JPH026023B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to an immune reaction stabilizer and a method for measuring an antigen-antibody reaction. For more information,
Stabilizers and antigens that enhance the specificity of immune responses
It concerns the improvement of antibody reactions. In recent years, in various fields such as medicine, biochemistry, hygiene, and epidemiology, quantifying different substances, especially antigens and antibodies, has become an extremely important issue. Conventionally, such a method has been widely used, in which latex sensitized with an antigen or antibody is reacted with the antibody or antigen on a glass plate, and the state of agglutination is observed with the naked eye. Additionally, measurement methods using turbidimetry and colorimetry, such as reacting a diluted antibody with a sample containing the corresponding antigen and utilizing light scattering of the antigen-antibody complex formed, have also been used for a long time. Furthermore, in the immune antigen-antibody reaction system, radioimmunoassay method [Radioimmunoassay Methods, p. 481, 1971] is used as a method for sensitizing and assaying the antibody to a water-insoluble carrier.
2010, UK reference], Oxygen immunoassay [Clinical Chemistry, vol. 22,
1243-1255, 1976], optical measurements by light scattering or turbidimetry, and agglutination reactions are known. [For example, Croatia Chemica Acta
(CROATIA CHEMICA ACTA) Volume 42, 457-
466 pages, 1970, Yugoslavia; European Journal of Biochemistry, Volume 20,
No. 4, pp. 558-560, 1971, German State and Immunochemistry, Vol. 12, 349
- p. 351, 1975, United Kingdom] In such an immune reaction system, the rapidity of the reaction,
Although it has advantages such as high sensitivity and ease of operation, it has the problem of unfavorable non-specific reactions other than the main reaction caused by non-specific aging factors contained in the specimen. Such non-specific reactions significantly inhibit the specificity of the immune reaction, often providing inaccurate results and requiring complicated manipulations for confirmation. In addition, in order to prevent non-specific reactions caused by the Fc portion, an immune reaction system in which only the F(ab') ₂ portion is used as an antibody and sensitized to a carrier is already known. (For example, Journal of Biochemistry, Vol. 81, p. 1557, 1977)
Year and European Journal of Immunology
5, p. 274, 1975) Antibodies frequently used in immunochemical reactions are of the immunoglobulin G (IgG) class, and the IgG molecule is composed of two parts called Fab and a part called Fc. There is. Regarding antibody activity, the Fab portion contains an antigen-binding site that indicates the antibody's specific activity, and the Fc portion is involved in complement fixation, human sensitization, and rheumatoid factor binding. Each fraction of IgG has F(ab′) ₂ ,
It is also indicated by symbols such as Fab', Fab, Fd.Fc. F(ab') ₂ is obtained by digesting immunoglobulin with pepsin, and when this is reductively cleaved with mercaptoethanol, it is dissociated into Fab'2 pieces. These are also regenerated to F(ab') ₂ by oxidation. Fab is obtained by decomposing immunoglobulin with papain, which is further reduced and alkylated to obtain Fd. However, the present inventors discovered that there are unfavorable non-specific reaction factors not only in immunoglobulin but also in reactions using F(ab') ₂ antibodies, and conducted extensive studies to prevent this reaction. As a result, we have arrived at the present invention. That is, the gist of the present invention is as follows: 1. F(ab') ₂ fraction of immunoglobulin, Fab' fraction,
An immunoreaction stabilizer containing a constituent fraction of Fab fraction or Fd fraction as an active ingredient; and 2. An antigen-antibody that reacts the antigen or antibody to be measured with the corresponding antibody or antigen in a reaction medium. In the reaction measurement method, the reaction between an antigen and an antibody is measured using the F(ab') ₂ fraction, Fab' fraction, or Fab of immunoglobulin that does not involve the antibody to be measured or the antibody corresponding to the antigen to be measured. It is carried out in the presence of the constituent fractions of the fraction or Fd fraction, or the sample containing the antigen or antibody to be measured is brought into contact with the constituent fractions, and the reactants of the constituent fractions are separated and removed. A method for measuring an antigen-antibody reaction is provided. The present invention will be explained in detail below. As an immunoglobulin, immunoglobulin M
(IgM), immunoglobulin A (IgA), immunoglobulin D (IgD), immunoglobulin E (IgE) and IgG
