JPS5883264A - Preparation of antigen for diagnosis - Google Patents

Abstract

PURPOSE:To permit prepn. of antigens easily without requiring any special aseptic operations by sampling the serous allantois of grown eggs after incubation, breaking the serous allantois cells and separating the same centrifugally. CONSTITUTION:EDS-76 Virus is inoculated to the grown eggs of 12-16 day age of ducks free from EDS antibodies and after 3-7 days of incubation, the serous allantois of the grown eggs are sampled, and a diluent is added thereto and the diluted allantois are pooled. The serous allantois cells are broken and are centrifugally separated. The supernatant is sampled. If the serous allantois are sampled after preservation in a refrigerator overnight since the incubation, the sampling is easy. Phosphoric acid buffers, physiological salt solns., distilled water and water are used for the diluent. Homogenization, freezing-thawing and ultrasonic treatments are suitable for breaking the serous allantois.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention provides a method for treating reduced egg production syndrome (Rgg) in poultry.
Drop 8 syndrome -1976, hereinafter rgD
The present invention relates to a method for producing an antigen for in-gel precipitation reaction, which is one of the serological diagnostics for s-76J.

gDs-76 is an infectious disease caused by infection with the EDS-76 virus, and since its first appearance in Europe in 1976, it has gradually spread to countries around the world. Chickens infected with this disease have a decline in egg production with abnormal eggs, resulting in significant economic damage. Therefore, it is necessary to diagnose this disease, and currently, HI reaction, gel precipitation reaction, etc. are used as serum diagnosis. Particularly, the precipitation reaction in a single container is quite widely practiced because of its simple operation. However, the preparation of antigens used in the in-gel precipitation reaction is complicated and requires fairly sophisticated techniques and equipment.

That is, the method for preparing antigens used in the conventional intraprecipitation reaction is described in Avian Pathol, J Vol. 7, No. 62.
pp. 9-639 (1978) and J, B, McF@
Edited by Mr. RRAN 5 studies on them
ig@nicRelationshipbetweenwee
n an l5olation (127) from
the EggDrop Syndrome 197
6 and a Fowl AdenovlruaJ, as follows.

1) Collect fetal liver cells from 8PF embryonated chicken eggs and perform cell culture.

2) Cell culture [gDS-76 virus is inoculated and the virus is propagated, the culture solution is centrifuged, an equal volume of saturated ammonium sulfate solution is added to the supernatant, and the mixture is left overnight at 4°C.

3) Collect the precipitate by centrifugation, and add 14
00 amount of distilled water is added to dissolve the precipitate.

4) Dialyze it against phosphate buffered saline.

5) Add formalin to a 2% ratio to inactivate the virus, adjust the titer to 4 units of Kr1il, and use it as an antigen.

The present inventors conducted research to resolve the difficulties in conventional methods, and as a result, the antigen used in gel precipitation reaction is easier than conventional methods and does not require advanced technology and equipment. I found a way to The method for producing the EDF3-76 diagnostic antigen according to the present invention will be described in detail below.

gDs antibody free embryonated duck eggs 12-16 days old KED
3-76 virus was inoculated, the eggs were incubated for 5 to 7 days, and the chorioallantoic membranes of the eggs were collected, a diluent was added to the eggs, the cells were pooled, the chorioallantoic membrane cells were destroyed, the chorioallantoic membrane cells were destroyed, and the supernatant was collected by centrifugation. . Virus inoculation sites include the chorioallantoic cavity and the chorioallantoic yolk sac, but chorioallantoic inoculation is also preferred. In addition, it is easier to collect the genus Chrysanthemum after the eggs have been incubated and stored in the refrigerator overnight. In addition, a phosphate buffer, physiological saline, distilled water, water, or the like is used as a diluent. In addition, homogenization, freeze-thawing, ultrasonication, etc. are suitable for destroying chorioallantoic cells.

The above-mentioned superior groove may be used as an antigen, but it is safer to use it by adding formalin to a ratio of [L2%] to inactivate it.
If a14 is set, it is possible to diagnose serum or egg yolk samples with a wide range of antibody titers. The optimum concentration referred to here is the antiserum obtained by diluting the antigen and antiserum one-fold with PB8, and performing a precipitation reaction on all combinations of each diluted antigen and each diluted antiserum. This is the concentration of antigen that reacts well.

Preparing antigens using the method of the present invention eliminates the need for tissue culture techniques and equipment required by conventional methods, and furthermore,
4, and no special aseptic procedures are required, making it possible for even beginners in virus handling to easily prepare antigens. In addition, the results obtained by performing a gel precipitation reaction using the antigen prepared by the method of the present invention were substantially the same as the results obtained using the antigen prepared by the conventional method. The procedure for in-gel precipitation reactions using diagnostic antigens is as follows.

1) Pour 1 to 2% agar solution or agarose solution ((Am in PBS containing 1116 NaN1) onto a slide glass so that the agar thickness is approximately 1111.

2) Once the 'III-sized pieces have hardened, punch them with a puncher, then remove the agar from the punched area and make holes. The distance between the holes is approximately 3 to 4 degrees.

6) Place the antigen into one hole and the test serum or test egg yolk into the other hole.

4) Place it in a humid box and leave it at room temperature, and observe the sedimentation line over time by shining a light source diagonally below the slide glass.

For the technique of further in-gel precipitation reaction, please refer to the description on pages 251-255 of "Revised 5th Edition Bacteriology Practice Summary" (Complete Edition of Alumni of the Institute of Medical Science).

Example ED8 Ui, A/, ((JAP-1 stock) 108-OT
CID5Q/m was inoculated into the chorioallantoic cavity of 10 14-day-old 8PF duck eggs at a dose of 0.11. 3'15℃ for 4 days
After incubating the eggs, the fetuses were killed by placing them in a refrigerator at 4°C overnight, and the eggs were broken and the chorioallantoic membrane was collected. Pool 10 chorioallantoic membranes,
An equal amount of PH1 was added and homogenized for about 3 minutes while cooling. The emulsion was repeatedly frozen and thawed three times and centrifuged for 10 minutes at about 5000 rpm to collect the supernatant [Sample (1)]. A formalin stock solution was added to the supernatant at a ratio of 0.2 to inactivate the virus [Sample (2)]. Substrates (1) and (2) were used as antigens for in-gel precipitation reaction. Sample (ri and (2)
) and gel-precipitated antigens prepared by conventional methods were subjected to gel-precipitation reactions with antiserum or diluted antiserum, respectively.

The results are shown in the table below, and the antigen obtained by the method of the present invention was fully comparable to that obtained by the conventional method.

Claims (1)

[Claims] Reduced egg production syndrome antibody-free embryonated duck eggs are inoculated with reduced egg production syndrome virus, the chorioallantoic membrane of the embryonated eggs is collected, and the cells of the genus Qinuria are destroyed and the supernatant obtained by centrifugation. A method for preparing an in-gel precipitated antigen for diagnosing serum antibodies or antibodies in egg yolk for reduced egg production syndrome, characterized by: