SU1650098A1 - Method for serodiagnosis of syphilis - Google Patents

Abstract

The invention relates to medicine and can be used to increase the sensitivity of serodiagnosis of syphilis in FTA-abs. The aim of the invention is to improve the accuracy of the method due to the fact that the disintegrate of ultra-sound cultures of pale treponems is additionally centrifuged, and the supernatant is used as a sorbent in RIF-abs. The sorbent prepared by the proposed method has a high sorption capacity for nonspecific serum antibodies, which increases the sensitivity of RIF-abs and, accordingly, the serodiagnosis of syphilis by 6.8%. and the specificity of the reaction is 22–2%.

Description

The invention relates to medicine and can be used to improve the accuracy of the serodiagnosis of syphilis in FTA-abs.

The aim of the invention is to improve the accuracy of the method of serodiagnosis of syphilis due to the fact that the disintegrate of ultra-sound pale treponems is additionally centrifuged, and the supernatant is used as a sorbent in the RIF-abs reaction.

The method is carried out as follows.

Cultured treponema I, V, VUI strains (one representative from each antigenic group) are grown under anaerobic conditions in liver broth with the addition of pieces of bovine liver. pH 8.2 for 8 days. The grown culture is filtered, centrifuged at 5.5 X 10 g for 30 minutes. The precipitate is washed twice with isotonic sodium chloride solution.

From the obtained precipitate, a suspension of treponem 10 microns is prepared. cl / ml according to the turbidity standard, then the mixture is disintegrated for 20 min and centrifuged at 103e for 30 min. The pellet obtained (cell walls and outer fibrils of pale treponema culture) is used for commercial production of treponemal antigen, and the supernatant (production waste) containing cytoplasmic cylinder proteins (cytoplasmic membranes, mesosomes, internal fibrils, cytoplasm of cells) is used as a sorbent for serodiagogyrostosterogyrosis, it is used as a sorbent at the serodiageology, at the serodiageology. in rif abs. The resulting sorbent (supernatant) is titrated; to do this, it is diluted with 0.85% NaO pH 7.2 in 2. 3. 4, 5 times and put in REEF-abs with eight serums: one (1) is reec-positive (). two (2. 3) weakly positive (2+). five (4-8) from people free from syphilitic infection, three of which give nonspecifically positive results in RIF (2+). All the sera are diluted with the proposed sorbent in a ratio of 1: 5 and examined in the REEF. For the titer of the sorbent they take such a dilution at which the sorbed patient serums retain a degree of positivity, and the sera of syphilis free individuals do not glow.

The titrated sorbent is standardized in series (the established titer, i.e., its working dilution is added to the passport), poured into 5 ml vials, lyophilized according to a known drying mode, and sealed for subsequent use in the RIF Abs serological laboratories.

For staging RIF-Abs, the following ingredients are preliminarily prepared: the test serum is inactivated at 56 ° C for 30 minutes; treponem antigen (suspension of pathogenic pale treponeum of Nichols strain, obtained from 7-day-old rabbit orchitis) should contain 40-60 treponems in the field of view during microscopy with a dark-field condenser; the sorbent (proposed) is diluted with an isotonic solution of sodium chloride to the specified titer on the passport; titrate dry luminescent serum against human globulins in RIF-abs with blood serum of people diluted by the proposed sorbent in a ratio of 1: 5 (5 serum of syphilis patients and 5 blood serum of persons free from syphilitic infection), and set the working titer by the most luminescence with positive blood sera and the complete absence of treponem cell luminescence with negative blood sera. Next, the obtained luminescent serum titer is tested on 100 human sera, of which 20% are serum from individuals with syphilis.

Thin defatted glass slides are taken, on the reverse side of which the boundaries of application of the reaction ingredients are determined (diameter 0.7). Then a treponem antigen is applied to the glass within a circle, dried in air and fixed for 10 minutes in chemically pure acetone. Next, the inactivated test serum was diluted with a sorbent previously diluted in a titer in the ratio of 1: 5, applied to a smear of antigen, placed in a moist chamber, which was then placed in a thermostat at 37 ° C for 30 minutes (reaction phase I). After a period of time, the chamber is removed from the thermostat, the slides with the first phase of the reaction are washed twice with phosphate buffer, and during the second

rinse to more completely remove proteins that are not involved in the reaction, the slides are kept in phosphate buffer for 10 minutes, then dried and applied

per smear luminescent serum, placed in a humid chamber and maintained at room temperature for 30 minutes (phase II of the reaction). After the expiration of the time of the sample, the test sample is washed with phosphate

0 with buffer in the same sequence as after the first phase of the reaction, after which the non-luminescent immersion oil (dimethyl phthalate) is dried, applied to the surface of smears, and the RIF5 abs is taken into account in a fluorescent microscope with a mercury-quartz lamp (DRSh-250). Accounting for the reaction is carried out according to the degree of lightening of pale treponemas.

Example 1. Patient B. clinical

0 manifestations of primary syphilis, however, serodiagnosis in the stationary complex of RSK for syphilis is negative. When formulating RIF-abs with an ultra-sound antigen, a weakly positive result was obtained.

5 result (2+). Staging RIF-abs with a sorbent prepared by the described method gave a sharp positive result (4+). This method makes it possible to establish the diagnosis: primary syphilis in RIF-abs.

0 A positive effect is achieved in order. which increases the sensitivity of rif-abs by half, which is extremely valuable for performing optimal therapeutic measures in a short time

5 of this category of patients.

Example 2. Patient C. clinical and serological studies at RAC for syphilis were diagnosed: syphilis, primary serologic serum Blood serum

0 patient C. in RIF-abs with ultra-sound treponemal antigen gave a positive result (3+), with a sorbent prepared as described, in RIF-abs the reaction was sharply positive (4+). This way, therefore, increases the degree of positivity (by 1-i), translating positive reactions into sharply positive ones.

PRI me R 3. Ill. G. with a disease of acute viral infection gave positive reactions in RSK to syphilis. No clinical manifestations of late syphilis were revealed, however, the study of spinal fluid (CSF) obtained during puncture gave a slightly positive result in RAC. In RIF-abs, serum of the patient and cerebrospinal fluid gave positive results () with a smiley treponemal antigen (), with a sorbent obtained according to the invention, in RIF-abs the reactions were sharply positive (4+).

The proposed method makes it possible to establish the diagnosis: late syphilis, in this example increases the degree of positivity (by 1+), translating positive reactions into sharply positive ones.

Thus, the sorbent prepared by the proposed method has an increased sorption capacity for nonspecific serum antibodies, which increases the sensitivity of RIF-abs and, accordingly, serodiagnosis0

syphilis stick by 6.8%, and reaction specificity by 22.2%.

Claims (1)

DETAILED DESCRIPTION OF THE INVENTION Claims for serodiagnosis of syphilis by setting up a REEF-abs reaction using ultra-sounding treponema cultures disintegrate as a sorbent, characterized in that, in order to improve the accuracy of the method, the disintegrate is additionally centrifuged and a supernatant is used as a sorbent.