JPH02673B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to reagents for solid-phase immunoassays. As is well known, in solid-phase immunoassays, antigen-antibody reactions are carried out on a reagent in which antibodies or antigens are adsorbed onto a solid support (carrier) such as polystyrene beads (globules) with a particle size of approximately 3 to 10 mm. After the reaction, the solid support is removed and the antibody or antigen on the solid support is detected and its activity is quantified.
This is essentially different from the latex agglutination method, which utilizes latex agglutination accompanying antigen-antibody reactions. The detection and quantification of hepatitis antigens and antibodies will be described below as an example, but the present invention is of course not limited thereto. Hepatitis A is characterized by a short incubation period, usually 2 to 6 weeks, mild prodromal symptoms, and a relatively mild clinical presentation. The disease is commonly transmitted by contaminated food and liquids, but has also been shown to be transmitted by systematic inoculation. Hepatitis A was previously called "infectious hepatitis." In the United States, more than 50,000 cases of non-B viral hepatitis, generally considered to be mainly hepatitis A, are reported annually, and the actual number of cases in the United States is estimated to be about 1.2 million higher. Currently, there is no convenient special test method for hepatitis A antigen (HAVAg) or its antibody (anti-HAV).
Diagnosis must depend on the clinical symptoms along with the time or cause of onset, the patient's medical history, liver function tests, and the absence of signs of hepatitis B infection. Two recent discoveries have facilitated the development of specialized tests for this disease or symptom. The major effort in determining animal typing for hepatitis A research was in 1973 when the monkey monkey and later chimps were identified as hepatitis A viral infectious disease (HAV).
The culmination was reached in 1973 with the discovery by Huynston et al. of virus-like particles 27 mm in diameter associated with hepatitis A. Using immunoelectron microscopy (IEM), Huinstone et al. found these particles in fecal extracts of patients with acute hepatitis A, and found that these particles were detected not by the patients' preinfectious serum but by the same patients' post-inflammatory serum. It was shown that the mixture was agglomerated. The same HAV particles were subsequently identified in the serum and liver of infected primates. This initial painstaking biological and IEM method was soon followed by HAAg and anti-HAV.
A more practical immunological method for the detection of Provost et al. published a total immobilization test and demonstrated its effectiveness in detecting anti-HAV. Müller et al. and Moritsug presented an sensitive immunoadherence assay (IAHA) that can be applied primarily to anti-HAV detection. Hollinger et al. and Purcell et al. A radioimmunoassay (RIA) that is highly sensitive for the detection of both HAVag and anti-HAV in biological specimens.
announcing the law. Anti-HAV
The IAHA and RIA tests appear to be the two most useful methods for laboratory application. Dean Stark et al.
A comparative study of IAHA and RIA found that the two methods were comparable in sensitivity and specificity.
Although other studies have substantiated this finding, RIA
In addition to those detected by IAHA,
It detects other early antibodies specific to HAV (Pradley et al., J. Clin. Microbiol. 5 , 521-530).
(1977)). Hepatitis B infections are commonly transmitted by blood products or contaminated equipment such as needles, but also by fecal-oral contact. Previously, hepatitis B infectious disease was thought to have an incubation period of 6 weeks to 6 months. However, a short incubation period of about two weeks has been reported recently.
The disease is mild or asymptomatic, but if symptoms are present, the disease can be particularly aggressive. Premonitory symptoms include joint pain,
There is arthritis, rash, fever, anorexia, fatigue, and pruritus with or without jaundice. At least two distinct antigen-antibody systems are associated with hepatitis B: surface ( _HBsAg : anti- _HBs ) and core ( _HBcAg : anti- _HBc ). Hepatitis B surface antigen (HB _s Ag), found in blood as 22 nm globules and elongated bodies 22 nm in diameter and varying in length, is believed to represent the coat protein of the hepatitis B virus. 42nm containing DNA and DNA polymerase
The particles appear to represent a contagious virus (Dain particles). In the detergent, Dane particles decompose into a 26nm electron-dense core, HB _c Ag. The latter is found in the nuclei of hepatocytes during the intense stage of infection in patients with serum hepatitis. Patients with viral hepatitis B can therefore be expected to produce antibodies against the protein coat surface antigen (anti- _HBs ) and against the protein core (anti- _HBc ). After exposure, anti- _HBc often forms antigenic blood ( _HBs
At the height of liver dysfunction with anti-HBs (Ag), the onset of anti- _HBs also appears for as long as 12-20 weeks. Anti-HB _c generally undergoes long-term circulation of HB _s Ag, suggesting that anti-HB _c is produced in response to viral activity. _HBsAg , anti- _HBs and anti- _HBc are present alone in serum or co-exist together in individual samples. All three types are important in determining the course of hepatitis B virus infection. Diagnosis of hepatitis B also includes various tests such as immunodiffusion or agar-gel diffusion, countercurrent electrophoresis, total volume fixation, hemmagglutination and radioimmunoassay. be. The diagnostic method of choice for hepatitis A and B antigen and antibody detection is the solid-phase radioimmunoassay (RIA) because of its simplicity and sensitivity. This method provides a simple and rapid separation of bound and unbound immunoreactants used in most immunoassays. A drawback of solid-phase RIA for general hepatitis diagnosis, however, is that the hepatitis immunoreactant is relatively unstable when coated directly or indirectly onto a solid material. U.S. Pat. No. 3,790,663 to Garrison et al.
