JPS61243363A - Highly sensitive assay of crp - Google Patents

Abstract

PURPOSE:To obtain a highly sensitive CRP measurement method, by differentiating the kind of animal to be immunized for obtaining a solid-phase antibody and a labelled antibody. CONSTITUTION:An anti-CRP rabbit IgG is used as solid phase antibody while an anti-CRP goat IgG as labelled antibody to react with a sample as measuring antigen (CRP serum) and then, the activity of the labelled substance involving the antigen-antibody reaction is measured to assay CRP. When a 1-step method is applied, a washing (b/f separation) is done after the simultaneous reaction of the solid-phase antibody and the labelled antibody with the measuring antigen (CRP serum sample) and then, the measurement of the activity of the labelled substance involving the antigen-antibody reaction. When a 2-step method is applied, initially, the solid-phase antibody is made to react with the measuring antigen and after a washing, the labelled antibody is added. Following a washing is done again, the activity of the labelled substance is measured.

Description

DETAILED DESCRIPTION OF THE INVENTION (Industrial Application Field) The present invention relates to a method for quantifying CRP, that is, C-reactive protein, and particularly to a highly sensitive method for quantifying CRP.

As is well known, CRP is an abnormal protein that appears in the blood as a result of inflammation and tissue necrosis, and when the above-mentioned diseases occur in the body, the amount increases within 6-24 hours and reaches a peak within 2-5 days. It has the property of reaching a certain level and gradually decreasing and disappearing as the disease recovers.

Therefore, the method of the present invention can be used for clinical diagnosis of the above-mentioned diseases.

(Prior Art) Conventional CRP testing methods include routine testing methods such as capillary tube method, latex agglutination method, and SRID method, and immunoturbidimetry.

Routine testing methods may not be able to quantitatively understand changes in CRP values over time, or may be difficult to detect due to measurement sensitivity, accuracy, or processing time, especially in low concentration ranges.
It is difficult to understand the fluctuation of RP value.

On the other hand, in the so-called Sanderutsch method, which is frequently used in enzyme-linked immunosorbent assay (EIA), a solid-phase antibody, a labeled antibody, and an antigen to be measured are reacted simultaneously, and then washed.
/f separation (immune complex).

The one-step sandwich method involves performing free #Affi antibody f that did not participate in the antigen-antibody reaction and antibody activity), and then measuring the iIA identification material that was involved in the antigen-antibody reaction to quantify CRP. The phased antibody and the antigen to be measured are first reacted, and then washed to perform the first b/f separation,
This is then reacted with a labeled antibody, followed by a second b/f
There is a two-step sandwich method in which CRP is quantified by separating and then measuring the activity of a labeling substance involved in the antigen-antibody reaction.

(Problems to be Solved by the Invention) The conventional one-step method is advantageous in that the b/f separation operation (washing treatment) is performed once, and therefore the time required is short; When a competitive reaction occurs between the phased antibody and the labeled antibody, accurate/f separation becomes difficult, resulting in a problem in that the measurement sensitivity decreases and the measured value fluctuates.

On the other hand, the two-step method is a method proposed to overcome the above-mentioned deficiencies in the one-step method. There is a problem in that the number increases and the time required increases accordingly.

Furthermore, although the measurement sensitivity of the conventional two-step method is relatively high, sufficient measurement sensitivity can be obtained even if the dilution rate of the labeled antibody is increased, thereby making it possible to accurately measure CRP in the low concentration range. desired.

(Objective of the invention and means and effects for solving the problems) Therefore, an object of the present invention is to provide a CR with even higher sensitivity than before.
The purpose of this invention is to provide a method for measuring P.

The present invention is based on different animal species to be immunized in order to obtain a homologous antibody and a labeled antibody, and according to the method of the present invention, anti-CRP rabbit IgG is used as the immobilized antibody and labeled Anti-CRP goat IgG is used as an antibody to react with the sample (CRP serum) that is the antigen to be measured, and then the activity of the labeling substance involved in the antigen-antibody reaction is measured.
The above objective can be achieved by quantifying RP.