, but those equivalent to IgG are usually used. Immunoglobulin can be introduced into its constituent fractions using conventional methods such as decomposition with proteolytic enzymes such as pepsin and papain, cleavage of S-S bonds with mercaptoethanol, and other chemical modification methods. You can do it by leaning. The constituent fractions of immunoglobulin used in the present invention (hereinafter referred to as "the constituent fractions") are antibodies or antigens (hereinafter referred to as "reactants") that are reacted with antigens or antibodies to be measured (hereinafter collectively referred to as "measurements"). It is preferable to use the same species as the animal from which the immunoglobulin was derived, but immunoglobulin components that are immunologically related may also be used. In addition to this constituent fraction, the stabilizer of the present invention includes a solvent,
It may contain a buffer, serum albumin, egg albumin, collagen fiber-derived protein, and the like. In particular, those containing the latter three proteins are preferable because they can maintain the collagen concentration and stabilize the reaction when diluting the specimen, and can also be expected to neutralize certain non-specific reactants in the specimen. It is. The stabilizer of the present invention is usually used by mixing it with a sample containing the analyte, but if the reactant is supported on blood cells, etc., the stabilizer is mixed with the supported material. It can also be freeze-dried in advance. The amount of the stabilizer of the present invention to be used varies depending on the magnitude of the non-specific reaction, but may be used when reacting the analyte and the reactant in the reaction medium or during the contact treatment between the sample and the component fraction. The amount of this constituent fraction used is usually
It is 0.005% to 1%. If the amount of the stabilizer of the present invention used is too small, the effect of suppressing undesirable non-specific reactions will be uncertain, and if the amount is too large, there will be no particular effect and the reactivity of the main reaction will actually decrease. This is undesirable because it causes problems and impairs economic efficiency. To measure the antigen-antibody reaction using the method of the present invention,
This can be done by a conventionally known method. These methods include the so-called solution system, in which both the analyte and the reactant are soluble, and the so-called carrier system, in which the reactant is sensitized (supported) on a particle carrier that is practically insoluble in the reaction medium. It can be broadly classified. For example, in a solvent system in which both the reactant and the analyte are reacted in a soluble form, it is preferable to contact the non-specific reaction factor present in the analyte with the insoluble or soluble main constituent fraction in advance, so that the reactant of the main constituent fraction is It can be separated and removed and subjected to the reaction, or it can be reacted in the presence of this constituent fraction. Further, in a carrier system, pretreatment of the analyte as in a solution system is preferable, but the reaction in the presence of the constituent fractions is also possible. Reaction media include aqueous media such as water and buffers, which may contain additives such as stabilizers, preservatives, chelating agents, surfactants, and the like. Examples of the buffer include tris[tris(hydroxymethyl)aminomethane]-hydrochloric acid buffer, tris-malate buffer, ammonia buffer, borate buffer, glycine buffer, and the like. Also,
It is also possible to directly use a specimen such as urine as a reaction medium. Examples of antigens to be measured and reactants include proteins, polypeptides, steroids, polysaccharides,
Various substances include lipids, flower powder, dust, and haptens. Furthermore, examples of antibodies include proteins that are antibodies against the above-mentioned antigens. Next, the carrier system will be explained as an example. The particulate carriers used in this method are usually particles that are substantially insoluble in aqueous media. Materials for such particulate carriers include organic polymeric substances such as polystyrene, styrene-butadiene copolymers, polyacrylic esters, polymethacrylic esters, acrylonitrile-butadiene-styrene copolymers, and polymers such as acrylic acid,
Latex of polymers activated by copolymerizing methacrylic acid, acrylamide, etc. and introducing carboxyl groups or amide groups; Inorganic oxides such as silica, silica-alumina, and alumina; Mineral powder; Metal powder; Blood cells or grapes Examples include coccus-type bacteria such as cocci and streptococci, Bacillus marcescens or Rickettsia and their fragments. The average particle size of these fine particle carriers is usually 0.05 to 10 μm, preferably 0.1 to 1.0 μm, and particularly preferably 0.2 to 0.8 μm. If the particle size of the particulate carrier is too large, the measurement range will be narrowed or it will become more unstable, and if the particle size of the particulate carrier is too small, there will be a large economic loss in reagent preparation.
