JPH0757178B2 - Biological particles coated with protein A and uses thereof - Google Patents

Description

TECHNICAL FIELD The present invention relates to a biological particle coated with protein A, more specifically, it has a function of being taken up by antibody-sensitized floating cells, and has a biochemical research potential. The present invention relates to biological particles having utility as materials and the like and uses thereof.

(Prior Art) As a method for incorporating foreign substances into various cells,
(1) So-called intracellular injection method using micromanipulator and injector scope [Journal of
Sciences (J.Sci.Ser.,) B. 9 , 307 (1914) and "Erythropoiesis and Differentiation in Friend Roychemia Cell"("Erythr.
opoiesis and differentiation in Friend leukemia ce
lls "), Elsvier / North-Holland BiomedicalPrc
e), B.V. Amsterdam (BVAmsterdam (19
80), in Press., Etc.], (2) Method of injecting substances in liposomes into cells using liposomes [“Method in Cell Biology”) Academy Press, New York (Acad. Press) , Nes Yor
k), 14 , 34-68 (1976)], (3) Method using HVJ virus [Procedure of National Academy of Sciences (Proc.Natl.Acad.Sc).
i.) USA, 72 , 4071 (1975)], and a method of incorporating a substance incorporated into erythrocytes based on the principle of cell fusion [Nature 249 , 449 (1974)] and the like. .

(Problems to be Solved by the Invention) According to the prior art, methods are selected according to the respective purposes, but each of them has a drawback that a substance in the unit of μm cannot be efficiently taken up by other cells. Have

Therefore, it has been impossible to examine the action / effect of various biological particles having a certain size on various cells at the cell level.

On the other hand, the present inventor has studied various actions of biological particles carrying protein A. As a result, the particles coated with protein A having a size which cannot be taken up by cells by the above-mentioned conventional techniques are antibody-sensitized and suspended. The present inventors have completed the present invention by finding that they are not only adsorbed on cells but also taken up by the cells.

It is known that protein A is bound to an appropriate carrier and used for quantification of antibody-producing cells, purification of IgG, etc. (JP-A-49-133518, etc.), but it is incorporated into antibody-sensitized floating cells. The particles to which protein A having the function described above is bound have not been described in the conventionally known documents.

(Means for Solving Problems) Accordingly, the present invention provides a biological particle coated with protein A that can be incorporated into antibody-sensitized floating cells, and its use.

The protein A referred to in the present invention is usually derived from the cell wall of Staphylococcus aureus , but it is prepared using a recombinant DNA method [eg, Journal of Bacteriology ( J.
Bacteriol.) 159 , 713, 1984; JP-A-59-113890, etc.], which is capable of specifically binding to the Fe portion of mammalian immunoglobulin G (IgG). .

Next, the particles referred to as biological particles coated with protein A refer to microbial cells, and examples thereof include pathogenic microbial cells such as Staphylococcus aureus, Escherichia coli, and Salmonella typhimurium.

In order to coat such particles with protein A, a method known per se, for example, (1) using a cross-linking reagent having a plurality of functional groups such as chromium chloride or glutaraldehyde and diisocyanates on the particles, the protein A and the particles are used. (For example, JP-A-49-133597), or (2) prepare an antibody against the antigen constituting the particle, first sensitize the particle with the antibody, and then directly or with soluble protein A. There is a method of binding via the next antibody. As such an antibody, a polyclonal antibody or a monoclonal antibody is used. As the polyclonal antibody, an antiserum obtained by immunizing a rabbit with an antigenic substance can be used as the moclonal antibody produced according to the method of Kohler & Milstein [Nature, 256 : 495-497, 1975]. .

The thus obtained protein A-coated biological particles have a function of adsorbing to antibody-sensitized floating cells and being taken up into the cells, even if they have a size of several μm. Not only is it useful as a biochemical research sample itself, but when incorporated with various physiologically active substances, it has the potential to be used as a cell killing agent or cell proliferation agent.

The floating cells referred to as antibody-sensitized floating cells refer to cells that can be suspension-cultured such as lymphoid cells and ascites cancer cells, and more specifically include mouse / Ehrlich cancer cells and mouse / myeloma cells. It is a concept. In addition, such cells can be made into antibody-sensitized cells using an antibody obtained by injecting each cell into a xenogeneic animal.

Hereinafter, the present invention will be described in more detail with reference to Examples.
Of course, the present invention is not limited to the embodiments.

Example 1 Salmonella typhimurium (derived from Tokyo Jikei University School of Medicine Hospital Central Laboratory) is cultured overnight on a Heart Infusion Agar plate at 37 ° C. and suspended in physiological saline. Bacteria with physiological saline 3
After washing twice, add formalin at a ratio of 0.6% and leave at 37 ° C overnight. After washing the formalin-treated bacteria with physiological saline, the number of viable bacteria is adjusted to a concentration of 100 million cells / ml. 1 ml of this bacterial solution once a week for a total of 5 times to ICR mice (female, 5 weeks old)
Inoculate each one intraperitoneally. One week after the final injection, whole blood was collected by cardiac blood collection to obtain antiserum. This antiserum was sensitized with 10 million freshly cultured Salmonella typhimurium diluted 1000 times and suspended in an equal amount of physiological saline at 37 ° C. for 30 minutes. The antibody-sensitized bacteria were washed 3 times with physiological saline and then reacted with anti-mouse IgG rabbit antiserum at room temperature for 30 minutes. After washing again with physiological saline three times, 100 μg / ml of soluble protein A was reacted at room temperature for 30 minutes. This was washed 3 times with physiological saline and mixed with 1 million antibody-sensitized cell suspension. After reacting at 37 ° C for 4 hours and then Giemsa staining, it was found that 100% of the cells incorporated 1 to 6 bacteria.

Example 2 Salmonella typhimurium obtained in the same manner as in Example 1 was treated with phosphate buffered saline (pH 7.2, 0.01M phosphate buffer + 0.15M NaC).
l) suspended at a rate of 100 million pieces / ml, add 1 ml of this to a beaker and add an equal amount of 100 μg / ml of soluble protein A, and stir with a magnetic stirrer while stirring 2.5% of glutaraldehyde to 3 ml. Addition and react for 30 minutes at room temperature.
This bacterium was washed three times with physiological saline and reacted with antibody-sensitized cells in the same manner as in Example 1. As for the uptake of bacteria by the cells, almost the same results as in Example 1 were obtained.

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Claims (5)

[Claims] 1. A microbial cell coated with protein A that can be incorporated into an antibody-sensitized floating cell. 2. The microbial cell according to claim 1, wherein Protein A is bound to the microbial cell via an antibody. 3. The microbial cell according to claim 1 or 2, wherein the microbial cell is Salmonella typhimurium. 4. A method for incorporating microbial cells into antibody-sensitized floating cells, which comprises coating the microbial cells with protein A. 5. The method according to claim 4, wherein the antibody-sensitized floating cells are mouse Ehrlich cancer cells or mouse myeloma cells.