SU1478122A1 - Method for determining antigens and antibodies - Google Patents

Abstract

The invention relates to the field of medicine and agriculture, namely immunology, and can be used in the analysis of immune preparations on a solid surface. The aim of the invention is to improve the accuracy of the method. This goal is achieved by forming a monolayer of specific antigens or antibodies on the surface of a sensor having reference and measuring delay lines on surface acoustic waves, a known amount of the analyzed solution is applied to the surface of the measuring line, incubated in a moist chamber, washed, dried, and measured The difference between the transit times of the acoustic wave in the reference and measurement delay lines and the number of specific antibodies or antigens are determined from the calibration curve. 1 tab.

Description

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FIELD OF THE INVENTION The invention relates to medicine and agriculture, namely immunology, and. can be used in the analysis of immune drugs on a solid surface.

The aim of the invention is to improve the accuracy of the method.

The method is carried out as follows.

A monomolecular layer of specific antigens or antibodies is formed on the surface of the surfactant sensor with the reference and measuring delay lines placed on the same substrate, and a known amount of the analyzed solution is applied to the surface of the measuring delay line. in a humid chamber

washed, dried, after which the difference in the transit time of the acoustic wave in the reference and measuring delay lines is measured and the amount of the antibody or antigen under study is plotted according to a previously measured calibration curve.

In order to prove the applicability of the proposed method, it is necessary to show that the increments in the thickness of the molecular layers due to the formation of immune complexes are sufficient to change the speed of the surface acoustic wave, and so that it is possible to measure this change.

The dependence of the wave propagation velocity on the layer thickness is

4i

sj

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to 1C

IV - V (h) J / V r 2ii Fh / Vs

(one)

where V, V (h) are the velocities of the surface wave on the free and covered surface;

Y is the coefficient characterizing the dispersion properties of the layered system; F - working frequency of the PAV sensor.

If the SAW sensor is used as the frequency-generating element of the auto-oscillator, then when the speed V changes, the relative frequency change of the generator F will be

F - F (h) V - V (h)

F v

Consequently, a relative change in speed can be recorded with the same resolution as the relative change in frequency, i.e. at the level .

The sensitivity to the thickness calculated by the formulas for layers C and piezoquartz sensors Y, X-cut (V 3159 m / s) with frequencies of 10 and 100 MHz is equal to 20 and 2000 Hz / them, respectively. It follows that the minimally recorded increments of the thickness LL MWH are t 10 - m, which is at the level of ellip. sometric method proposed in the prototype.

The sensitivity can be increased by increasing the frequency of the SAW sensor. Modern technology provides the ability to create SAW sensors, whose frequencies can lie in the range of up to 1 GHz.

Example 1 Determination of the amount of insulin in solution.

SAW sensors with two delay lines on a piezoquartz crystal Ґ, X-slice, basic purity 10 MHz were used.

The dilution of all reaction components is carried out in 0.01 M phosphate buffer (PB) with a pH of 7.4.

Step A. A surfactant sensor is introduced into a solution of immunoglobulins with a concentration of 10 µg / ml, isolated from anti-insulin serum of the guinea pig, incubated overnight at + 4 ° C, the sensor is washed without removing it from the solution

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five

0

five

0

100 ml of FB containing 0.05% tween-20 (TPB) are removed from the solution and the working surface is flushed with a stream of inert gas to remove TPB droplets.

Stage B. 100 µl of the analyzed solution, diluted, is applied to the surface of one of the delay lines.

Incubate in a humid chamber at 37 ° C for 2 hours. Then, the surface of the sensor is washed off 100 ml of PB and 100 ml of distilled water from unbound insulin molecules. The sensor is dried in an environment dried with silica gel until the measured frequency of the difference signal in the open and measuring delay lines repeats at least 10 times the order. The measurement is carried out by including the sensor in the feedback circuit of the amplifier driving at the frequency determined by the delay time of the SAW sensor.

The table presents the results of the analysis performed (the values given are average values of two measurements).

One sensor can be used to perform two analyzes, not repeating step A, for which an aliquot of the analyzed solution is applied to the surface of the reference delay line, and the measuring one is used as a reference. Then you need to clean the surface of the sensor from the reaction products. The purification is carried out by keeping the sensor in a 10% aqueous solution of trisodium phosphate for 1 hour at room temperature, followed by washing in distilled water.

Example 2. Determining the amount of antibodies in serum.

Used surfactant sensors Y, X-slice, with a frequency of 10 MHz and the classes IgG, IgM, IgA, produced by IEM them. P.F. Gamale AMS USSR.

Stage A. A solution of pure IgG in PB with a concentration of 10 µg / ml is prepared to adsorb antigen to the surface of the sensors. Surfactant sensors are introduced into the prepared solution and incubated overnight at + 4 ° C. Wash the sensors, do not remove from the solution, 100 ml of TPB, then remove the sensors from the solution and blow the working surface with a clean stream of inert gas.

Stage B. 100 µl of the test solution of different dilutions of serum are applied to the surface of the measuring delay lines. Dilution of sera is prepared in TFP. Incubate the sera with adsorbed immunoglobulin for 1 hour at 37 ° C. To eliminate the effect of washing quality on the measurement result, and to control the specificity of the reaction, 100 µl of a control serum solution without analyzed antibodies was injected into the reference delay lines. After incubation, the sensor surface is washed of 100 ml of PB and distilled water from unbound antibodies. Drying and measurement are carried out as in Example 1.

The level of antibodies in the studied sera is estimated according to the calibration graph of the dependence of the frequency of the difference signal on the dilution of the control sera.

Thus, from the above examples, it follows that the sensitivity of the proposed method when using SAW sensors with a frequency of 10 MHz and a 1 Hz frequency detection accuracy was 0.5 μg / ml, which is not worse

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-.

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than in the prototype. However, as follows from the description of the invention, increasing the sensitivity by at least 2 orders of magnitude is a purely technical task associated with increasing the operating frequency of the SAW sensor to 1 GHz.

Claims (1)

In addition, preliminary preparation of sensors for stage A, for example, the use of ready-made immunological kits, significantly reduces the analysis time. If we compare the time of stage B, spent on the study of eight solutions in two replications for the prototype and the proposed method, it will turn out that the prototype requires 5 hours more. Therefore, the technical and economic advantages of the invention over the prototype are obvious. Invention Formula The method of determining antigens or antibodies by immobilizing antibodies or antigens on the solid surface of the sensor, followed by applying the test solution, incubating, washing, drying, measuring acoustic characteristics and determining the amount of antigens or antibodies by calibration curves, which are accuracy of the method, the test and control solutions are applied respectively to the sensor surface in the area of the measuring and reference delay lines of the acoustic waves, and the measurement The bushy characteristics are carried out according to the difference in the travel time of the acoustic wave in the reference and measurement delay lines.