JP3098970B2 - Peptide elution immunoassay for protein - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a protein elution immunoassay for a protein, which can accurately measure the amount of protein in a sample by utilizing an antigen-antibody reaction.

[0002]

2. Description of the Related Art As a method of measuring the amount of a protein to be measured in a sample with high sensitivity by utilizing an antigen-antibody reaction, a sandwich ELISA (ELISA) method has been conventionally known. Further, by utilizing this principle for an HPLC column, an HPLC immunoassay method in which the measurement time is short and which can be repeatedly measured has been developed. In this method, as shown in FIG. 3, first, a specific antibody 12 for a measurement protein 11 is bound to a carrier 13,
The sample is passed through the surface of the sample to allow the measurement protein 11 in the sample to be adsorbed to the specific antibody 12, washed, and the unadsorbed impurities 14 are discharged. In this method, the measurement protein 11 and the fluorescently labeled antibody 15 are eluted with hydrochloric acid, and the amount of the fluorescently labeled antibody 15 is measured by a fluorescence detector.

However, in this method, as shown in the upper right of FIG. 3, the fluorescently labeled antibody 15 easily adsorbs not only on the target measurement protein 11 but also on the carrier 13 and the specific antibody 12. Due to such a large amount of non-specific adsorption, the amount of the fluorescently labeled antibody 15 at the time of elution increases as shown in the lower part of FIG. 3, and there is a problem that the amount of the measurement protein 11 cannot be measured accurately. Was. In addition, the pH of the eluent changes due to hydrochloric acid used for elution, and the fluorescence intensity greatly changes with the change. Therefore, there is also a problem that an accurate fluorescence intensity cannot be obtained.

[0004]

SUMMARY OF THE INVENTION The present invention solves the above-mentioned conventional problems and provides a method for immunoassay of peptide elution of a protein, which can perform highly accurate measurement even when nonspecific adsorption of a labeled antibody is present. It was made to provide.

[0005]

SUMMARY OF THE INVENTION The present invention has been made to solve the above-mentioned problems.
First , the method is based on the peptide constituting the measurement protein.
To elute all or part of the measurement protein
Monoclonal or polyclonal antibodies
Immobilize the body on a carrier and pass the sample through the surface
The protein to be measured in the sample is
Is adsorbed to the polyclonal antibody, and then
The labeled product of the specific antibody is the monoclonal antibody.
Or the measurement protein adsorbed by the polyclonal antibody
After washing , the labeled antibody is eluted together with the measurement protein with an eluent containing an antigen peptide, and the amount thereof is measured. Second, the measurement protein
The specific antibody is immobilized on a carrier, and the sample is
Pass the solution to adsorb the measurement protein in the sample to the specific antibody,
Next, an antibody against the peptide that constitutes the measurement protein
The labeled product of the anti-peptide antibody was adsorbed to the specific antibody.
After adsorbing to the measurement protein and washing, the antigen peptide is
The labeled antibody is eluted with an eluent containing
It is characterized by the following.

[0006] Labeling substances include fluorescent substances, enzymes, radioisotopes, luminescent substances, metal atoms, metal sols, and the like.
Either a stable free radical, latex, or bacteriophage can be used. In addition, as the peptide constituting the measurement protein, it is preferable to use a peptide having several residues at the C-terminal or N-terminal of the measurement protein.
Hereinafter, preferred embodiments of the present invention will be described.

[0007]

DESCRIPTION OF THE PREFERRED EMBODIMENTS First Embodiment First, an anti-peptide antibody which is an antibody of a peptide constituting a protein to be measured (measurement protein) is prepared. The measurement protein is, for example, insulin or amylase,
From the peptides constituting the measurement protein, a portion having a high degree of freedom that can be easily used for measurement is selected as an antigen peptide. For example, in the case of bovine insulin, the eight residues at the C-terminal of the B chain are peptides having an amino acid sequence of (Gly-Phe-Phe-Try-Thr-Pro-Lys-Ala), and in the case of rice α-amylase, The eight C-terminal residues are (Arg-Val-Pro-Ala-Gly-A
rg-His-Leu), and these peptides may be selected.

As described above, according to the present invention, the measured protein C
It is preferable to use a peptide having several residues at the terminal or N-terminus. The reason is that in the protein, the C-terminal portion or the N-terminal portion has a high degree of freedom, and the portion has high reactivity with the anti-peptide antibody. Conversely, the peptide in the central part of the measurement protein has a low degree of freedom and is not very suitable for use in the present invention. However, by specifically degrading the measurement protein with a protease such as trypsin, a peptide having several residues at the C-terminal or N-terminal of the fragment can also be used. The number of residues in the peptide must be at least 5 for immunogenicity and specific recognition.
There is no upper limit on the number of residues, but if it is too large, it will not be clear where it will be recognized.