Furthermore, it becomes difficult to increase the sensitivity, so neither is preferable. The concentration of these particulate carriers is usually at the final concentration.
It is 0.03-0.3%, preferably 0.05-0.2%. If the concentration of the particulate carrier is too high, the measuring device using the reagent of the present invention becomes complicated and there is no particular effect, and if it is too low, the sensitivity will be low and the reactivity will be weakened, which is not preferable. For antigen or antibody sensitization to the fine particle carrier,
Any of the well-known methods, ie, physical adsorption of the antigen or antibody to the microparticle carrier or chemical bonding of the antigen or antibody to the microparticle carrier, may be used, or a combination of both may be used. The amount of antigen or antibody to be sensitized to the carrier may be appropriately selected and determined depending on various conditions such as the type thereof and the accuracy of measurement intended for the reagent. The particulate carrier sensitized with antigen or antibody may be treated with other stabilizers prior to suspension in an aqueous medium. As such stabilizers, amino acids, polypeptides, proteins, etc. that are not involved in the target immune reaction are used, and serum albumin is particularly suitable. Such treatment with a stabilizer can be performed by a conventionally well-known method. Stabilizer treatment is preferred in terms of stability during storage and reaction;
The effect is particularly great when the antigen or antibody is physically adsorbed onto the carrier. The pH of the aqueous medium is usually 5 to 10, preferably 6 to 10.
9.5, most preferably 6.5-8.5. of aqueous medium
If the PH is too low, the sensitivity will increase, but it may become unstable, and if the PH of the aqueous medium is too high, the sensitivity will be low and it may become unstable during storage, which is not preferable. The concentration at which the particulate carrier sensitized with an antigen or antibody is suspended in an aqueous medium is usually 0.01 to 20%, preferably 0.05 to 10%. If the concentration is too low, the stability will be low, which may be inconvenient and restrictive in use, and if the concentration is too high, the stability will be decreased, which is undesirable. Examples of methods for measuring the immune reaction include a method in which the reaction is performed on a slide or in a test tube and observed with the naked eye, and a method in which the reaction is performed in an optical cell and optically measured. To measure the immune reaction, the following methods may be used. First, if necessary, the above-mentioned microparticle carrier solution is diluted with a buffer solution, and the concentration of the antigen or the antibody-sensitized carrier, or both, is adjusted to be convenient for measuring the immune reaction. The concentration varies depending on the type, concentration, reactivity, and other conditions of the antigen or antibody to be measured, so conditions must be determined appropriately, but in general, immune reactions are measured optically. In this case, the final concentration is usually 0.03% by weight or more, preferably 0.05 to 1% by weight, particularly preferably 0.1 to 0.5% by weight. In addition, when attempting to observe the immune response with the naked eye,
Usually the final concentration is 0.05-0.8%, preferably 0.1-0.5
%. Buffers used for dilution include Tris-hydrochloric acid buffer, Tris-malate buffer, ammonia buffer, borate buffer, glycine buffer, and the like. The pH of the buffer solution is usually 7.6 to 9.6, preferably 8.0 to
Set it to 9.0. If the pH of the buffer solution is too low, the sensitivity will be high, but it may tend to aggregate and become unstable, and if the pH is too high, the sensitivity will be low and the shelf life at high temperatures may deteriorate. Undesirable. In this way, a conventionally well-known sensitized particle reagent for immune reactions can be obtained. The immune reaction can be measured using this reagent by a conventional method. That is, when observing an immune reaction with the naked eye, for example, a test solution containing a predetermined amount of a reagent, a predetermined amount of the stabilizer of the present invention, and an antigen or antibody is placed on a slide, in a test tube, or on a microplate. What is necessary is to mix the sensitized particles and observe the aggregation of the sensitized particles. In addition, when trying to optically measure an immune reaction, a predetermined amount of reagent and a test solution containing an antigen or antibody are mixed and changes in light transmittance, scattered light, or turbidity are measured in an optical cell. Just measure it. The light may be monochromatic or polychromatic. The thickness of the optical cell used for measurement is usually 0.2 to 10
