Stepanov A, Sviridov L, Korotkina SA, Achmeteli GG, Kriganivski EV.
Research Center, St. Petersburg, Russia
At the present moment the spheres of application of Gas Discharge Visualization (GDV)
technique in biology and medicine are widely searched. Basing on the well-known fact that this
technique can detect changes of physico-chemical characteristics of solutions of non-organic
substances and biological liquids, we made an attempt to study the possibility of application of
gas discharge glow for the registration of specific interaction of an antigen with a complimentary
antibody – the so-called agglutination reaction. This very fact defined the aim of the present in-
vestigation.
In order to realize this aim the authors developed and applied the method of estimation of
the characteristics of gas discharge glow around a drop of liquid. The method assumed that the
drop of liquid should be pressed out of a disposable insulin syringe and placed in its end. Each
sample was tested 10 times with the frequency of 30 shots a second and the duration of influence
of the electromagnetic field – 10 seconds. Summary data of the GDV tests were compared with
the results obtained by standard visual method.
Specific antibodies were received by the way of immunization of rabbits by ovalbumen
or tularemic vaccine and the white mice – by specific vaccine. Apart from that the blood serum
of a person recovered from the infection caused by conditionally-pathogenic bacteroid B.fragilis
was used.
In order to carry out the main experiments it was first of all necessary to optimize the
scheme of GDV analysis. Particularly, the following question had to be solved: was it necessary
to stir the antigen-antibody complex laid-down on the bottom of the test tube? This part of the
work was performed using the following complementary pairs of reagents:
• Ovalbumen + antibodies (blood serum of rabbits immunized by albumen);
• Vaccine strain of the specific virus+ antibodies (immunoglobulin against virus, ob-
tained from ascetic liquid of white mice, inoculated with the corresponding vaccine);
• Tularemic antibody + antibodies (blood serum of rabbits inoculated with tularemic
vaccine).
Using the abovementioned systems similar data were received. It turned out that even
when the sediment (agglutinate) was carefully stirred the results of GDV analysis didn’t agree
with those of the visual test; moreover, they often contradicted the latter.
In case of GDV investigation of the supernatant when the sediment wasn’t previously
stirred, the results coincided with the data of visual registration of agglutination reaction. We
suppose that under positive reaction the antigen-antibody complex formed sediment and the su-
pernatant “got cleaned” from the components of reaction. When agglutination was not per-
formed, the components of reaction were found in the suspension state, i.e. no cleaning (clarifi-
cation) of the supernatant took place. The GDV technique registered visible (clarification of su-
pernatant) and invisible (change of physico-chemical properties) differences in experimental and
control (known negative) samples appearing at that; thus the technique indicated positive agglu-
tination reaction.
Considering these data detailed experiments proving this generally formulated aspect
were carried out further. One of these experiments was performed with the application of the fol-
lowing reacting components:
1. Human blood serum with antibodies to B.fragilis in titer 1:640 (serum N 1);
2. Human blood serum without B.fragilis antibodies (serum N 2);
3. Suspension В.fragilis (antigen);
4. Physiological solution (0,85 % solution NaCl).
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The scheme of experiment is shown in table 1.
Table 1. Scheme of experiment
Combination of
components in
24 h before
investigation
4
0,85 %
NaCl+В.fragilis
+blood serum
N1
5
0,85%
NaCl+B.fragilis+b
lood serum N1 in
dilution 1:1000
Interspecific
(complimentary)
components of
reaction
Combination of
components
right before
investigation
6
same 7
same
1*
0,85
%
NaCl
2
0,85 %
NaCl +
B.fragilis
3
0,85 %
NaCl +
antibodies
to
B.fragilis
Internonspecific
(non-
complimentary)
components of
reaction
Combination of
components in
24 h before
investigation
8
0,85 %
NaCl+В.fragilis
+blood serum
N2 in dilution
1:10
9
0,85 %
NaCl+В.fragilis+
blood plasma N2
in dilution 1:1000
Comment: * – shows the number of a sample.
Investigation of samples 1,2 and 3 (physiological solution NaCl, the same solution with
B.fragilis or blood serum N1, respectively) was aimed at determining if the GDV technique
could disclose such microscopic objects as antibodies and microorganisms in the solution.
Comparative assessment of GDV-grams of these three objects determined that sample 1
reliably differed from sample 2 in the intensity of glow and in background area, and from sample
3 – not only in these criteria, but also in the form coefficient and length of isoline. Samples 2 and
3 reliably differed from one another in all the abovementioned parameters. To our mind, all that
indicates that the applied method enables to disclose if such biological objects as microorgan-
isms, being the model of antigen in this case, and immune serum, containing specific antibodies
to these microorganisms, are present in physiological solution. Moreover, differences between
the blood serum and suspension of microorganisms can be revealed.