[0009] The selected peptide (antigen peptide)
It can be chemically synthesized using an automatic device by a known peptide synthesis method, and this is injected into an animal body to produce an anti-peptide antibody. Antibodies are generally Y-shaped, have binding sites for antigens on their N-terminal two arms, and are immobilized on a carrier with the remaining C-terminal foot as shown in FIG. The type of the carrier is not particularly limited, and may be a column packed or fixed to a tube wall or a membrane. In particular, if a perfusion type carrier is used, the measurement can be performed in a short time. The antibody is a monoclonal antibody,
Any of polyclonal antibodies may be used, as long as the peptide can be eluted.

A sample containing the measurement protein 3 is passed over the surface of the carrier 2 on which the anti-peptide antibody 1 is immobilized as described above. Although various impurities 4 are contained in the sample, only the measurement protein 3 is adsorbed to the anti-peptide antibody 1 by a selective antigen-antibody reaction between the anti-peptide antibody 1 and the peptide of the measurement protein 3.

Next, the labeled substance 5 (labeled antibody 5) of the specific antibody to the measurement protein 3 is adsorbed to the measurement protein 3 adsorbed to the anti-peptide antibody 1. In this case, labeled antibody 5
As the labeling substance, any one of a fluorescent substance, an enzyme, a radioisotope, a luminescent substance, a metal atom, a metal sol, a stable free radical, a latex, and a bacteriophage is used. As described in the section of the prior art, the labeled antibody 5 is not only adsorbed on the measurement protein 3 but also inevitably partially non-specifically adsorbed on the carrier 2 and the anti-peptide antibody 1. afterwards,
The surface of the carrier 2 is washed by flowing the mobile phase liquid.

Next, in the present invention, an eluent containing the antigen peptide is passed through the surface of the carrier 2. As a result, the anti-peptide antibody 1 binds to the antigenic peptide in the eluant having a higher affinity and elutes the measurement protein 3. Therefore, the measurement protein 3 is eluted together with the labeled antibody 5. However, as shown in FIG. 1, the labeled antibody 5 non-specifically adsorbed to the carrier 2 and the anti-peptide antibody 1 is not eluted by the antigen peptide. Therefore, if the amount of the labeled antibody 5 in the eluate is measured,
The amount of the measurement protein 3 can be measured.

When the labeling substance of the labeled antibody 5 is a fluorescent substance, the amount of the labeled antibody 5 may be measured by a fluorescence detector, and the enzyme, radioisotope, luminescent substance, metal atom, metal sol, In the case of free radicals, latexes, and bacteriophages, known measuring instruments or measuring methods according to their types may be used. Thus, in the present invention, since the eluent containing the antigen peptide was used,
The measurement can be performed without eluting the nonspecifically adsorbed labeled antibody 5. Further, since no hydrochloric acid is used for elution as in the conventional case, the fluorescence intensity does not fluctuate depending on the pH. Therefore, the amount of the measurement protein 3 can be measured with high accuracy.

[Second Embodiment] In the first embodiment described above, an anti-peptide antibody which is an antibody of a peptide constituting a measurement protein is immobilized on a carrier. However, in the second embodiment, a specific antibody of the measurement protein is immobilized on a carrier. Then, similarly to the first embodiment, the sample containing the measurement protein is passed through to allow the measurement protein to adsorb to the specific antibody, and then the labeled antibody is adsorbed to the measurement protein adsorbed to the specific antibody. In this case, an anti-peptide antibody is used as the labeled antibody. As described above, the labeling substance of the labeled antibody is provided with a fluorescent substance, an enzyme, a radioisotope, a luminescent substance, a metal atom, a metal sol, a stable free radical, a latex, a bacteriophage, and the like. Next, an eluent containing the antigen peptide is passed through the surface of the carrier to elute the labeled antibody. Also in this case, the labeled antibody non-specifically adsorbed to the carrier or the specific antibody is not eluted by the antigen peptide. Therefore, if the amount of the labeled antibody in the eluate is measured by a known means, the amount of the measurement protein can be accurately measured.

[0015]

[Example] (Measurement of bovine insulin) Eight residues (Gly-Phe-Phe-T) at the C-terminal of B chain of bovine insulin
The peptide of ry-Thr-Pro-Lys-Ala) was chemically synthesized by a known peptide synthesis method. This was injected into guinea pigs to produce anti-peptide antibodies. This anti-peptide antibody was immobilized on the surface of the carrier. Type of carrier is Formyl-Cellu
lofine (manufactured by Seikagaku Corporation), packed in a column with an inner diameter of 4.6 mm and a length of 30 mm. The amount of the anti-peptide antibody immobilized on the carrier was 3.5 mg per 1.5 g of the carrier.