mm. According to the stabilizer and method of the present invention, non-specific reactions that occur when attempting to immunochemically measure an antigen or antibody can be suppressed, and the specificity of the measurement can be increased. EXAMPLES The present invention will be described in more detail with reference to Examples below, but the present invention is not limited by the Examples unless it exceeds the gist thereof. In the examples, "%" is based on weight unless otherwise specified. Example 1 Surface area of latex particles 1.52×10 ^-9 cm ² 800
1400 anti-alpha-fetoproteins (AFP) from
A rabbit antibody (F(ab') ₂ K=5.40×10 ⁹ L/M) was sensitized and a latex reagent was prepared. AFP samples of various concentrations were prepared with physiological saline containing 0.1% bovine serum albumin (BSA), and reactivity was examined using near-infrared turbidimetry. This standard reaction curve is shown in FIG. This optical measurement method (LPIA or simply PIA)
The turbidity of the reaction solution changes depending on the strength of the reaction with the corresponding antigen, and the antigen-antibody reaction rate can be quantitatively determined by measuring absorbance at a specific wavelength, for example, 940 nm. Since a non-specific reaction occurs in substantially the same way, a negative specimen that does not substantially contain antigen or is below the detection limit will be judged as if it were positive. Example 2 Anti-human fibrinogen antibodies were prepared using immunoglobulin and F(ab') ₂ fractions as in Example 1, and a standard curve was created in the same manner. Standard fibrinogen was prepared in 0.1% BSA-physiological saline containing 0.02% purified gelatin. The results are shown in Figure 2. Example 3 An anti-human myoglobin antibody was also prepared in the same manner as in Example 1, and a standard curve was created. The results are shown in Figure 3. Example 4 An anti-human β ₂ -microglobulin rabbit antibody was prepared as in Example 1. A β ₂ -microglobulin preparation was prepared in a physiological saline solution containing 0.05% purified egg albumin. Example 5 Non-rheumatoid serum, patient serum requested for rheumatoid agglutination test, and patient serum clearly diagnosed with rheumatoid arthritis and determined to be () by rheumatoid agglutination test were diluted 4 times with 0.1% BSA-physiological saline solution. the
Put 200 μl into a 7 mm x 7 mm cuvette and prepare Example 1.
50 μl of the latex reagent prepared above was injected and reacted for 1 minute, and the change in the amount of transmitted light (wavelength 940 nm) per hour was measured during that time. The results are shown in Table 1.
Positive cases were confirmed by radioimmunoassay (RIA) using a two-antibody method. Example 6 Immunoglobulin constituent fractions from the same animal in which the antibody was produced were added to the specimen that gave a false positive result in Example 5 at a final concentration of 0.005 to 2%, and the reaction was carried out in the same manner as in Example 5. (The results are shown in Table 2.)

【table】

[Table] * Same sample ** See Table 3 When using antibody IgG sensitized latex reagents, a positive coagulation reaction is usually seen in 20 to 30% of healthy people, but F
Even with the latex reagent prepared with the (ab') ₂ antibody, false reactions appeared in approximately 1 to 4%. Inject immunoglobulin fractions from normal rabbits into these samples.
When 0.005 to 0.5% was added, false positives disappeared and the specificity of the latex agglutination reaction could be highly improved (Table 2). Example 7 It was also possible to react qualitatively the latex reagent prepared in Example 3 on a glass slide. That is, one drop (approximately 40 μl) of the sample that showed a strong pseudoreaction with PIA was dropped onto a glass slide, and further F
(ab′) ₂ fractions of 0.2% BSA-physiological saline solution
One drop of myoglobin (Mb) latex reagent was added and mixed, and the mixture was allowed to react for 3 minutes while shaking before and after, and the presence or absence of aggregation was examined. Since the amount of Mb in normal human serum is as low as approximately 30 ng, a slide reaction will not produce a positive reaction with a normal sample. but
When the sample that showed a reaction equivalent to 550 ng with PIA was reexamined on a slide, it showed an agglutination reaction in 1 minute and 30 seconds.