Reagents for detecting antigens for use in RIA are disclosed. Their technique coats an organic polymer with antiserum and air-dries it, creating an attractive shelf-stable reagent for antigens that can be compared to reagents coated with freshly prepared antiserum that has not been dried. It includes. Garrison et al. reported on antibodies attached to solid supports, which are convenient for detecting antigens. The present invention involves adsorbing an antigen or antibody onto an inert, non-suspending solid support, immersing it in a sugar-containing solution, separating the solid support from the sugar-containing solution, and leaving it in a non-frozen state. The present invention provides a method for producing a reagent for solid-phase immunoassays, which is characterized in that the reagent is dried for several months. The storage stability of the reagents according to the invention obviates the need for freshly prepared solid phases coated with immunoreactants, thus allowing their use in practical solid phase immunoassays in routine clinical measurements. The present invention relates to storage stable reagents useful in immunoassays for detecting and measuring antigens and antibodies. This reagent ideally consists of a solid support coated directly or indirectly with the antigen or antibody and stabilized with a sugar coating to impart storage stability. Indirect application of antigens or antibodies to a solid support generally involves coating the solid support with the antigen or antibody in advance to enable attachment of the corresponding antibody or antigen. For example, if a solid support is to be ultimately coated with a hepatitis A antigen, the support may first be coated with a hepatitis A antibody. In attaching an antigen or antibody to a solid support, either directly or simply, it is desirable to preserve either the binding capacity of the antibody or the antigenicity of the antigen. The method of the present invention provides a way to preserve the binding capacity or antigenicity of a bare immunoadsorbent by applying a sugar coating. The present invention has solved the stability problem that occurs when antigens and antibodies are attached directly or indirectly to solid phase materials. It is recognized that naked antibodies or antigens do not lose their binding capacity or antigenicity within a few hours and are essentially unusable in tests for the detection of the corresponding antibodies or antigens. One way to solve this problem has been to keep the coated solid support moist or moist by storing it in a buffered salt solution. Although this is a suitable way to preserve desire and anti-genie, the inconvenience and expense of maintaining this environment will be readily apparent. In immunoassays for the detection of antibodies, antigens with affinity for antibodies are attached to an easily handled solid support. Antigens can be attached directly to the solid support surface if they are obtained in a suitable pure state to give the appropriate concentration. Also, some antigens attach more easily to solid support surfaces than others. Antibodies with low affinity for the surface of the support can be attached using a pre-antibody membrane. In this case, the solid support is coated with antibody by conventional methods and then exposed to a source of antigen. In general, the antigen attaches more easily to the attached antibody, and the antigen does not require purification before exposure to the antibody membrane.
The affinity between antigen and antibody selectively secures antigenic substances, and antigen-carrying debris can be washed away. Solid support materials in the present invention include globules, tubes, wells and rods fabricated from materials such as glass, metal or plastic. Since the solid support is a support for solid-phase immunological tests, its size is essentially different from that of latex particles and the like used in agglutination reactions. In other words, they have a size that allows the number of solid supports in the reaction system to be easily determined, and unlike latex particles, they are not suspended in a static reaction system. A preferred embodiment of the invention uses polystyrene spherules. This material is readily available, easy to apply immunoadsorbents to, and easy to handle. It is important to remember that this invention demonstrates the stability of immunoadsorbent attached to a solid support and is not concerned only with solid supports. Immunoadsorbents contemplated within the scope of this invention include all antibodies and antigens that exhibit immunoreactivity. A most preferred embodiment of the present invention is to use antigens or antibodies with affinity for antibodies and antigens of either hepatitis A or B that are used as immunoadsorbents and are attached directly or indirectly to polystyrene globules by the methods described below. characterize. Sugars contemplated within the scope of this invention include monosaccharides, disaccharides and polysaccharides. Although the examples below demonstrate the specific effects of sucrose, other sugars have also been formulated and used in antigen-coated supports and have been found to be effective in preserving appetite and anti-antigenicity. For example, the following sugar solutions were made: Table % in Phosphate Buffered Salt Solution (PBS) Polystyrene globules exposed to spring water were washed three times in PBS.
This was then immersed in the formulation shown in the table for 30 minutes at room temperature. Except for a small amount of globules soaked in PBS only.
