JPS6122262B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a detection reagent for HB _s A _g .
More specifically, specific antibodies against _HBsAg related to hepatitis B (hereinafter referred to as anti _{- HBs} ) are obtained from plasma or serum immunoglobulin ( _IgG ) or purified globulin obtained by fractionating these. We selectively obtained electrophoretically "slow" immunoglobulin fractions, which were newly found to have a concentration of
The present invention relates to the production of an HB _{s Ag} detection reagent for reverse passive antibody agglutination reaction with less nonspecific agglutination by sensitizing a particulate carrier with an immunoglobulin fraction of the present invention. HB _s A _g exists in human serum and is thought to be a so-called transfusion hepatitis virus or a substance closely related to the same virus. As a preventive measure against transfusion hepatitis, it is important to check the presence or absence of HB _s A _g in the blood to be transfused. To detect,
It is an important clinical testing method that is currently widely used.
Conventionally, _HBsAg in serum has been determined by the Ouchterlony method using anti- _HBs serum, or by counter electrophoresis (Counter-electrophoresis) _.
electrophoresis), complement fixation reaction, and sensitized hemagglutination inhibition reaction. however,
These conventional methods require a long time for determination and require complicated procedures. Recently, when latex adsorbed with anti- _HBs globulin was mixed with serum to be tested and gently shaken for several minutes, agglutination of the _latex occurred in serum containing _HBsAg , and _HBsAg was _removed . It has been reported that it is possible to distinguish between the two because it does not occur with serum that does not contain it, and it was created using this principle.
_{HBsAg detection} reagents are commercially available. However, this reagent also has the following drawbacks, and it is considered that it cannot at least replace the various testing methods described above.
It is not particularly sensitive than other methods, and sera that clearly contain _HBsAg by _other methods often test negative. _Human serum that does not contain _HBsAg may be determined to be positive due to an unspecified nonspecific agglutination reaction. As described above, there is still no detection method that can simply, quickly, and accurately detect HB _s A _g , and there has been a demand for the establishment of such a HB _s A _g detection method and detection agent. Based on this technical background, the present inventors investigated a method for obtaining high-concentration anti _-HBs globulin, and as a result, the objective specific antibody,
We have discovered that a reagent obtained by obtaining anti- _HBs and sensitizing it to a granular carrier is a detection reagent for _HBsAg for reverse passive carrier agglutination reactions that causes less _nonspecific agglutination than ever before, and has thus arrived at the present invention. did. The present invention involves electrophoretically separating "slow I _g G" belonging to the γ position (particularly preferably the more basic γ position) from immunoglobulin obtained from anti- _HBs positive plasma or serum. , a specific antibody (anti- _HBs ) _{against HBsAg} is obtained, and the specificity consists of a particulate carrier sensitized with the specific antibody against _{HBsAg .
