JPS6161069B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to an antigen for agglutination reactions and a method for producing the same, and its purpose is to provide an antigen for agglutination reactions with high sensitivity. Currently, a suspension of killed pathogenic bacteria is used as an antigen for an agglutination reaction in the diagnosis of various infectious diseases and as a means of testing whether immunity has been acquired. For example, Haemophilus gallinarum used for antibody testing for infectious coryzasis, Mycoplasma gallisepticum and Marcoplasma synoviae for mycoplasmosis, Brucella abortus for diagnosing Brucella, Vibrio fetus for diagnosing Vibrio disease, and Salmonella for paratyphoid fever diagnosis. , Abortus equi, Salmonella prorum for diagnosing brood dysentery, and Escherichia coli. Bacterial cells that cause agglutination reactions are used as antigens for agglutination reactions. However, these bacteria are susceptible to spontaneous agglutination, in other words, and the agglutination reaction is unclear, so even though they are not infected and do not have immune antibodies, the agglutination reaction does not occur. As a result of a positive test result, it is sometimes mistakenly determined that the patient has antibodies and is infected. Conversely, an agglutination reaction may not occur immediately and a test result may be determined to be negative even though the patient has antibodies. Recently, the present inventor has discovered that by treating bacteria that tend to cause spontaneous agglutination, which are used as antigens for agglutination reactions, with trypsin, it is possible to make an excellent antigen for agglutination reactions that does not cause spontaneous agglutination and has a clear reaction. discovered this and completed the present invention. The antigen for agglutination reactions of the present invention is produced as follows. The selection of the production strain should be carried out by considering the use of a strain with a high infection rate and a strain with good antigenicity that has the same serotype as the infecting strain. For example, when producing a trypsinized antigen for agglutination reaction of Haemophilus gallinarum, Page; Am.J.Vet.Res., 20 , 85 (1962)
Strains with good antigenicity may be selected based on the serotypes described in
It is recommended to use the 0211 strain. Examples of suitable strains of bacteria used as antigens for agglutination reactions are Mycoplasma gallisepticum.
S6 strain, Mycoplasma shinobies WVU1853 strain,
Brucella abortus 99 strains, Brucella abortus 125 strains, Vibrio fetus Venereal type
UM strain, Salmonella abortus equi type S, Salmonella prorum standard type S
type bacteria, E. coli Umezawa strain, etc. The production and cultivation of bacterial cells is carried out according to the method commonly used when culturing the above-mentioned bacterial strains. The culture is preferably liquid culture, and the medium is preferably a medium normally used for culturing the bacteria. As a medium for Haemophilus gallinarum, for example, Kato medium, chicken serum-added chicken broth medium, DPN-added brain heart infusion medium, etc. are used. In addition, Kato medium is
Iritani and Hidaka; Avian Dis. 20 , 614
(1976), chicken serum supplemented chicken broth medium was used by Otsuki and
Iritani; Avian Dis. 18 , 297 (1974), DPN-added brain heart infusion medium was
Yamamoto and Somensett; Avian Dis. 8 , 441
(1964). Examples of the medium for Mycoplasma gallisepticum include chicken PPLO medium, for Brarula abortus, such as Brasella dialysis culture medium, for Vibrio fetus, such as bovine blood agar, and for Salmonella abortus, for example, For Salmonella prorum, for example, CS agar medium, YCC agar medium, medium for continuous culture of Shigella chinensis, etc., and for Escherichia coli, heart infusion medium, nutrient medium, etc. are used. Culture conditions such as culture temperature and culture time follow conventional methods. Bacterial cells are collected from the culture solution by, for example, centrifugation, and after washing and concentration adjustment as necessary, they are treated with trypsin. Saline, phosphate buffered saline, etc. are used for washing. For trypsin treatment, if desired, the bacterial cells are brought to an appropriate concentration with an appropriate buffered saline solution, then trypsin is added, and the trypsin is suitable for performing a peptide decomposition reaction.
Maintain the cell-containing liquid at pH and temperature. In particular, P.H.
The temperature is preferably around 6 to 8 degrees Celsius, and the temperature is preferably around 25 to 40 degrees Celsius. Trypsin treatment is stopped by adding trypsin inhibitor when the bacterial solution becomes a homogeneous suspension, which is easy to do. Examples of the buffer used for concentration adjustment include physiological saline, phosphate buffered saline, and Tris-HCl buffered saline. The trypsinized product thus obtained is separated and collected by a method such as filtration or centrifugation, if necessary. The conventional centrifugation method is particularly convenient. When trypsin-treated products are used for diagnosis as antigens for agglutination reactions, they should be diluted to an appropriate concentration and treated with disinfectants (e.g., thimerosal, phenol, formalin, etc.) and dyes (e.g., crystal violet, malachite green, etc.) as necessary. etc.) to make it easier to confirm the agglutination reaction. Diagnostic reagents can be used in the usual manner; blood is collected from the test animal, the diagnostic reagent is mixed with serum in an appropriate ratio, and the agglutination properties are examined. In the following Examples, an example of producing a trypsin-treated product of Haemophilus gallinarum is shown, but the present invention is not limited to these Examples. Example Haemophilus gallinarum 221 strain was grown in Kato medium (0.5% polypeptone, 0.5% sodium chloride, 0.1% casamino acids (no vitamins), 0.5% monosodium glutamate, 0.1% glucose, 0.2% potassium phosphate, 1% yeast extract) , containing 0.5% chicken serum) at 37°C for 48 hours. Culture solution at 2-5℃
After continuous centrifugation at 10,000 r.pm4/hour at Add acid buffered saline. Add an equal volume of 0.25% trypsin solution to this concentrated solution and sensitize at 37°C for 30 minutes. Then add an equal volume of 0.25% trypsin inhibitor solution to the trypsin solution. The trypsinized bacterial cells obtained by centrifugation at 10,000 rpm for 30 minutes are centrifugally washed twice with phosphate buffered saline. 540 m
The concentration was adjusted with phosphate buffered saline so that the OD of μ was 10 times 0.35, crystal violet was added to make it 0.01%, and it was used in the following experiment (hereinafter referred to as the trypsinized bacterial cell). . Experimental example 1 Spontaneous agglutination reaction inhibition test Bacterial culture solution (1) of Example 1, bacterial body fluid treated with trypsin but not treated with trypsin inhibitor
(2), and 0.05 ml each of trypsinized bacterial cells (3) and 0.05 ml of phosphate buffered saline (PBS) or Haemophilus gallinarum antibody-negative serum (negative serum)
0.05 ml was mixed on a glass plate, and those that formed clear aggregates within 2 minutes were considered positive.

