JPH03257367A - Serological diagnostic method - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

DETAILED DESCRIPTION OF THE INVENTION Field of Industrial Application The present invention relates to a serum diagnostic method characterized by using cytochrome C.

Conventional technology Tumor markers that have been used in cancer serological diagnostic tests include CEA (carcinoembryonic antigen) and AFP.
Examples include human-derived substances such as fetal proteins such as (α-fetoprotein), cancer-related antigens, hormones, and isoenzymes. Especially in lung cancer, CEA, S(
, C antigen (squamous cell carcinoma antigen), N5E (nerve-specific enolase), etc. are used. Testing methods using these tumor markers are used not only for cancer screening, but also for
It is also becoming important for the therapeutic effect of cancer or for monitoring after surgery. Furthermore, this method has advantages such as being simple and being able to process a large number of samples.

Problems to be Solved by the Invention However, with conventional tumor markers alone, the positive rate of cancer was still low and the incidence rate was high. Especially for lung cancer, CEA, S (, C.

Even for markers that give a higher positive rate than others such as NSE, the positive rate is low at 30-40% of all lung cancers.
In addition, there was a disadvantage that the biased positive rate was as high as about 10 to 20%.

These testing methods require tumor markers as standard substances, but since these markers are human-derived substances, it is difficult to obtain them in large quantities. Furthermore, since these markers are human-derived substances, they are also present in healthy people, causing a high rate of false positives.

Furthermore, with the method of measuring the amount of tumor markers in the blood, in the case of early-stage cancer, the amount of markers (antigen amounts) is small, making early diagnosis difficult.

The present invention has been made in an attempt to solve these problems in the prior art.

As a result of searching for marker substances derived from organisms other than humans that are effective for serological diagnosis of cancer, the present inventors found that cytochrome C1, especially those derived from lower organisms, are effective for this purpose. The present invention was completed based on this finding.

One feature of the cancer serodiagnosis method of the present invention is that instead of measuring the amount of a marker in blood as in the conventional method, the amount of antibody against the marker is measured. In other words, even if there is only a trace amount of a marker in the blood that cannot be measured, the amount of the antibody is amplified in the living body, and as a result, it is possible to measure the amount of the antibody. This will enable early diagnosis of cancer.

The present invention will be explained in detail below.

(1) Search for marker substances Using 5 samples of serum from lung cancer patients and 5 samples from healthy individuals, we conducted a search for marker substances that exist in organisms other than humans. It was found that it reacts with patients' serum antibodies at a high rate.

The following measurement method was used to search for marker substances.

Measurement method: Various substances derived from organisms other than humans were mixed with 0.05% FC8 (
Fetal bovine serum) added sodium carbonate buffer (0 mouths 9.5)
Adjust the concentration to 50 pg/d. This is dispensed into a 96-well immunoplate in 100 pi portions and treated at 37°C for 1 hour to coat the plate with the substance. After washing the plate twice with PBS (phosphate buffered saline), blocking is performed with 10% FC3/PBS for 1 hour at 37°C to suppress non-specific adsorption. Then, without washing, when examining the reactivity of the substance with serum antibodies of the IqG class, multiply by 500 times;
A serum sample diluted 1,000 times was added to the wool in 50 l portions, and the reaction between the antibodies in the serum and the substance was incubated at 37℃ for 1 hour.
Do time. If an antibody that reacts with the substance is present in the serum, the antibody binds to the substance and is immobilized in the well. After washing the plate three times with PBS/Tween 20, the amount of immobilized antibody is measured by a conventional enzyme antibody method using a peroxidase-labeled anti-human IqG antibody or anti-human IqM antibody. That is, when examining the reactivity of an I (IG class antibody) with the substance, a peroxidase-labeled anti-human IqG antibody is used, and in the case of a 1 gM class, an anti-human ICIM antibody labeled with the same enzyme is used at 1 to 10
Dilute with 0.1% BSA (PBS/Tween 20 supplemented with bovine serum albumin) to a concentration of pH/-, add 100 samples to each well, and react at 37°C for 30 minutes.

After washing three times with PBS/1 to Vienna 20, ABTS
E2. A usual substrate solution containing 2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) ammonium salt] is added in 100 increments, and a coloring reaction is carried out at room temperature for 15 minutes. When the amount of immobilized antibody is large, that is, when the serum contains a large amount of antibodies that react with the substance, a strong color develops. After terminating the reaction by adding 100 μg of a 1.5% oxalic acid solution, the absorbance is measured at 415 nm.

The above-mentioned method for measuring reactivity with the substance can be similarly used in the present serum diagnostic method using cytochrome C derived from lower organisms.

Using this method, we investigated the reactivity of various substances with serum samples from lung cancer patients and healthy individuals.
It has been revealed that cytochrome C derived from lower organisms strongly reacts with serum samples from lung cancer patients, but does not react with serum samples from healthy individuals.

