JPS6145777B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention utilizes the nephelometric method to detect haptens (haptens: antigens that do not exhibit antigenicity in vivo when used alone, and for which antigen-antibody reactions cannot be demonstrated in vitro using conventional methods). Author, Clinical Serology, page 2, Uchudo Yagi Shoten, 1966
year) on how to measure it. A wide variety of high-molecular weight and low-molecular weight substances exist in body fluids, and knowing the abundance of these substances has important clinical significance as well as for research purposes. Therefore, it is extremely important to develop a simple measurement method with excellent measurement sensitivity and accuracy. In general, high molecular weight substances with a molecular weight of 10,000 or more have in vivo antigenicity, so when administered parenterally to a foreign animal, immunoglobulins (antibodies) that specifically react against the substance are produced. . It is known that when this antibody is mixed with a corresponding high molecular weight substance in a test tube, the two bind to form a large molecular weight complex. The Hiro method is a method in which the reaction mixture is irradiated with light and the intensity of light scattered by the large molecular weight complex (light scattering intensity) is measured. The amount of large molecular weight complexes formed will change depending on the amount of the substance being used, and the light scattering intensity will change accordingly, so by using a standard curve prepared in advance for substances with known concentrations, It is possible to measure substances with unknown concentrations. Ritchie (1967) was the first to measure proteins in body fluids using the immunofluorescence method, and he measured IgG and albumin using antibodies prepared by immunizing domestic rabbits. A comparison method using a photometer or laser beam has been developed and is being widely applied in the field of clinical testing. Currently, substances that can be measured by the hyphenometric method include IgG, IgA, IgM, albumin, α ₁ -antitrypsin, α ₁ -acid glycoprotein, α ₂
- Covers almost all major plasma proteins such as macroglobulin, haptoglobin, ceruloplasmin, Gc-globulin, transferrin, β-lipoprotein, C ₃ , C ₄ , and fibrinogen, but is limited to high molecular weight substances. ing. On the other hand, in addition to the above protein components, there are many low molecular weight components in body fluids, and it is clinically extremely important to measure these substances. For example, it has been confirmed from many basic and clinical results that the amount of estrogen in pregnant maternal fluids reflects well the function of the fetoplacental system and is the most reliable fetal information. Measuring the concentration of administered drugs in body fluids is important for determining drug effectiveness and preventing side effects, and is also important for managing drugs whose use is prohibited by law. is important. Generally, low molecular weight substances are called haptens, and since no antibodies are produced even if they are administered alone to a living body, special consideration is required to prepare antibodies against haptens. In other words, rather than using hapten alone, a hapten bound to a high molecular weight carrier such as a protein (hereinafter simply referred to as carrier) must be administered to animals, and the antibodies produced in this way have a strong anti-hapten effect. Since it contains not only antibodies but also antibodies against the carrier moiety, the antibodies against the carrier moiety must be absorbed and removed to produce hapten-specific antibodies. Even if the antibody prepared in this way is mixed with a sample containing a hapten in a test tube, unlike when the sample is a plasma protein, a large molecular weight complex cannot be formed. This is because haptens have small molecules and are thought to have only one site on each molecule that can react with antibodies, so even if they react with the corresponding antibody, the resulting hapten-antibody conjugate cannot bind further, so large molecular weight complexes are not formed. Therefore, it has not been possible to measure haptens using the conventional pyrometry method that measures the intensity of light scattering by large molecular weight complexes. Under these circumstances, the present inventors have conducted extensive research and found that even if a hapten has only one antibody-binding site per molecule, it can be used to bind proteins, synthetic polymers, and other high-molecular substances in large numbers. By combining 1 to several dozen or more haptens, the properties in nephelometry will show reactivity similar to that of so-called multivalent substances, which have reactive sites equivalent to the number of haptens bonded to one molecule. Based on this knowledge, the present invention was completed. Adding an anti-hapten antibody to a fixed amount of a conjugate of a hapten and a high molecular weight substance (hereinafter abbreviated as hapten conjugate) while increasing the amount sequentially and measuring the light scattering intensity at each step shows the amount of antibody added. Up to a certain amount, the light scattering intensity increases as the amount added increases, but
When a larger amount of anti-hapten antibody was added, the light scattering intensity was found to gradually decrease depending on the amount added (FIG. 1). In Figure 1, the quantitative ratio of the hapten conjugate and anti-hapten antibody showing the highest light scattering intensity (point C in Figure 1) and to the right side (range a in Figure 1)
When a hapten corresponding to an anti-hapten antibody is added to a reaction system consisting of a quantitative ratio of a hapten conjugate and an anti-hapten antibody, a large molecular weight complex is formed by the hapten conjugate and the anti-hapten antibody. It was found that the amount of body, that is, the light scattering intensity decreased, and that the decrease in the light scattering intensity was inversely proportional to the amount of hapten added. Furthermore, to the left of point C in Figure 1 (first
When the corresponding hapten is added to a reaction system composed of the quantitative ratio of the hapten conjugate to the anti-hapten antibody under the conditions (range b) in the figure, up to a certain amount, the amount of hapten increases depending on the amount added. Although the light scattering intensity increased, when the amount of hapten added was further increased, the light scattering intensity gradually decreased depending on the amount of hapten added. The present invention applies the relationship between the amount of hapten added and the light scattering intensity as shown above to the determination of hapten in an unknown sample in a reaction system configured under the conditions a and b in Figure 1. be. The measuring method of the present invention is generally carried out as follows. That is, a certain amount of a hapten conjugate and a standard dissolved at an appropriate concentration are placed in a test tube, a certain amount of an anti-hapten antibody is added thereto, and the mixture is allowed to react for a predetermined period of time. After the reaction is completed, the light scattering intensity of the reaction mixture is measured using a nephelometer, and the relationship between the concentration of the standard product and the light scattering intensity is plotted on a graph to create a standard curve. Next, the light scattering intensity is measured in the same manner using a sample solution instead of the standard product, and the amount of sample is calculated from the standard curve prepared previously. In the present invention, the high molecular weight substance to which the hapten is bound is not particularly limited as long as it is water-soluble, but animal body fluid proteins such as bovine serum albumin, human serum albumin, and bovine γ-globulin, moderately polymerized polystyrene, polypropylene, etc. Synthetic polymers can be used. In addition, hapten conjugates are used to produce anti-hapten antibodies and to configure a reaction system in combination with anti-hapten antibodies; The hapten in the hapten conjugate does not necessarily have to be the same as the hapten to be measured;
They may be different substances that exhibit cross-reactivity with each other as long as the specificity of binding is not lost. Therefore, in this specification, the term hapten includes not only the hapten but also substances having the same identity as the hapten. Furthermore, the high molecular weight substance of the hapten conjugate for antibody production and the high molecular weight substance of the hapten conjugate for constructing the reaction system are not necessarily the same. The temperature and time for reacting the hapten conjugate, anti-hapten antibody, and sample solution are not particularly limited, but considering the sensitivity and accuracy of measurement and practical convenience, the reaction temperature is 15 to 45 °C, and the reaction time is 5 to 120 °C.
It is preferable to set it to about minutes. The measurement reagent kit of the present invention comprises at least the following reagents: a hapten conjugate solution and b anti-hapten antibody solution. As mentioned above, the hapten conjugate is
A hapten that is identical to the hapten to be measured in terms of reactivity with anti-hapten antibodies is bound to a water-soluble high molecular weight substance such as a protein or synthetic polymer, such as bovine serum albumin or polystyrene, and the method of Erlanger et al. (J.Biol.Chem.,
234 , 1090 (1959)). Anti-hapten antibodies are obtained by removing antibodies against the carrier moiety from antiserum obtained by immunizing animals with hapten-conjugated substances and specifically purifying only antibodies against haptens. To specifically purify only antibodies against haptens, the method of Imanishi et al. (Biochemistry of Immunology p40, Kyoritsu Shuppan, (1967)) can be used. The measurement reagent kit of the present invention may be partially or entirely composed of lyophilized reagents, and may further include a dissolving solution or a sample for dissolving the lyophilized reagents when the kit is used. dilute to an appropriate concentration, and set the reaction field to an appropriate
A buffer solution may be included to maintain pH and ionic strength. Furthermore, accessories such as reagent tubes and pipettes may be attached to make the use of the reagent kit convenient. EXAMPLES Next, the present invention will be specifically explained using Examples, but the present invention is not limited thereto.
In addition, for ease of explanation, in this specification, an antigen-antibody reaction system will be described as an example of a reaction system, but it is not limited to the antigen-antibody reaction system, but also includes substances that have the property of specifically binding to a hapten, such as a receptor. body (receptor) and binding protein (binding protein)
It goes without saying that it is also possible to utilize a reaction system constructed using . Example 1 Measurement of estrogen A Preparation of estriol (E ₃ )/bovine serum albumin (BSA) conjugate Method of Erlanger.BF et al. (J.Biol.Chem.234;
1090, 1959), E ₃ was converted into 16,17-dihemisuccinate and combined with BSA. B Preparation of estrone (E ₁ )/BSA conjugate Weliky, N. et al. (Immuno chemistry 2; 293,
E ₁ -17-carboxymethyloxime BSA was prepared by the method of (1965). C Preparation of anti- _E3 serum Physiological saline solution of _E3・BSA conjugate (1 mg/
ml) and an equal amount of Freund's complete adjuvant were mixed into rabbits and immunized at two-week intervals. The obtained antiserum was mixed with a BSA solution at an optimal concentration, left at 4°C for 3 days, and then centrifuged at 6000 rpm for 10 minutes, and the supernatant was used as E _3- specific antiserum. Measurement of D E ₃ A predetermined amount of E ₁ / BSA conjugate solution (concentration 10 μg) determined by the preliminary test (concentration 10 μg) was placed in a test tube, and 10 μ of the standard E ₃ solution with the concentration shown in Figure 2 was placed in a test tube. 1 ml of anti- _E3 serum diluted and prepared was added and reacted at 37°C for 60 minutes. After the reaction was completed, the light scattering intensity of the reaction mixture was measured using a laser nephelometer, and the relationship between the concentration of standard _E3 and the light scattering intensity was plotted on a graph to obtain the standard curve shown in Figure 2a. . Next, the effective scattering intensity was measured in the same manner using a sample solution instead of the standard _E3 solution, and from this value, the _E3 concentration in the sample solution was determined using the previously determined standard curve. . This result is the first
Shown in the table.

