JPS6231301B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a rubella virus antigen, a method for producing the same, and a reagent for immunochemical assay using the antigen. The antigens of the present invention are used in the development of specialized immunoassay reagents useful for the rapid detection and quantification of rubella antibodies in test fluids. The antigens and reagents of the present invention are useful in establishing the immunological status of a patient (eg, a pregnant woman) and are also valuable in diagnostic planning.

The method commonly used for the detection of anti-rubella antibodies in test liquids is based on the antibody suppression effect of infant red blood cell agglutination by insoluble rubella virus particles. The essential steps of this method include absorption of the test fluid by kaolin to remove non-specific fatty protein inhibitors (all before testing for aggregation inhibition) and infant absorption to remove cross-reactive antibodies present in the fluid. There is serum absorption by red blood cells. This type of hemagglutination inhibition (HAI) test is quite reliable but is time consuming due to the serum pretreatment step mentioned above. Final results are usually not available until at least 5-7 hours after test fluid collection. Other antibody detection methods for rubella include, for example, H.
M. Mayer et al., Am.J.Clin. Pathol., 57 :803.
813 (1972).

Apart from the insoluble hemagglutinin used in the HAI test, conventional tests have been performed to obtain soluble rubella virus antigen. The method describes the identification of two major "soluble" antigens (termed theta and iota), but attempts to definitively isolate and characterize these antigens from rubella-infected cell cultures have been largely unsuccessful. Soluble antigens useful for the development of antigen-sensitized particles useful for the detection and quantification of antibodies against rubella have not previously been isolated.

According to the present invention, soluble rubella virus antigen is isolated from a medium that supports the growth of tissue culture cells infected with rubella virus. The antigen has a molecular weight of 40,000 to 60,000 daltons, is insoluble in 50% saturated ammonium sulfate, and exhibits beta mobility in immunoelectrophoresis.

More specifically, the novel antigen is characterized by the formation of a single line precipitate (as shown by the hemagglutination inhibition test) with human serum reactive with rubella virus. Furthermore, this antigen is characterized by having a specific rubella antigen activity (SRAA) of 500 to 10,000.

Purified antigens are isolated by processes including affinity chromatography; gel permeation chromatography and reciprocal reverse passive hemagglutination (RPHA) activity-based separation methods.

Immunological reagents of the invention include stabilized red blood cells, bentonites, collodium, cholesterol crystals, quartz, synthetic resins, various synthetic latexes, and immunologically inert substances such as liposomes made from phospholipids and sterols. This can occur when an antigen is used to sensitize particulate matter. The sensitized particles are used in a direct agglutination test, in which rubella antibodies present in a given test liquid are rapidly detected by observing particle agglutination phenomena and quantified by standard dilution methods. This passive aggregation method is similar to traditional HAI
It does not require removal of non-specific inhibitors or anti-erythrocyte antibodies from the test fluid as required by the method.

The sensitized particles of the present invention can also be used in radioimmunoassays (RIA).
It can also be used in enzyme immunoassay (EIA) methods. Furthermore, the soluble antigens of the present invention are expected to be useful in performing well-known immunoprecipitation assays.

The benefits associated with the use of the antigens and reagents of the present invention and the performance of immunological test methods will become apparent from the following detailed description.

In the present invention, soluble antigens are isolated from the culture medium of cells infected with rubella virus. Suitable cell types for growing tissue cultures to obtain antigens include baby hamster kidney (BHK-21), porcine stable kidney (PS), Serum Institute rabbit cornea (SIRC), and others well known in the art. be. Generally HAI
Tissue culture used for production of insoluble rubella hemagglutinin for testing (Stewart et al., NNJour Med. 276, No.
10 554-7 (1967)) are well suited for use in accordance with the present invention.

Separation of the antigen is accomplished by a two-step chromatographic separation of growth media components. As mentioned above, the culture medium is first concentrated, preferably by forced dialysis, and first coated with a solid phase conjugated or covalently bound with IgG derived from human serum known to contain antibodies that react with rubella virus. by affinity chromatographic separation through a column consisting of: Preferred column specific materials include agarose beads.

After washing away unbound substances, the antigen bound to IgG is eluted with a suitable buffer. A glycine-sodium hydroxide buffer with a pH of 8 or higher is preferred. Especially when using a buffer with a high pH, it is better to neutralize the eluted material.

Antigens can be separated from high molecular weight substances bound to an affinity column and eluted with a buffer by gel permeation chromatography using, for example, a Sephadex G-150 column. The effluent from the column is examined with an ultraviolet spectrophotometer at 280 nm and a reverse passive hemagglutination standard is used to identify fractions containing purified antigen. For agglutination, for example, red blood cells sensitized with human IgG from the same source as used to make the affinity column are used.

The antigen thus obtained is insoluble in 50% ammonium sulfate and has a precipitation coefficient of approximately 3.4S, as measured by Sephadex G-150 chromatography.
It has an estimated molecular weight of 40,000 to 60,000 and exhibits β mobility in immunoelectrophoresis.