The} present invention provides a reagent for detecting _HBsAg . In general, immunoglobulins (I _g G) mainly belong to the γ position electrophoretically, but some of them extend to the α position, and those belonging to the α and β positions are called "first I _g
G (Fast I _g G)", those belonging to the γ position are "Slow I _g
It is called ``G (Slow I _g G)''. For example, according to the findings of the present inventors, it has been found that tetanus antibodies have the highest antibody titer in the basic portion of slow _IgG (Presentation at the 23rd Society of Blood Transfusion). As described above, even when viewed electrophoretically, antibodies are localized specifically depending on their type, and some antibodies have antibody activity concentrated in the first _IgG . On the other hand, with regard to anti- _HBs , the electrophoretic distribution of its activity has been unknown until now, and in particular the distribution from the viewpoint of specificity in antigen-antibody reactions has been completely unknown. In order to solve this problem, the inventors developed a hyperimmune anti-
After repeated research using HBs _- positive human plasma, anti-inflammatory
We have discovered for the first time that _HBs is localized in slow _IgG , and is particularly concentrated at the γ position, which is electrophoretically highly basic. Furthermore, when a particulate carrier is sensitized with this antibody fraction and an antibody agglutination reaction is performed through an antigen-antibody reaction, the reaction is extremely highly specific compared to sensitization with antibodies obtained by conventional methods. I found out. As is well known, immunoglobulin is produced using plasma or serum as a starting material, using ethanol fractionation, ammonium sulfate fractionation,
It can be obtained by ribanol fractionation, ion exchange chromatography, isoelectric focusing, or a combination of these methods. In general, immunoglobulins are divided into fractions with different electrophoretic behavior, and α
Fractions such as ₂ , β ₁ , β ₂ , γ ₁ and γ ₂ are known. Fractionation methods for these α, β, and γ positions include cation exchange chromatography [Immunochemistry 4 , 101 (1967)] and anion exchange chromatography [Transfusion]. (Trans fusion) 6 , 146 (1966)] and block electrophoresis [Electrophoresis Experimental Methods, p. 303, edited by the Electrophoresis Society (Bunkodo)] and isoelectric focusing [Biophysical Chemistry 14 , (3) ), 21, 209, (1969)] are well known. In the present invention, immunoglobulin obtained from human anti- _HBs positive plasma or serum according to a conventional method is fractionated by a known method, and the γ-position (γ ₁ ,
γ ₂ ) fraction, preferably a more basic fraction consisting of γ ₂ . Among the above-mentioned known fractionation methods, the most preferred fractionation method in terms of the reagents, operations, and conditions used is ion exchange chromatography. However, immunoadsorption methods can also be used, provided one uses the highly purified HB _{s Ag} required and anticipates some specificity of the product and attenuation of the antibody titer. Although the conditions for fractionation by ion exchange chromatography are selective depending on each carrier,
In any case, a fraction in which anti- _HBs is concentrated and is electrophoretically localized in slow _IgG can be collected from the fractionated fractions. For example, when using a cation exchanger, 0.2 to 0.005M;
Immunoglobulin is adsorbed onto a carrier equilibrated with a phosphate buffer solution of pH 4 to 7, and the immunoglobulin is eluted while increasing the salt concentration. From the eluate, the fraction containing the above-mentioned target antibody is examined and separated. Also, when using an anion exchanger, 0.1~
The carrier is equilibrated with a phosphate buffer of 0.005M and pH 7 to 10, and immunoglobulin is adsorbed and fractionated and eluted while increasing the salt concentration. The thus obtained slow fraction of γ-globulin is then sensitized to a particulate carrier.
A reagent _for detecting _HBsAg is provided. As particulate carriers, conventionally known latex resins, rubbers, inorganic adsorbents, and red blood cells of animals (e.g., sheep, guinea pigs, type O humans, chickens, etc.) fixed with formalin, glutaraldehyde, etc. are used. Good too. Such particulate carriers have a particle size of less than about 20μ, especially 5 to 15μ
It is advantageous to use something of the order of μ. These particulate carriers are generally preferably used suspended in an aqueous solvent. Examples of the aqueous solvent include water, physiological saline, and various buffer solutions (eg, glycine buffer, borate buffer), and the like. It is preferable to suspend the particulate carrier in an aqueous solvent in an amount of usually about 0.3 to 1% (by volume) and adjust the pH to about 7.0 to 8.9, particularly 7.2 to 8.0. Treatment for sensitizing such particulate carriers with antibodies can be carried out according to methods known per se. It is preferable to contact the particulate carrier and the antibody in an aqueous solvent (e.g., water, physiological saline, various buffer solutions, etc.),