The reaction was reversed by the above treatment. Example 8 Enzyme immunoassay (solid-phase method) also uses Fab or F(ab') ₂ fractions as sensitizing antibodies, but in such cases, false reactions occur with certain types of specimens. In such specimens, false reactions are completely eliminated by treatment of the specimen with normal immunoglobulin, preferably Fab fraction or F(ab') ₂ fraction. Specifically, a buffer solution prepared so that normal rabbit F (ab') ₂ fraction is 0.05 to 0.2% is placed in a 0.5 μl test tube, 20 μl of pseudo-reactive serum is added and mixed, and anti-AFP rabbit is added to the treated serum sample. Add one F(ab') ₂ antibody-sensitized bead and heat to 37°C for reaction. The following operations depend on conventional methods. (Ishikawa, Kato, History of Medicine
(Vol. 102, p. 58, 1977) Example 9 Cyanodiene bromide-activated cepharose (trademark of Pharmacya Fain Chemicals Co., Ltd.) 4B1
PIA using anti-Mb guinea pig F(ab′) ₂ antibody combined with 15 mg of normal guinea pig F(ab′) ₂ fraction to
The supernatant was mixed with the sample that showed a false positive result.
A re-examination was conducted at PIA. Treatment with goat F (ab') ₂ constituent fractions slightly reduced pseudoreactivity, but the guinea pig F
(ab′) The pseudo-reaction completely disappeared by treatment with _{the two} component fractions.

[Brief explanation of drawings]

Figure 1 shows PIA using anti-AFP sensitized latex.
It is a graph showing a standard curve of. Figure 2 shows PIA using anti-human fibrinogen antibody sensitized latex.
This is a graph showing the standard curve for antibody F(ab') ₂ sensitized latex, and ○... for antibody F(ab')2.
Results for IgG sensitized latex are shown. Third
The figure is a graph showing a standard curve of PIA using anti-human myoglobin antibody F(ab') ₂ sensitized latex.

Claims (1)

[Claims] 1. F(ab') ₂ fraction of immunoglobulin, Fab' fraction,
An immune reaction stabilizer containing constituent fractions of Fab fraction or Fd fraction as active ingredients. 2. The stabilizer according to claim 1, characterized in that it also contains serum albumin or egg albumin. 3. The stabilizer according to any one of claims 1 to 2, characterized in that it also contains a protein derived from collagen fibers. 4. In an antigen-antibody reaction measurement method in which an antigen or antibody to be measured reacts with the corresponding antibody or antigen in a reaction medium, the reaction between an antigen and an antibody is The antigen or antibody to be measured may be carried out in the presence of constituent fractions of the F(ab') ₂ fraction, Fab' fraction, Fab fraction, or Fd fraction of immunoglobulins that are not associated with antibodies corresponding to the antigen or antibody. 1. A method for measuring an antigen-antibody reaction, which is carried out after contacting a specimen containing the constituent fraction with the constituent fraction, and separating and removing the reactant of the constituent fraction. 5. The measuring method according to claim 4, characterized in that the reaction between the antigen and the antibody is carried out in the presence of serum albumin or egg albumin. 6. The measuring method according to any one of claims 4 to 5, characterized in that the reaction between an antigen and an antibody is carried out in the presence of a protein derived from collagen fibers. 7. The measuring method according to any one of claims 4 to 6, characterized in that a soluble antigen is used as the antigen to be measured, and a soluble antibody fraction is used as the antibody corresponding to the soluble antigen. 8. The measuring method according to any one of claims 4 to 6, characterized in that the antibody or antigen corresponding to the antigen or antibody to be measured is sensitized to a biological cell carrier. How to do it. 9. The measuring method according to any one of claims 4 to 6, characterized in that an antibody or antigen corresponding to the antigen or antibody to be measured is sensitized with a synthetic carrier. how to. 10. The measuring method according to any one of claims 4 to 9, characterized in that the reaction between an antigen and an antibody is optically measured. 11. The measuring method according to any one of claims 4 to 9, characterized in that the reaction between an antigen and an antibody is measured radioimmunologically. 12. The measuring method according to any one of claims 4 to 9, characterized in that the reaction between an antigen and an antibody is measured enzyme-immunologically. 13. The measuring method according to any one of claims 4 to 9, characterized in that the reaction between an antigen and an antibody is measured fluorescently immunologically.