A mobile phase solution (phosphate buffer) is added to this column.
Was flowed at a flow rate of 1 mL / min, and 200 μl of a sample containing bovine insulin as a measurement protein was passed. The sample was prepared by dissolving bovine insulin (SIGMA, I-6383) as a standard reagent in a liquid having the same composition as the mobile phase liquid. After one minute, 10 to 40 μl of the fluorescently labeled antibody was injected onto the column. This fluorescent-labeled antibody was Ig-labeled by the FITC method.
G. After that, the mobile phase solution was allowed to flow for 15 minutes to wash the inside of the column, and 1 mL of the eluate obtained by dissolving the peptide (antigen peptide) at a concentration of 0.2 mg / ml in a solution having the same composition as the mobile phase solution was injected into the column. Then, the fluorescently labeled antibody was eluted. The fluorescence intensity of this eluate was measured with a fluorescence detector having an excitation wavelength of 490 nm and a measurement wavelength of 525 nm to obtain a fluorescence peak area.

FIG. 2 is a graph showing the results of repeating the measurement while changing the concentration of bovine insulin in the sample. The difference between the white circle and the black circle in the graph indicates the difference between the measurement dates.
From this figure, it can be seen that the concentration of bovine insulin and the fluorescence peak area correspond to 1: 1 and that the measurement is reproducible. Therefore, the concentration of bovine insulin in the sample can be accurately determined from the fluorescence peak area. The column is then rinsed by passing 1 mL of 0.1N hydrochloric acid once, then passing the peptide solution once, and again passing 1 mL of 0.1N hydrochloric acid twice, and moving on to the next measurement operation. .

[0018]

The effects of the present invention are summarized as follows. Even when there is non-specific adsorption of the labeled antibody, the measurement can be performed without being affected by the non-specific adsorption. Since the elution is performed using the antigen peptide, the measurement accuracy does not decrease due to a change in pH as in the case where the elution is performed using conventional hydrochloric acid. Since there is no effect of the nonspecifically adsorbed labeled antibody, the background can be low, and measurement can be performed up to a low concentration by using an anti-peptide antibody having a high binding constant. The total measurement time is as short as about 30 minutes, and the measurement can be performed in a shorter time if a perfusion type carrier is used. The carrier can be used repeatedly, and the amount of the peptide used for elution may be small.

[Brief description of the drawings]

FIG. 1 is a process explanatory view of the present invention.

FIG. 2 is a graph showing the relationship between bovine insulin concentration and fluorescence peak area in Examples.

FIG. 3 is an explanatory diagram showing steps of a conventional HPLC immunoassay method.

[Explanation of symbols]

1 anti-peptide antibody, 2 carrier, 3 measurement protein, 4
Impurities, 5 labeled antibodies, 11 Proteins measured by conventional methods, 12 specific antibodies, 13 carriers, 14 impurities, 15 fluorescently labeled antibodies

──────────────────────────────────────────────────続 き Continuation of the front page (56) References JP-A-2-73158 (JP, A) JP-A-61-217765 (JP, A) JP-A-2-198361 (JP, A) JP-A-4-19761 130364 (JP, A) (58) Fields studied (Int. Cl. ⁷ , DB name) G01N 33/543 525 G01N 33/543 515 G01N 30/26 G01N 30/88

Claims (5)

(57) [Claims] Depending on the peptide constituting the 1. A measuring protein
To elute all or part of the measurement protein
A possible monoclonal antibody or polyclonal antibody is immobilized on a carrier, and the sample is passed through the surface thereof to measure the protein in the sample to the monoclonal antibody or polyclonal antibody.
Adsorbed to polyclonal antibodies, then walk the monoclonal antibody labeling of specific antibodies to measure protein
Is a method for immunoassay of peptide elution of a protein, which comprises adsorbing and washing a measurement protein adsorbed on a polyclonal antibody , washing the labeled antibody with an eluent containing an antigen peptide, together with the measurement protein, and measuring the amount. 2. A specific antibody of a measurement protein is immobilized on a carrier, and the sample is passed through the surface thereof to adsorb the measurement protein in the sample to the specific antibody. Then, an antibody against a peptide constituting the measurement protein is used. A protein peptide characterized in that a labeled product of a certain anti-peptide antibody is adsorbed on the measurement protein adsorbed on the specific antibody and washed, and then the labeled antibody is eluted with an eluent containing the antigen peptide, and the amount is measured. Elution immunoassay method. 3. The peptide-eluting immunity of the protein according to claim 2 , wherein a monoclonal antibody or a polyclonal antibody capable of eluting all or a part of the measurement protein by a peptide constituting the measurement protein is used instead of the anti-peptide antibody. Measuring method. 4. The protein according to claim 1, wherein the labeling substance is any one of a fluorescent substance, an enzyme, a radioisotope, a luminescent substance, a metal atom, a metal sol, a stable free radical, a latex, and a bacteriophage. Peptide elution immunoassay method. 5. The method according to claim 1, wherein a peptide having several residues at the C-terminal or the N-terminal of the protein to be measured is used as the peptide constituting the protein to be measured.