[Table] Example ₂ Measurement of estrogen The concentration of E ₁・BSA conjugate was 10 μg/ml,
The standard curve shown in FIG. 2b was obtained in the same manner as in Example 1, except that the dilution factor of the serum was 400 times. In the present invention, as shown in Examples 1 and 2, by changing the amount ratio of the hapten conjugate and the anti-hapten antibody constituting the reaction system, the standard curve (FIG. 2 b) that rises toward the right and A standard curve (FIG. 2a) that descends toward the right is obtained. Therefore, the most appropriate reaction system can be set according to various conditions such as the type of the object to be measured, its abundance, or the antibody titer of the anti-hapten antibody. Example 3 Measurement of pentobarbital A Preparation of barbiturate (B)/bovine gamma globulin (BGG) conjugate Method of Spector S., et al. (Science 174; 1036;
(1971), 5-allyl-5-(1-carboxyisopropyl)barbituric acid was converted into 5-allyl-5-(1-p-nitrophenyloxycarbonylisopropyl)barbituric acid, and this was converted into dicyclohexylcarbodiimide. 10mg in the presence of
Combined with BGG. B. Preparation of anti-B serum 100 μg of the B/BGG conjugate was dissolved in 1 ml of physiological saline, mixed with an equal amount of Freund's complete adjuvant, and administered intradermally to approximately 50 sites on the back of a rabbit. The same administration was performed a total of 3 times at 5-week intervals. The obtained antiserum was treated according to the method of Example 1-C.
It was absorbed with BGG and used as a B-specific antiserum. Measurement of CB Anti-B serum and B/BGG in a pre-tested ratio
A reaction system was constructed using the conjugate. That is, 10μ of the anti-B serum diluted 1200 times and 10μ of the standard B solution shown in FIG. 3 were placed in a test tube and allowed to react at room temperature for 10 minutes. Next, 10μ of B/BGG conjugate solution (concentration: 21μg/ml) was added, and after reacting for 10 minutes at room temperature, 1.0ml of physiological saline passed through a Millipore filter with a pore size of 0.45μm was added to determine the light scattering intensity. It was measured. An example of the obtained standard curve is shown.
Using this standard curve, 0.1 μg/ml of pentobarbital can be measured. Example 4 An estrogen measurement reagent kit was constructed from the following reagents. a. 4 bottles each containing 0.2 ml of standard E ₃ dissolved at concentrations of 2.0, 0.5, 0.1 and 0.025 μg/ml, b. A bottle containing 0.5 ml of E ₁ /BSA conjugate (concentration 10 μg/ml). 4 bottles, c 4 lyophilized ampoules containing 0.5 ml of anti- _E3 antibody solution (antibody titer 16-fold dilution), d 4 bottles containing 50 ml of phosphate buffer (PH7.2)
Book.