All other globules were removed and dried overnight at room temperature. The next morning, the globules were placed in an incubator at 37°C for 2 hours. Three negative control samples and three positive control samples for each set were tested for antigenicity using radiolabeled antibodies. The average count per minute ratio of the negative control sample to the positive control sample indicates the antigenic activity remaining on the globules. The following table shows these ratios showing the relative effectiveness of the sugar coatings used. Table 1 Wet PBS 36.9 6 Dextrin 26.8 2 Glucose 32.7 7 Sorbiturate 25.9 3 Lactose 29.8 8 Mannite 22.4 4 Sucrose 28.5 9 Dry PBS 7.9 5 Xyrite 26.8 The table above shows that various sugars help coat antigens and attached solid support It has been shown to protect and preserve the activity of The following examples will further demonstrate the utility of the invention. Example 1 Anti-HAV containing antiserum was diluted 1:500 to 1:6000 with 0.01M Tris buffer, pH 9.5.
Polystyrene pellets approximately 0.7 cm in diameter were added to this dilution. The coating operation was carried out for about 2 hours at room temperature.
The globules were then washed in Tris buffer, which
Positive for HAV-Ag and in 0.3M saline
Extracts of either liver or feces were diluted 1:5 to 1:100 with 0.01M phosphate buffered solution (PBS) at pH 7.5. HAV-Ag can be inactivated by formalin and heat treatment in the usual manner before use. HAV other than clarification by centrifugation
- No need for purification of Ag-containing extracts. The globules are HAV bound to the globules by the anti-HAV reserve membrane.
- coated with Ag. This HAV-Ag coating operation was carried out at room temperature for 24 hours. The globules were then washed in PBS in which they were stable and stored until use. To obtain stable dry globules, globules washed with PBS were coated with a 5% sucrose solution for approximately 30 minutes at room temperature and then air-dried. - Manufacture of labeled antigen reagent ¹²⁵ - Labeled antibody (anti-HAV) is prepared in the usual way, PBS, normal human serum 2% and Tween - 50% containing 200.2% and 0.005M.
It was diluted in fetal bovine serum and cultured at 45°C for 24-48 hours. Incubation can be shortened to 0.5-3 hours using high temperatures such as 56°C. Test method Serum or remineralized plasma is preferably used as a test sample for hepatitis A antibodies (anti-HAV). The antibody detection test against hepatitis A antigen is based on the principle of competitive binding between serum anti-HAV and radiolabeled anti-HAV against hepatitis A antigen (HAV-Ag). After mixing anti-HAV ¹²⁵ with patient serum in a test tray, a stable solid phase reagent to which HAV-Ag was conjugated was added. Overnight at room temperature or 45℃
After incubation for 1 hour, the cells were washed, and the count ratio of the globules was measured and recorded using a gamma radiation counter. A count ratio higher than the set cut-off value is negative for the antibody, whereas a lower count ratio indicates competitive binding between the added radiolabeled antibody and the indigenous antibody in the serum. The presence of anti-HAV can also be determined by calculating the percent neutralization of a patient sample compared to a test control sample. 50%
Percent neutralization greater than the cut-off value is indicative of the presence of antibody using the same logic as described above. Example 2 Preparation of a Stable Solid Phase Reagent Dean particle formulations of varying purity were treated with 2-mercaptoethanol (0.30-0.75%) and ca.
Treated with a nonionic detergent such as 2.5% Triton X-100 Nonidet P-40 for 1 hour at 37°C. The purpose of this treatment was to remove the fatty protein membrane of the Dean particles and reveal their core antigens. The conditions described above are preferred for this process, but may be varied without adverse effects.
After treatment, the mixture is buffered, e.g. 0.001 MEDTA
0.01M Tris-HCl in physiological salt solution containing
(PH7.1) and diluted appropriately. This solution was immediately used to coat solid surfaces such as spheres, tubes or wells made of plastic or glass. The Dein core (HB _c Ag) produced in this way is very sticky and easily adsorbs to solid surfaces.
If the dein grains produced are highly impure and contain large amounts of extraneous proteins, it may be necessary to pre-coat the solids with anti-HB _c before reacting with the dein cores as described above. Polystyrene spherules were mixed with Dain wick solution 24
When incubated for 72 hours, this method produces anti-
There was more dein wicking on the plain globules than on the HB _c precoated globules, especially when lower concentrations of dein wicks were used. It has been discovered that if the Dane wick is applied directly to a solid surface, the detergent concentration in the coating liquid must be very low (preferably less than 0.005%). To stabilize the obtained HB _c Ag-coated globules, 5-
A 10% sucrose solution was used. HB _c Ag globules were incubated in the above sucrose solution at room temperature for about 20 minutes and then air-dried. ¹²⁵ -Labeled Antibody Reagent Production Example A ¹²⁵ -labeled Deincore antibody (anti- _HBc ) against HB _c Ag was produced by a conventional method using 50% fetal bovine serum, 2% remineralized normal human plasma, and Tween. −20 in 0.04M EDTA buffer containing 0.4%
Diluted in 0.005M Tris with pH 7.3. Test Method Serum or remineralized plasma were preferred as test samples for hepatitis B antibodies (anti- _HBc ). Anti- _HBc detection test is hepatitis B core antigen ( _HBc Ag)
¹²⁵ -radiolabeled anti-HB c to globules coated with anti-HB _c and anti-HB _c present in patient samples.