Generally, this is carried out by mixing an antibody-containing solution and the above-mentioned particulate carrier suspension. This sensitization treatment is generally preferably carried out at a pH of about 4.5-8.6 and a temperature of about 20-37°C. The reagent for detecting HB _s A _g obtained in this way is
It has extremely high specific reactivity and provides great benefits in detecting _HBsAg , and can be said to be extremely effective in preventing post-transfusion _hepatitis , estimating the prognosis of hepatitis patients, and determining therapeutic efficacy. EXAMPLES Below, the present invention will be explained in more detail with reference to Examples. Example Starting from anti- _HBs positive human plasma 1, 8.2 g of crude immunoglobulin fraction was obtained according to Cohn's ethanol fractionation method. The anti- _HBs antibody titer of this product using the PHA method (passive hemagglutination assay) was 1:8000 when measured using a 1% solution. Next, 1 g of this immunoglobulin was dialyzed overnight against 0.01M phosphate buffer (PH6.0), and then a CM-cellulose (manufactured by Serva) column (3 cm in diameter and 30 cm in length) equilibrated with the same buffer. It was then adsorbed on the plate and thoroughly washed with the same buffer used for equilibration. Next, 0.01M PH6.0 phosphate buffer and
Concentration gradient elution was performed using a solution prepared by adding 0.4M NaCl to a 0.01M, PH6.0 phosphate buffer. The early eluting fraction was identified as the fast _IgG fraction, the late eluting fraction as the slow _IgG fraction, and the intermediate between them as _{the middle IgG fraction} . After pooling each, they were concentrated under reduced pressure as necessary to standardize the concentration to 1%, and when the anti- _HBs antibody titer was measured using the PHA method, it was only 1:64 in the first _IgG fraction, but Slow I _g G
The fraction showed 1:64000 and was purified 8 times. When immunoelectrophoresis is performed using a 1% solution of each fraction, the slow I _g G fraction, in which anti- _HBs activity is concentrated, migrates toward the cathode side among the immunoglobulins, and the fast I _g G fraction has concentrated anti-HBs activity. The fraction migrated to the most anode side. The middle _IgG fraction migrated to a position intermediate between the two. Slow I _g G fractionated with the above CM-cellulose
_Ig purified by affinity chromatography [vox Sang, 29, 319, (1975)] using the _G fraction and the crude immunoglobulin shown above as starting materials.
G [affinity I _{g G} ] was used to sensitize sheep erythrocytes treated with glutaraldehyde with a certain amount, and the specificity of the obtained reagents was compared. That is, sheep red blood cells are washed four times with phosphate buffered saline and suspended in a 5% concentration solution. Add 2.5% solution of glutaraldehyde to this
%, the reaction is carried out at room temperature for 60 minutes, and the unreacted glutaraldehyde is removed by washing four times with the above-mentioned physiological saline to obtain fixed red blood cells. This fixed red blood cell was made into a suspension with a concentration of 5%, and the method of Imai et al. [Igaku no Ayumi, 78 , 759-760,
(1970)] and sensitized with various immunoglobulins. Table 1 summarizes the presence or absence of positive sera from 2,605 adult sera and the presence or absence of false positives in agglutination inhibition reactions using purified _HBsAg in order to investigate the specificity of antibody-sensitized _red blood cells. Among those sensitized with the first I _g G fraction, the number of false positives was extremely high (707), and among immunoglobulins, the first I _g G fraction contains components that cause such non-specific reactions. I know that there is. On the other hand, in those sensitized with _IgG purified by conventional affinity chromatography, there were 11 false _positives ;
When G is used, the improvement is even more remarkable,
It has decreased to about 1/3. The fast I _g G fraction removed in this way _has a high non-specific reactivity, while the slow I _g G fraction has about 1/57th of that non-specific reactivity. The effectiveness of the method of fractionation into a first I _g fraction and a first I g fraction is clear. Furthermore, the anti- _HBs fraction that was highly purified using conventional techniques also suggests the effectiveness of fractionating into slow _IgG and fast _IgG . 【table】

Claims (1)

[Claims] 1. Using a specific antibody against _HBsAg obtained by fractionating "slow _IgG " which belongs to the electrophoretically more basic γ position from immunoglobulin obtained from anti _{-HBs positive} plasma or serum. _A specific _HBsAg detection reagent consisting of a granular carrier that has been sensitized.