[Brief explanation of the drawing]

Figure 1 is a graph showing the relationship between anti-hapten antibody amount and light scattering intensity, Figure 2 is a graph showing the standard curve of estriol in Examples 1 and 2, and Figure 3 is a graph showing the relationship between the amount of anti-hapten antibody and light scattering intensity.
The figure is a graph showing the standard curve of pentobarbital in Example 3.

Claims (1)

[Claims] 1 a) A step of causing a reaction between an object to be measured and a substance that reacts with the object in a solution; b) Measuring the amount of the reaction product produced in step a) by a comparative method. In the method for measuring a analyte, the analyte is a hapten, and the reaction in a) involves bonding a plurality of molecules of hapten to a water-soluble carrier per molecule of the hapten, and A method for measuring a hapten, characterized in that the hapten to be measured prevents or suppresses a precipitation reaction caused by a substance that reacts with the hapten. 2. The measuring method according to claim 1, wherein the amount of the substance that reacts with the hapten is an amount corresponding to or an excess amount of the hapten to be measured. 3 At least a comparative method consisting of a combination of a reagent consisting of a solution of a hapten to be measured bound to a water-soluble carrier in a plurality of molecules per molecule of the carrier, and b a reagent consisting of a solution of a substance that reacts with the hapten. Hapten measurement reagent kit. 4. The reagent kit according to claim 3, wherein at least one of the reagents constituting the kit is lyophilized.