The method of the invention can be applied to both one-step and two-step methods. When applied to the one-step method, the immobilized antibody, labeled antibody, and antigen to be measured (CRP serum sample) are reacted at the same time, then washed (b/f separation), and then involved in the antigen-antibody reaction. The activity of the labeled substance is measured. In this case, the measurement sensitivity may decrease slightly, but this can be done by providing a slight time lag between the reaction between the immobilized antibody and the antigen to be measured and the reaction between the antigen to be measured and the labeled antibody. This can be prevented by causing a reaction between the labeled antibody and the antigen to be measured, and then adding a labeled antibody and causing a further reaction.

When applied to a two-step method, the immobilized antibody and the antigen to be measured are first reacted, then washed, a labeled antibody is added, another reaction is carried out, and then washed again. Thereafter, the activity of the labeling substance involved in the antigen-antibody reaction is measured in the same manner as in the one-step method.

As the substance used for labeling the antibody, any commonly used substance can be used, such as enzymes such as alkaline phosphatase, fluorescent substances such as fluorescein isocyanate, dyes, radioactive substances, etc. However, ease of handling,
From the viewpoint of availability, it is convenient to use an enzyme such as alkaline phosphatase. As a method for attaching a label, a method known per se can be adopted.

The activity of the labeling substance involved in the antigen-antibody reaction can also be measured by methods known per se. For example, when alkaline phosphatase is used as a labeling substance, a substrate solution for its measurement is added and reacted, a coloring solution is added to stop the enzymatic reaction and color is developed, and this is measured photometrically (absorbance) using a measuring device. measurement). CRP quantification of the antigen to be measured can be performed by comparing this absorbance value with a standard calibration curve.

(Effects of the invention) C by the so-called Sanderutsch method using antigen-antibody reaction
According to the present invention, in the determination of R and P, anti-CRP is used as an antibody obtained by immunizing a specific animal species, that is, as an immobilized antibody.
Rabbit IgG and anti-CRP goat IgG as a labeled antibody
By selecting ℃, it is possible to improve the measurement sensitivity. This means that if the measurement sensitivity is kept constant, the dilution rate of the labeled antibody can be increased. Therefore, by setting a high dilution rate, the influence of nonspecific adsorption due to the labeled antibody can be significantly reduced. Because you can
It is also possible to improve measurement accuracy in the low concentration range.

(Example) Next, an example of implementing the method of the present invention will be described. I will clarify.

A) Preparation of antibodies Rabbits and goats were immunized by intravenously injecting human CRP in serum, blood was collected to obtain anti-human CRP serum, which was subjected to ammonium sulfate salting out and ion exchange to prepare an IgG fraction.

B) Preparation of immobilized antibody The anti-human CRP antibody (IgG fraction) obtained in Section A above was diluted with 0.05 M carbonate buffer (pH 9,6) +: to make an antibody solution of 50 μg/ml, which was prepared by Nunc Co., Ltd. A 200 μm aliquot was dispensed into each well of a microtiter plate made by Manufacturer Co., Ltd. After the wells were left at 4-10°C for 24 hours, the contents of the wells were washed with distilled water. Next, phosphate buffered saline (pH 7, 2) containing 1% albumin was added to each well.
) was dispensed into 200 μm portions and left at 4-10° C. for an additional 24 hours to obtain immobilized antibodies.

Note that this immobilized antibody is washed with distilled water before use.

C) Preparation of labeled antibody Alkaline phosphatase (derived from calf small intestine, manufactured by Beringa) 3 mg ttl containing 0.0 mg ttl mM magnesium chloride.
Add and dissolve in 1 ml of 1M N,N-bis(2-hydroxyethyl)-glycine buffer (pH 8,5), then add 20 mg of potassium periodate, and heat at 37°C.
I left it for 2 hours. Thereafter, insoluble materials were removed by centrifugation at 80 rpm for 5 minutes, and the supernatant was dialyzed overnight against 0.1 M carbonate buffer (pH 9.5) containing 1 mM magnesium chloride. Next, 1.5 mg of the anti-human CRP antibody (IgG fraction) according to section A above was added, and the mixture was incubated at 4-10°C for 1.5 mg.
The mixture was left for 4 days, diluted 50 times with phosphate buffered saline containing 1% bovine albumin, and then stored frozen.