The results of assessment of samples 4-9 should have answered the main question: could
the GDV technique identify not only antigen or antibodies to it in the physiological solution, but
also specific reaction between them – agglutination reaction? This is possible theoretically, as
new biological structures (immune complexes) are formed at such interaction. These structures
possess different physico-chemical properties as compared to other initial reagents (antigens and
antibodies), and most probably change the characteristics of the solution.
The main (pilot) sample of all was sample 4, which consisted of human blood serum with
antibodies to B.fragilis, diluted by physiological solution up to titer 1:10, and suspension
B.fragilis. Agglutination reaction was inevitable in such a system in view of specificity of the
biological reagents taken, which was visually proved in 24 hours (when GDV-grams were
taken).
Control sample 5 differed from sample 4 only by the fact that it had blood serum diluted
at 1:1000. In this case no agglutination reaction took place, as the serum dilution (1:1000) ex-
ceeded its maximal titer (1:640).
Comparing GDV-grams of samples 4 and 5 it was found that they significantly differ in
the intensity of glow. The only difference between them consisted in the fact that agglutination
reaction took place in one sample (sample 4), and was absent in the other (sample 5), which fact
proved that the GDV technique could register new physico-chemical state of sample 4, which
took place as a result of specific interaction of antigen and antibodies in it. In other words, the
technique could visualize the agglutination reaction.
The following aspects prove this conclusion, as well:
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- reliable differences in the intensity of glow (fig.1) between samples 4 and 6 (there was
no agglutination in sample 6 as a result of insufficient exposure within the mixture of compo-
nents: the components had been mixed right before the GDV-grams were taken, i.e. agglutina-
tion reaction hadn’t yet taken place);
- reliable differences in the background area (fig.2), intensity of glow (fig. 3) and length
of isolines (fig.4) between the samples 4 and 8 (agglutination in sample 8 didn’t take place be-
cause of the fact that there was no specific antibodies to B.fragilis in blood serum N2);
- absence of differences between samples 6 and 7 (agglutination was absent in both sam-
ples because of insufficient exposure of antigen with antibodies);
- absence of differences between samples 8 and 9 (agglutination didn’t take place in both
samples as a result of heterologicality of antigen and antibodies).
Thus, as follows from the data above, comparative analysis of GDV-grams of pilot and
control samples enables to reveal antibodies in the investigated material.
It was also considered important to study the informativeness of the GDV as a method of
titration of immune sera. Special experiment on the determination of titer of antibodies in the
blood serum of rabbits, inoculated with ovalbumen, was planned for that. The scheme of the ex-
periment is represented in table 2.
Table 2. Results of titration of the blood serum of rabbits, inoculated with ovalbumen
NN of samples 1 2 3 4
Dilution
of serum 1:10 1:20 1:40 1:4000
Titer of antibodies according to
the data of visual assessment ++++ +++ + -
Comparative analysis demonstrated that the GDV-grams of samples 1 and 2, similar in
visually registered agglutination (4+ and 3+ respectively), didn’t differ reliably. At the same
time, sample 1 reliably differed in area and intensiveness of glow from sample 3 (agglutination
+), and in area, intensity of glow, and also in the quantity of fragments (fig. 5) from sample 4,
where agglutination was absent at all. Analogous differences were found comparing GDV-grams
of sample 2 with the characteristics of sample 3 and 4. At the same time, computer analysis of
samples 3 and 4, where agglutination was expressed weakly (sample 3) or was absent at all
(sample 4), didn’t reveal differences between them.
Therefore, the titer of antibodies determined by visual and GDV methods turned out to be
the same: 1:20. This fact forms the basis that the GDV technique gives an opportunity not only
to register antibodies in liquids, but also to determine their titer.
CONCLUSION
Based on the presented materials we can conclude that the GDV technique enables to
identify specific reaction of antibodies with a complimentary antigen, called agglutination reac-
tion. The technique is based on the registration of dynamics of parameters of gas discharge glow
in time – from the moment of combination (mixing) of specific components (antigen and anti-
bodies to it) to the moment of completion of their interaction and formation of the so-called im-
mune complexes. As a results of such interaction, physico-chemical characteristics of the inves-
tigated material, and consequently, GDV-gram parameters change.
The results obtained are of course preliminary and need to be specified further. But al-
ready now we realize the significance of this direction of scientific search.
The technique can be applied for the investigation of nontransparent biological liquids
when it is not only difficult, but even impossible to implement the agglutination reaction in its
classical form (visual registration of results); for example, researching the blood with the pur-
pose of revealing etiology of human allergies.
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Fig. Example of dynamical trend for two samples of blood: 1 – initial; 2 – with allergen.
