental and control tests is sequentially placed in a photographic or scanning unit to obtain a digitalized image of the lysis zones, then each digitized image is processed using an adaptive filter to obtain contoured separate lysis zones of various sizes, as well as merged lysis zones, then op They determine the average total area of lysis zones for experimental and control samples, and when the average total area of lysis zones in the experiment exceeds the value of the average total area of lysis zones in the control by at least 5%, the phage immunoassay is considered positive.

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