SU1529119A1 - Method of determining neuroviruses in cerebrospinal fluid - Google Patents

Abstract

This invention relates to medicine, in particular to the diagnosis of neuroinfections. The goal is to increase the sensitivity of the method. In order to detect neuroviruses in the cerebrospinal fluid, primary cultures of the neurons of the spinal cord and brain of rats are used, after having previously been treated with Eagle's medium with a pH of 6.0-6.5 for 30-60 minutes. After incubation in the neurons, virus antigens are detected by the immunoperoxidase method. Pre-treatment of cell cultures leads to an increase in the sensitivity of the method by 20%.

Description

This invention relates to medicine.

The aim of the invention is to increase the sensitivity of the method

The method is carried out as follows.

Dp detection of the virus was used primary cultures of neurons of the spinal cord and brain of embryos (12-14 and 16-18 days, respectively) rats prepared as follows. Pregnant rats of appropriate terms were placed in an atmosphere of carbon dioxide; Anesthesized and blocked with a fracture of the neck. After the embryos were removed, they were placed in Hanks solution, washed in it, then the cerebral canal was opened or the embryos were removed.

skull, removed the spinal cord or brain. The brain was purified from the membranes, the crushed brain tissue was repeatedly pipetted with Pasteur pipettes of various diameters (from larger to smaller). For 5 minutes, the tube with the cell suspension was settled to precipitate large pieces of tissue onto the bottom, the supernatant was collected and nl cover slides pre-coated with collagen were applied. Then the culture was placed in a thermostat at 37 ° C. Cultivation was carried out in Petri dishes.

The study of spinal-urinary fluid (CSF) on a cell culture was carried out as follows:

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In 10-14-day-old cell cultures, the growth medium was withdrawn and the Eagle's Minimal Environment MEME was added with glucose added up to 600 mg /% (pH 6.0-6.5) for 1 hour. Then the cultures were transferred to the growth medium (MEM 80% j human placental serum 10%; horse serum 10%, glucose 600 mg /%) and 10% CSF was added to it. After 1-5 days, the glasses with cultures were removed from the growth medium, washed for 5 minutes in saline, dried with a hairdryer and fic. silyat in chilled to + A ° C of adeton 7 Min. After fixing, the glass was washed 2 times for 3 minutes in physiological solution and immersed for 10 minutes (cells up) in a solution of sodium azide (0.001 M) with the addition of 0.01% hydrogen peroxide. Then the glasses and cells were washed 3 times for 10 minutes and dried hair dryer Necessary rabbit antisera (against herpes simplex virus or amyotrophic leuko-spongiosis) were applied to cell cultures at a working dilution of 1:50 and placed in a moist chamber for 40 minutes at 37 C. Then the cells were washed again and the rabbit-labeled anti-rabbit antibodies were applied. - sidase) at a dilution of 1: 100. After 40 min of contact in a humid chamber at 37 ° C, the washing procedure was repeated, and the glasses with the cell cultures under investigation were immersed in a dark chamber in a substrate solution (35 mg of 3,3-diaminobenzidine in 50 ml physiological p The solution and 0.03% hydrogen peroxide per 30 min. Then the glasses were removed, washed, passed through a series of alcohols and xylene, and placed in a Canadian balsam. The result was taken under a light microscope, according to the presence of brown colored structures in the infected brown cells. the absence of those in non-infected cells.

In order to establish optimal conditions for the entry of pathogens from the CSF into cells, a number of experiments have been carried out. The cell culture of neurons was treated with medium with a pH of 6.0-6.5 for 30-60 minutes. This was done to change the permeability of the cell membrane.

Example 1. The CSF of patient K was investigated with suspected herpetic encephalitis. The embryonic cell culture of dissociated neurons was infected after being treated with medium with a pH of 6.0 for 30 minutes. Two days after the infection, the herpes simplex virus antigens were detected using the immunoperoxidase method. Diagnosis - Herpetic Encephalitis

PRI mme R 2. The study of CSF of patient D with suspected amyotrophic leuko- spongiosis infected a cell culture of dissociated neurons after their treatment for -. 40 min medium with a pH of 6.0. Three days after cultivation, an antigen of the pathogen was diagnosed in cells;

PRI me R 3. CM} To patient G., with suspected amyotrophic leukospongiosis, infected the embryonic cell culture of dissociated neurons after their pretreatment with medium with a pH of 6.5 for 15 minutes. When observing and examining this culture for 2 weeks, the development of a viral infection has not been established.

Pretreatment of cultures of cells of dissociated neurons leads to an increase in the sensitivity of the method - out of 53 samples of CSF suspected of having a herpetic infection using the proposed method, the virus was detected in 23 cases, and in the prototype method - in 11 cases

Claims (1)

Invention Formula A method for determining neuroviruses in cerebrospinal fluid, including the cultivation of viruses in a cell culture followed by an enzyme immunoassay, characterized in that, in order to increase the sensitivity of the method, 3M6prf-the primary neurons of the brain are used as cell cultures Before the determination of neuroviruses, the needle is treated with a minimal medium.