This antigen has the characteristic of forming a single line precipitate in human serum that reacts with rubella virus in a hemagglutination inhibition test. Antigens are also clearly characterized by approximately
It has a specific rubella antigen activity (SRAA) of 500 to 10,000. The SRAA value referred to herein is indicated by the following criteria. Any crude tissue culture medium from the growth of rubella-infected cells represents the absorbance at 280 nm. A typical crude medium from infected BHK-21 cells exhibits an absorbance of approximately 1.1 when compared to water. The crude medium also has a relatively fixed titer as determined by RPHA. A typical crude medium from infected BHK-21 cells has a titer of 1:32. infected
A representative crude medium from BHK-21 cells shows a titer of 1:32. The "total A ₂₈₀ units" of material found in the crude culture medium is defined as the product of volume (ml) and absorbance at 280 nm. The reciprocal of the RPHA titer is
A SRAA is determined by dividing by ₂₈₀ units. Therefore,
SRAA is equal to the reciprocal of the RPHA titer divided by ₂₈₀ units of total A.

The following examples include (1) the production of "enriched" cell culture media containing the antigens of the invention; (2) the production of human IgG for use in affinity chromatography and reverse passive hemagglutination; (3) affinity (4) Preparation of a gel permeation chromatography column; (5) Purification of antigen from a "concentrated"medium; and (6) Preparation of red blood cells sensitized with rubella antigen.

EXAMPLE Preparation of Concentrated Medium Containing Antigen BHK-21 cells were plated as a monolayer in 20 roller bottles and inoculated with Gilchrist sp. rubella virus.
After 3-4 days of culture, the medium was harvested, zonally centrifuged, and the effluent was pooled. The effluent was concentrated 100 times in an Amicon DC-2 hollow fiber dialysis concentrator at 2-8°C. Centrifuge the concentrated material at 9000 rpm for 6 to 18 minutes.
It was made transparent by time processing. The obtained concentrated substance is at -20℃
It was stored in

EXAMPLE Preparation of IgG Human remineralized plasma with a rubella titer of approximately 1:640 was purified by precipitation and dialysis with ammonium sulfate according to the following sequence.

(1) Equal volumes of 150 ml of saturated ammonium sulfate and rubella-positive human remineralized plasma were mixed at room temperature with magnetic stirring at a rate of 6 to 10 ml per minute. The pH was adjusted to approximately 7.3 with sodium hydroxide.

(2) The mixture was stirred for about 1 hour, and the resulting precipitate was
The mixture was collected by centrifugation at 8° C. and 9000 rpm for 30 minutes.

(3) Put the separated precipitate into a dialysis tube with a pH of 8.0 at 0.01M.
Dialysis was performed against K ₂ HPO ₄ /0.01M KH ₂ PO ₄ buffer 2 for 48 hours with 2 changes of the same buffer 5 times.

(4) Collect the dialyzed substance and further
IgG purified and collected through DE ₅₂ diethylaminoethyl cellulose microgranules (pre-swollen) anion exchange resin was concentrated, clarified by centrifugation and stored.

(5) The final yield of recovered IgG is per 70ml of total serum.
It is 150 to 160 mg.

EXAMPLE Preparation of affinity gel Human IgG after activation of Sepharose 4b (Pharmacia) or a suitable agarose solid phase with 97% (Aldrich) cyanogen bromide in acetonitrile.
(produced in the example). The binding of IgG to the solid phase was performed using Kyuatrecasus (J.Biol.Chem.,
245 :3059–65 (1970), March SC et al. Analyt.
Biochem., 60 :149-52 (1974) (modified method).

Example Manufacture of Gel Permeation Column A gel permeation column used for purification of the antigen eluted from the affinity column was manufactured by the following method.

(1) 18 g of Cephadex G-150 (Pharmacia) was added to 1 part of 0.15M NaCl/0.02% NaN ₃ and 0.05M Tris-HCl pH 8.0 buffer, mixed, and allowed to swell at room temperature for 3 days to degas.

(2) A swollen gel slurry was added to the column, which had been filled with a buffer solution and partially drained, to create a hydrostatic pressure of 3-5 cm during packing.

(3) The column is operated using a peristaltic pump, and 4.8
Equilibrate with 2 bed volumes of buffer at a flow rate of ml/cm ² /hr and 6-8 of 0.2% Blue Dextran 2000.
A homogeneity test was conducted using 12 ml of the sample, and 12 ml fractions were collected.

Examples Purification of Antigen from Concentrated Media Purification of soluble antigen from concentrated solutions in Examples was carried out by (A) affinity chromatography and (B) gel permeation chromatography as follows: (A) Affinity Chromatographic Method (1) The affinity column of the example was warmed to room temperature and the buffer was injected from the top of the bed.

(2) Three bed volumes of the ultracentrifugation concentrate of the example were loaded into the column at a flow rate of about 1 ml/min.