[Table] Note * - Not performed + Performed ** - Negative Strongly positive The above results confirm that the trypsin-treated bacterial cells do not cause spontaneous agglutination. Experimental Example 2 Cross-aggregation reaction test Cultured bacterial cells before trypsin treatment obtained in Examples (trypsin-untreated bacterial cells), trypsin-treated bacterial cells, and Haemophilus gallinarum 221 strains obtained by culturing in chicken serum-added chicken broth medium. 30 bacterial cells (CMI bacterial cells)
Vaccinate LS white leghorns and collect blood after 30 days. Using these sera and serum obtained from chickens that have recovered from infectious coryza disease (HG infection recovery serum), trypsinized bacterial cells and CMI bacterial cells as antigens, the agglutination reaction is examined. The agglutination titer was expressed as the highest dilution factor of the serum in which aggregates were observed using the method shown in Example 1.

【table】

[Table] The above results show that trypsin-treated bacterial cells can be used as an antigen for agglutination reactions in exactly the same way as CMI bacterial cells, and the trypsin-treated bacterial cells are more clearly used than CMI bacterial cells. This indicates that aggregation reactions occur. Experimental Example 3 Cross-absorption test Antigen (bacterial trypsin) adjusted to the same concentration was added to 1 volume of antiserum obtained by inoculating LS White Leghorn with trypsin-treated bacterial cells and CMI bacterial cells in the same manner as in Experimental Example 2. Absorption of 540 mμ for treated material, trypsin-untreated bacterial cells, and CMI bacterial cells was 0.37.
Adjust to 10x. However, crystal violet was not added), the mixture was reacted at 37°C for 2 hours, and then left at 2-5°C overnight, and the antigen was removed by centrifugation. Experimental example 2 is used to measure the agglutination value.
Follow the method shown in .

[Table] From the above results, it is recognized that the homology between CMI bacterial cells and the trypsinized bacterial cells as antigens is extremely high. Experimental Example 4 Antibody Production Test in Artificially Infected Chickens Haemophilus gallinarum was inoculated into five 6- to 7-week-old LS white leghorns through the nostrils. Blood is collected before and at 7-day intervals after inoculation, and the presence or absence of an agglutination reaction is determined according to Experimental Example 1 using trypsin-treated bacterial cells as an antigen. As shown in Figure 1, no agglutination reaction was observed in the non-inoculated group, but an agglutination reaction was observed in the serum of all chickens from the 3rd week onwards in the bacteria-inoculated group. Therefore, it was confirmed that the bacterial trypsin-treated product can be used as a diagnostic agent for infectious coryza infection. As confirmed in the above examples, the trypsin-treated Haemophilus gallinarum obtained in the examples is an excellent antigen for agglutination reactions that does not cause spontaneous agglutination and exhibits clear agglutination properties. This excellent property is similarly observed when other naturally flocculating bacterial cells are treated with trypsin.

[Brief explanation of the drawing]

FIG. 1 shows the results of measuring antibody production in artificially infected chickens by an agglutination reaction using trypsin-treated Haemophilus gallinarum as an antigen. The vertical axis indicates the positive rate (%), the horizontal axis indicates the number of weeks after artificial infection, 〇 indicates the infected group, and ▲ indicates the non-infected group.

Claims (1)

[Scope of Claims] 1. A method for producing an antigen for an agglutination reaction, which comprises treating bacterial cells having spontaneous agglutination properties with trypsin and a trypsin inhibitor. 2. An antigen for an agglutination reaction characterized by containing a trypsin- and trypsin inhibitor-treated bacterial cell having spontaneous agglutination properties.