DE2126766A1 - Process for the production of carrier particles - Google Patents

Description

Process for the production of!

The invention relates to a method for treating bacterial cells treated with iOrmaline with a cyanide halide and coupling with an antigen, resulting in antigen-sensitized carrier particles which are only present in the presence agglutinate the antibody specific for this antigen can.

Many diseases are characterized by generation of antibodies in response to a microbiological infection. If these antibodies in an early state after " their presence is a diagnostic clue to the disease. Antigens are commonly used on carrier cells such as erythrocytes, polystyrene or gramnega-

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tive bacteria are gel-coupled so that it is convenient to bring these antigens into contact with the corresponding antibody. One of the major drawbacks of these immunological detection systems is the non-specific agglutination of coupled Antigens and antibodies that lead to incorrect test results leads. To detect bacterial particles coupled with an antigen for the detection of homologous antibodies, the in. human serum are available, may be used the particles agglutinate or flocculate only in the presence of the specific protein to be detected.

It has been shown that E. eoli cells that have been treated with formalin, Bis-diazobenzidine or protein or combinations of these substances are treated in the presence of $ 60 normal agglutinate human serum. Such E. coli cells, which are sensitized with an antigen, cannot for Detection of homologous antibodies can be used as long as the non-specific binding sites are on the bacterial Surface are not destroyed or covered.

It has now been shown that the treatment of with Formalin treated bacterial cells such as E. coli with a Cyan halide such as cyan chloride iodide fluoride or bromide before coupling with an antigen gives carrier particles, which are free of nonspecific binding sites «if an antigen is coupled with bacterial cells treated in this way, the resulting antigen can be sensitized Cells only in the presence of the antigen specific for this antigen .Agglutinate or flocculate antibodies and incorrect test results are avoided,

E. coli cells were placed in a nutrient solution at 37 ° C. for 3 h

(brain heart infusion broth) and then was the culture by treatment for 30 min at 37 ° C with a

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36.8 $ -ig € m aqueous solution of treated with calcium carbonate Forma! Denyd killed. The killed cells were obtained by centrifugation and by alternate centrifugation and resuspending in physiological saline solution washed. The washed E. coli cells were then with a 1 # aqueous formaldehyde solution in physiological Treated with saline solution and left to stand for 2 days at 26 ° C with occasional shaking. Those treated with formalin E. coli cells were washed in physiological saline solution, in 0.2% aqueous formaldehyde solution, in physiological Resuspended saline and adjusted the cell concentration to $ 2 by hematocrit.

The following examples show one method of treatment formalin-treated E. coli cells prepared as indicated above with a cyanide halide and coupling with various proteins.

example 1

10 ml of a 2 $ suspension of formalin treated E. coli cells in physiological saline solution was washed thoroughly with distilled water and distilled in 2 ml Water resuspended. An equal volume of cyanbroinide at a concentration of 50 mg / ml distilled Va-aser was added quickly with stirring. The reaction mixture was immediately washed with 4 liters of sodium hydroxide solution for about 5 minutes Treated at room temperature in order to adjust the pH to 10 to and then centrifuged at 5,000 rpm. The supernatant liquid was decanted and the E. coli cells treated with cyanogen bromide were in 10 ml of a 0.1 M sodium bicarbonate solution adjusted to a pH of was buffered at a temperature of 4 ° C, resuspended. Subject to centrifugation, the treated cells were in 2 ml resuspended this buffered sodium bicarbonate solution.

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Then 0.4 ml of a physiological saline solution., containing 10 mg bovine serum albumin with stirring was added and the reaction mixture was left at 4 ° C for 24 hours

shaken slightly. The resulting E. coli cells, which were coupled with bovine serum albumin, then became physiological Washed saline solution and resuspended in 10 ml of the solution for testing purposes. The cells thus obtained reacted strongly with anti-beef albums from rabbits, but did not agglutinate in the presence of normal human serum.