(3) 5 bed volumes of 0.05M Tris-HCl PH8.0 buffer in 0.15M NaCl-0.02% NaN ₃
The column was washed at a flow rate of ml/min.

(4) Then 0.1M glycine in 0.15M NaCl-
Approximately 1 using 6 bed volumes of NaOH PH12 buffer
The column was eluted at a flow rate of ml/min.

(5) Immediately, 1N HCl was added dropwise to the eluted material and neutralized to pH 8.0 with constant stirring.

(6) The neutralized material was concentrated to 5 times the original volume in a single hollow fiber concentrator and clarified by centrifugation.

(B) Gel Permeation Chromatography The material eluted from the affinity column was chromatographed on a Sephadox G-150 column as described in the Examples. Molecular weight 40000
The eluted fraction, which was thought to contain substances with a concentration of 60,000 to 60,000, was collected and the RPHA titer was examined. Fractionated portions with titers equal to or greater than 1:6400 were combined. The SRAA value is the combined fractional part.
It can be determined based on A ₂₈₀ value and RPHA titration value. Generally, the antigen is 80 to 100% of the original growth medium.
The volume is concentrated from 3 to 8 ml and the volume is 500 to 500.
It has an SRAA value of 10000.

Example: Red blood cells sensitized to rubella antigen Human red blood cells
3715427 and/or 3925541. 2.0ml of 10% suspension
2 to 3 centrifugation at 500-1000rpm
It took a minute. Drain the buffer and bring the cells to pH4.0.
Resuspend in 2.0 ml of 0.01M acetate buffer.
Add chromium chloride aqueous solution (10mgCrCl・
6H ₂ O/ml) was added. After adding 0.05 to 0.50 ml of the antigen obtained from Example 5 to red blood cells, the suspension was
The cells were incubated at -32°C for 2 hours with stirring every 30 minutes.
The sensitized red blood cells were centrifuged to form small spheres and the supernatant was discarded. Red blood cells were washed twice by resuspending them in 0.1M phosphate buffer and centrifuging as described above. The pellets were resuspended in 0.1M phosphate buffer to give a 0.125% (V/V) suspension of sensitized red blood cells.

EXAMPLE Sensitized red blood cells essentially according to the example were used in antigen titer assays of serum samples from unspecified blood donors and the results were compared with titers obtained by the HAI method. 1336 serum samples were tested and the correlation coefficient (γ) was determined to be 0.99 by linear regression analysis. If sensitized red blood cells are used, there is no need to preabsorb the serum sample to remove antibodies that cross-react with foreign red blood cells. Furthermore, there is no need to pre-process the serum sample to remove non-specific fat protein inhibitors.

Numerous modifications and variations of this invention will occur to those skilled in the art. For example, antigens that sensitize various types of immunologically inert particles well known in the art, such as those useful in antigen-antibody assays, can be used. Particles sensitized in this way can be used for antibody detection by agglutination, radioimmunoassay, fluorescence and enzyme immunoassay. Additionally, particles such as red blood cells and liposomes can be sensitized to create a test article based on complement-mediated lysis.

Claims (1)

[Claims] 1. Forms a single line precipitate with human serum reactive to rubella virus in a hemagglutination inhibition test,
It has specific rubella antigen activity of 500 to 10,000, and 40,000
A method for producing purified rubella virus antigen having a molecular weight of 60,000 to 60,000, insoluble in 50% saturated ammonium sulfate, and exhibiting beta mobility in immunoelectrophoresis, the method comprising: chromatographic separation is carried out through the growth medium of a tissue culture of cells infected with rubella virus onto a column consisting of a solid phase to which IgG derived from human serum is covalently bound; (c) subjecting the separated eluate to gel permeation chromatography to separate substances based on molecular size;
and (d) a method for producing rubella virus antigen, which comprises the steps of isolating rubella antigen from the above-mentioned separated and eluted substance based on mutual reverse passive hemagglutination activity. 2. The method according to claim 1, wherein the solid phase in step (a) consists of agarose beads. 3. The method of claim 1, wherein in step (b) the eluting substance is a buffer solution with a pH greater than 8 and the method comprises the step of neutralizing the eluting substance before step (c). 4 Formed a single line precipitate with human serum reactive to rubella virus in a hemagglutination inhibition test,
Has specific rubella antigen activity of 500 to 10,000, and 40,000
A purified rubella virus antigen having a molecular weight of 60,000 to 60,000, being insoluble in 50% saturated ammonium sulfate, and exhibiting β-mobility in immunoelectrophoresis. 5 Formed a single line precipitate with human serum reactive to rubella virus in a hemagglutination inhibition test,
Has specific rubella antigen activity of 500 to 10,000, and 40,000
Immunochemistry characterized by consisting of individual particles having a molecular weight of 60,000 to 60,000, insoluble in 50% saturated ammonium sulfate, and whose surface portion has been activated by purified rubella virus antigen that exhibits β-mobility in immunoelectrophoresis. Measurement reagent. 6. The reagent according to claim 5, wherein the particles are red blood cells.