In addition, at the time of use, the required dilution rate is made with phosphate buffered saline containing 0.1% bovine albumin.

D) Measurement method a) One-step method Dispense 200 μm of the diluted solution of the labeled antibody according to the above section into each well of an antibody-immobilized microtiter plate, add 20 μl of sample (CRP serum), and react at room temperature for 2 hours. After that, wash the contents with distilled water. Next, a substrate solution for measuring alkaline phosphatase activity (containing 45 mM disodium phenylphosphate and 2 mM 4-aminoantipyrine) was added.
．． 025 M carbonate buffer, PH10,2) at 200 μm
Add and react for 20 minutes at room temperature.

After that, 1 color developer (0.8% sodium periodate) 10
0 μm was added to stop the enzyme reaction and cause color development.

Next, absorbance measurement is performed using an ELIsA analyzer (ETY-96).

b) 1-step improved method Dispense 100 μm of phosphate buffered saline containing 0.1% bovine albumin into an antibody-immobilized microtiter plate, and prepare sample 2.
Add 0 μm and react for 1 hour at room temperature.

Next, 100 μm of a diluted solution of the labeled antibody according to item 0 above is added, and after reacting for 1 hour at room temperature, the plate is washed with distilled water. Thereafter, the activity of alkaline phosphatase involved in the antigen-antibody reaction is measured in the same manner as in section a above.

c) 2-step method Dispense 100 μl of phosphate buffered saline containing 0.1% bovine albumin into each well of a microtiter plate immobilized with anti-human CRP antibody, add 20 μm of sample, and react for 1 hour at room temperature. let Then, it was washed with distilled water and 0.1
Add 200 μl of a diluted solution of the labeled antibody according to item 0 above diluted with phosphate buffered saline containing % bovine albumin, and react at room temperature for 1 hour. Thereafter, it is washed with distilled water, and then the activity of alkaline phosphatase involved in the antigen-antibody reaction is measured in the same manner as in section a above.

E) Results Samples with various CRP concentrations were prepared, and rabbit Ig was used as the immobilized antibody according to the operating procedure described in the section ``F measurement method'' above.
Tests were conducted using G and goat IgG, and goat IgG and rabbit IgG as labeled antibodies, and the relationship between CRP concentration and absorbance (500 nm) was investigated, and the results shown in Figures 1-3 were obtained.

Figures 1-3 show graphs obtained by plotting the one-step method, one-step improved method, and two-step method, respectively, and it can be seen that the method according to the present invention has extremely good measurement sensitivity. It has been found that the one-step method is particularly sufficient and effective for measurements in the low concentration range.

In these figures, RG, RR, GG, and GR each represent the antibody combination as shown in the table below.

[Brief explanation of the drawing]

The drawings are graphs showing the relationship between CRP concentration and absorbance when carrying out the method of the present invention. Figure 3 shows the case where the two-step method is applied.

Claims (4)

[Claims] (1) Using anti-CRP rabbit IgG as the immobilized antibody and anti-CRP goat IgG as the labeled antibody, react with the CRP sample that is the antigen to be measured, and then measure the activity of the labeled substance involved in the antigen-antibody reaction. A highly sensitive method for quantifying CRP, which is characterized by quantifying CRP. (2) The method according to claim 1, characterized in that the immobilized antibody, the labeled antibody, and the antigen to be measured are simultaneously reacted, washed, and then the activity of the labeled substance is measured. Sensitivity quantification method. (3) A patent claim characterized in that the immobilized antibody and the antigen to be measured are first reacted, then a labeled antibody is added and the reaction is further carried out, followed by washing, and then the activity of the labeled substance is measured. The high-sensitivity quantitative method according to item 1. (4) Washing after reacting the immobilized antibody with the labeled antibody, and then washing it again after reacting with the labeled antibody,
characterized in that the activity of the labeled substance is then measured;
A highly sensitive quantitative method according to claim 1.