Example 2

According to the procedure given in Example 1, formalin-treated E. coli cells were treated with cyan chloride treated under the same conditions and then mixed with human / - globulin. Those treated like that Cells accordingly agglutinated with anti-human V ^ globulin from .Rabbits, . but not with normal human serum.

Example 3

10 ml of a 2% suspension contains formalin-treated substances E. coli cells in physiological saline were washed and treated with cyano iodide as described above. The emerging Cells were washed once with 0.1 M aqueous sodium bicarbonate solution which had been buffered 4 ° C and resuspended in physiological saline solution three times washed with this saline solution before being put into 5 ml of sodium phosphate saline solution, buffered to pH 7.2 were resuspended. 9 ml of buffered sodium phosphate saline solution, containing approximately 9 mg of insulin were added to the above cells and immediately before the addition of 25 ml of a solution containing 1 mg of bis-diazobenzidine per ml of sodium phosphate saline solution, mixed. The emerging

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Cells were rotated at room temperature for 30 minutes and then "centrifuged at 2,000 rpm. They were then used with Washed physiological saline solution and resuspended in 10 ml of the saline solution prior to performing the tests. Lasine non-specific agglutination with human was shown Serum. .

Although the above examples relate specifically to the treatment of formalin treated E. coli cells, can other bacterial cells with lipo- or mucopolysacarides on the surface in a similar way with formalin

"V" and treated with a cyanide halide. Such Bacteria are i.a. subtilis, S. marcesens, B. abortus, P. fragi, L. casei and A. aerogenes. The.cyanhalide is in the form of an aqueous concentrate containing 25 mg / ml of water to saturation, but preferably does not contain less than 50 mg / ml water is used. The antigens used are in a solvent such as a physiological one Saline solution at concentrations of 0.5 mg / ml solvent to saturation, but preferably no more than 5 mg / ml Solvent, dissolved.

The temperature for the treatment of bacterial cells with the cyanogen halide can vary from 15 to 30 ⁰ C, although about 25 ⁰ C and advantageously are convenient. The antigens are commonly coupled to the cyanogen halide-treated cells at about 4 ⁰ C, although temperatures may optionally be applied! Of about 0 to 40 ⁰ C as well. In any case, it is beneficial to stabilize the surface of the bacterial cells with formalin before they are treated with a cyanide halide in order to ensure that all sites are blocked which would otherwise agglutinate such cells by unknown components of the human serum would lead. As shown in the examples, the formalin treated bacterial cells are treated with a cyan halogen

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nid treated and then treated with an antigen via the iminocarboxylic acid Coupled with ester groups or, alternatively, the cells treated with cyanide halide can be reacted with an antigen that previously with a usual coupling miniirfcel how BLs-diazobenzidine has been coupled.

The cyanide halide treatment of those treated with Eormalin bacterial cells should be kept in an alkaline medium at a pH of about 10 to 12 and preferably Tbei at a pH of about 11. Even though Cyanobromide is preferred, other gyanhalides such as chlorine, iodide or fluoride can optionally also be used be used.

Claims

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Claims (7)

Claims 1. Process for the preparation of carrier particles which are free of unspecific "binding sites," characterized in that they were treated with formalin Treated bacterial cells in an aqueous medium with a cyanide halide. 2. The method according to claim 1, characterized in that there is used as cyanogen halide cyanogen bromide. 3. The method according to claim 1 or 2, characterized in that the cyanide halide in a concentration of approximately 25 Ms 100 mg / ml water used. 4. The method according to claim 1 Ms 3, characterized in that the bacterial cells treated with formalin are brought together with the cyanide halide at a pH of approximately TO to 11 and a temperature of approximately 15 Ms to 30 ^° C. 5. The method according to claim 1 Ms 4, characterized in that E. coli cells are used. IC 98 SO / 185 0 - 8 - 1A-39 637 6.. Method according to claims 1 to 5 »thereby g e ken η show that the carrier particles are coupled with an antigen. 7. The method according to claim 6, characterized in that "at a temperature of 0- Ms 40 ⁰ C is used. 1 O 98 !; O / 18 5 0