That is, by using cytochrome C derived from lower organisms, it has become possible to perform serodiagnosis of lung cancer with a high positive rate. Furthermore, a low value was obtained for the random positive rate.

In addition, when implementing the serum diagnostic method of the present invention, it is preferable to use, for example, the kit shown below.

This kit is for 96 measurements and consists of the following components.

■, Components to Kit 1 Components 1, Microplate 2, Cytochrome C (2 my/d) 3, Carbonate buffer (0 mouth 9.5) 4, Phosphate physiological saline 5 Powder for buffer solution 5, Tobean 20 solution 6, fetal bovine serum 7, standard serum 8, horseradish peroxidase-labeled anti-human IOG or anti-human I (JH antibody solution 9, bovine serum albumin 10, 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) ) Niammonium salt 11, citric acid/phosphate buffer 12, reaction stop solution (1.5% oxalic acid solution) Accessories 13, 1 lid for Microplay 1 - 375 1 bottle with Id 15 1 bottle with D 11. 1 bottle containing 3f?2 - 1 bottle containing 10 - 1 bottle containing 5 pieces 4 bottles containing 20 pieces 2 bottles containing 2011g 2 bottles containing eRg 1 bottle containing 20rd 1 bottle 1 bottle containing 15d 1 bottle or more each configuration The products can be obtained or manufactured by methods known to those skilled in the art.

Examples of the present invention are shown below.

Example 1 When we investigated the reactivity of various substances derived from non-human organisms with serum samples from lung cancer patients and healthy individuals, we found that cytochrome Q (derived from Candida) reacted with IgG or IqM class antibodies in the serum of lung cancer patients. (Table 1).

Next, the reactivity of Ti1 derived from various organisms with chrome C was investigated. These cytochrome C are commercially available,
For example, those manufactured by Sigma are easily available. As shown in Table 2, cytochrome C derived from lower organisms such as Candida and yeast was found to be particularly effective for serodiagnosis of lung cancer.

lung Ken disease but always given name Table 1 blood yeah Man Serological diagnosis using cytochrome C anti Response sex (OD415oI11) Qing IgG 1 (IM 0.240 0,087 1.343 0.218 0.385 0.049 0.205 0.046 0.053 0,151 0.066 0.053 0.064 0.044 0.036 0.057 0.045 0.059 0.043 0.039 Example 2 yeah Man Table 2 -3 ni-45 ni-55 -5 -9 -11 -4 Serum diagnostic reaction using cytochrome C derived from various organisms Response sex (OD415nm) qG qM qG 0gM 1.134 0.392 0.498 1.456 1.236 0.062 0.047 0.056 0.048 0.061 0.055 0.043 0.143 0,237 0.964 0.529 0.321 1.580 1.379 0.216 0,153 0.122 0.139 095 0.113 0.088 217 0.304 071 0.098 0.087 0.098 0.073 0.0?2 0.089 0.051 0.041 0.040 0,196 0.294 239 0.223 0.126 0.136 0.167 0.109 o,io.

Using cytochrome C from Candida Kruset, the positive rate was determined for each tissue type of lung cancer. However, the reactivity of healthy subjects (average OD>+28.D) or higher was defined as positive.

As shown in Table 3, in the serum of lung squamous cell carcinoma patients, 56%, 80% in lung small cell carcinoma, and 1 in lung large cell carcinoma.
It showed a very high positive rate of 0.00%.

The rate for lung adenocarcinoma was also high at 37%. Conventional C
The positive rate was greatly improved compared to cases such as EA, SCC, and NSE. The biased positive rate was also low, less than 5% (5 out of 110 samples were positive).

Table 3: Positive rate for lung cancer histology Table 4: Number of samples for serum diagnosis of cancer histological type: Positive rate (%) Squamous cell carcinoma Small cell carcinoma Large cell carcinoma Adenocarcinoma Example 3 Using this serological diagnosis method We investigated the positive rate of sera from various patients. As shown in Table 4, lung cancer.

High positive rates were shown for bile duct cancer and esophageal cancer.

disease given name lung cancer bile duct cancer esophageal cancer liver cancer pancreatic cancer rectal cancer stomach cancer colon cancer breast cancer benign disease The number of samples 2 Yang sex rate (%)

Claims (4)

[Claims] (1) A serum diagnostic method characterized by using cytochrome C. (2) The serum diagnostic method according to claim 1, wherein the cytochrome C is derived from Candida or yeast. (3) The serum diagnostic method according to claim 1 or 2, which comprises measuring the amount of antibodies in blood that bind to the cytochrome C. (4) Claim 1 characterized in that an enzyme antibody method is used.
The serological diagnostic method according to any one of items 3 to 3.