It is based on the principle of competitive binding with Patient samples were incubated with ¹²⁵ -radiolabeled anti-HB _c and HB _c Ag coated globules in test trays for approximately 20 hours at room temperature. After incubation, the count ratio of washed globules was measured and recorded using a suitable gamma radiation detector.
A count ratio higher than the set cut-off value indicates the absence of anti- _HBc or an undetectable amount of anti- _HBc in the sample.A count ratio lower than the set cut-off value indicates the presence of anti-HBc in the serum. This indicates the existence of HB _c . The same method of solid phase stabilization described above can be performed when the hepatitis B surface antigen is coated directly or indirectly onto a solid phase material and the RIA is for an antibody against the hepatitis B surface antigen. The two methods in the examples depend primarily on the purity of the antigen coated on the solid phase. Highly purified antigen can be coated directly onto the microspheres without the need to pre-coat the antigen with antibody. Example 3 Guiana pig antiserum containing anti- _HBs was prepared in phosphate buffered salt solution (PBS, 0.01 M sodium phosphate, 0.15 M sodium chloride, PH 7.2) at 1:
Diluted to 1750. This solution was added to a flask containing polystyrene pellets with a diameter of 0.64 cm. This coating solution containing the small spheres was placed in a 45°C bath and heated to 45°C for 2 hours. The pellets were then washed twice with PBS (which was warm at room temperature). Then PBS containing 2% sucrose
The solution was used to coat the pellets at room temperature for approximately 15 minutes and allowed to air dry. Using this method, other globules were prepared with 2% lactose and 2% lactose.
It was immersed in glucose and PBS alone for about 15 minutes and coated in the same manner. All pellets were air-dried. Example 4 A radioimmunoassay for the detection of hepatitis B surface antigen was performed using radiolabeled antibodies on negative control samples, samples with HB _c Ag/ad antigens, and samples with HB _s Ag/ay antigens. The ratio of antigen-containing sample counts to that of negative control samples is shown in the following table for polystyrene globules coated with various sugars.

TABLE The data in the table show that various saccharides aid in coating and preserve the activity of the antibody-coated solid support. In addition, the data in the table shows that antibody-coated globules without protective sugar coats are HB _s Ag/ad and HB _s after heat treatment.
It shows that the activity against Ag/ay antigen is decreased and the activity is decreased even before heat treatment. Example 5 An enzyme immunoassay for detecting hepatitis B surface antigen was conducted using an anti-hepatitis B surface antigen-peroxidase conjugate. The samples were a negative control sample, a sample containing HB _s Ag/ad antigen, and a sample containing HB _s Ag/ay antigen.
The optical density at 492 nm of the antigen-containing sample minus that density of the negative control sample is shown in the following table for polystyrene globules coated with various sugars.

[Table] Sugar-free
Sucrose (in water) 0.513 0.420 0.496 0.374
The data in the table shows that various saccharides aid in coating and in protecting and preserving the activity of the antibody-coated solid support. Furthermore, from the data in the table, antibody-coated globules without protective sugar coating were observed before and after heat treatment.
It shows that the activity against HB _s Ag/ay antigen is decreased.

Claims (1)

[Scope of Claims] 1. After adsorbing an antigen or antibody to an inert, non-suspending solid support, this is immersed in a sugar-containing solution, and then the solid support is separated from the sugar-containing solution. 1. A method for producing a solid-phase immunological test reagent, which comprises drying the reagent in a non-frozen state. 2. The method according to claim 1, wherein the adsorbent is an antigen. 3 Antigen is hepatitis A antigen, hepatitis B surface antigen, hepatitis B
3. The method of claim 2, wherein the method is selected from the group consisting of core antigen and hepatitis e antigen. 4. The method according to claim 1, wherein the adsorbent is an antibody. 5 Antibodies are hepatitis A antibody, hepatitis B surface antibody, hepatitis B
5. The method of claim 4, wherein the antibody is selected from the group consisting of core antibodies and hepatitis e antibodies. 6. The method according to claim 1, wherein the solid support is plastic, glass or metal in the form of beads, tubes, wells or rods. 7. The method according to claim 1, wherein the solid support is polyethylene beads.