JPH0814578B2 - Apolipoprotein B measurement method and reagents - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION (1) Field of Industrial Application The present invention relates to a method and a reagent for measuring blood apolipoprotein B. Therefore, the present invention has a field of biochemical or medical significance in the measurement of blood apolipoprotein B,
For example, it is used in the field of clinical diagnosis.

(2) Prior Art and Problems Hypercholesterolemia is one of the important causes of arteriosclerotic diseases, and its mechanism has been clarified in detail.
A study by Gold Stein and Brown with familial hypercholesterolemia IIa revealed that the disease was caused by an abnormality in the receptor for apolipoprotein B, and thus the patient's blood apolipoprotein. It is known that the B value becomes high. That is, hypercholesterolemia can be diagnosed by measuring blood apolipoprotein B. The following document (a) is referred to as a material for explaining the above conventional knowledge in more detail.

(A) Goldstein, J .. Elle, And Brown, M. S., The LDL Receptor Defect In Familiar Hyper Cholesterol Remia. Mede. Clini. North. Am. 66: 273, 1982 (Goldst
ein, JL, and Brown, MS, The LDL recepter defect in
familial hyper cholesterolemia.Med.Clin.North.Am.
66: 273, 1982) There are various methods for measuring blood apolipoprotein B, but an immunoassay is recommended as a sensitive method. For example, using anti-apolipoprotein B antibody
The method of performing the ELISA method can be mentioned.

By the way, it is known that hypercholesterolemia has already developed from an early stage in a newborn baby and leads to arteriosclerosis as a result of progression over a long period of time thereafter. That is, if a risk factor for hypercholesterolemia is discovered as early as possible in a newborn baby and the cholesterol level can be controlled by removing the risk factor, arteriosclerosis after adulthood can be significantly reduced.
In this sense, it is understood that the measurement of blood apolipoprotein B in newborns is very important.

However, since there is a limit to the amount of blood collected from newborns, there is a problem that the measurement of blood apolipoprotein B in newborns cannot be easily performed. In particular, there is a problem that the reproducibility of measured values deteriorates when it is necessary to measure several days after blood collection.

In view of such problems, the present inventor has studied to provide a method capable of measuring apolipoprotein B in blood with high sensitivity even with an extremely small amount of collected blood sample.

(3) Solution Means As a result of various studies, a very small amount of blood was collected from a newborn baby using dry paper and eluted in a solution containing a surfactant.
The inventors have found that apolipoprotein B can be measured by an appropriate immunological assay method using this as a measurement data, and have completed the present invention.

 Hereinafter, the present invention will be described in detail.

Apolipoprotein has AI, A-II, B, C-II, C-II
Although there are I, E, etc., the present invention covers only apolipoprotein B among them. For a description of apolipoprotein B, reference is made to the description of the above-mentioned document (a) for the present invention.

In the present invention, the dry paper is DBS (Dried Blood Spot).
For commercial use, a commercially available product may be obtained and used. For example, a circular paper with a diameter of 3 mm may be used to collect an extremely small amount of blood from a newborn baby.

The collected dry paper blood is eluted into the solution, but the present invention is characterized in that the solution contains a surfactant. As shown in the experimental examples below, the inclusion of a surfactant makes the elution rate extremely stable and enables highly reproducible and highly sensitive measurements. Here, for example, polyethylene glycol p-isooctylphenyl ether is recommended as the surfactant, but the present invention is not limited thereto.

Next, the immunological assay method according to the present invention may be any method utilizing a specific reaction between apolipoprotein B and an anti-apolipoprotein B antibody. That is, the enzyme immunoassay, the radioimmunoassay, the passive hemagglutination method, etc. may be arbitrarily selected and applied. For example, the two-antibody sandwich type ELISA method is a preferable application example. The outline of the case of applying this method is as follows.

First, the anti-apolipoprotein B antibody (IgG) prepared in advance is adsorbed to the wells of the microtiter plate. To this, a measurement sample obtained by elution of dried paper blood was added and incubated, then an enzyme-labeled anti-apolipoprotein B antibody (Fab) was added and incubated, and finally, a substrate solution was added and the reaction was performed after a certain time. Stop the measurement and measure the amount of decomposition of the substrate by the absorbance value.

The anti-apolipoprotein B antibody may be prepared by a combination of conventionally known methods. The outline is as follows. Apolipoprotein B is a low-density lipoprotein (LD
Since L) occupies 98%, LDL is fractionated from normal human serum by ultracentrifugation by the method of Havel et al. (Reference (b)), and then Con A by the method of McConathy et al. (Reference (c)).
Purify apolipoprotein B using a Sepharose column. A rabbit is immunized with the obtained apolipoprotein B and antiserum is taken. Next, anti-apolipoprotein B antibody (rabbit IgG) is purified and fractionated from the antiserum using affinity chromatography. Here, CNBr-activated Sepharose 4B was used as the affinity column, and 1 mg of apolipoprotein B was added to Cuatrecasas per ml of the swollen gel.
What was coupled by the method (reference (d)) may be used. Further, 0.2M glycine hydrochloric acid pH 2.2 may be used for elution. The purified anti-apolipoprotein B antibody (rabbit IgG) was further treated with pepsin, and the F (a
b) ² can be further reacted with mercaptoethylamine to give an anti-apolipoprotein B antibody (rabbit Fab). This Fab may be labeled with peroxidase by the method of Ishikawa et al. (Reference (e)).

The following documents (b) to (e) can be referred to as materials for explaining the above method.

(B) Harbel Earl. J. D. Tar., J. Clini. Invest. (1955) 34: 1345-1353 (Hav
el RJet.al., J.Clin.Invest. (1955) 34: 1345-135
3) (c) Maconacy double. J. D. Tar., FBS, Letter. (1974) 41: 174-179 (M
cConathy W Jet.al., FEBS lett. (1974) 41: 174-17
9) (d) Quatrecasus SP., J. Biolo. Chem. 245 (1970) 3059-3065 (Cuatrecasas P., J. Biol. Ch
em.245 (1970) 3059-3065) (e) Yoshitake S., d. Tar., J. Biochem. 92 (1982) 1413 (Yoshitake S.et.al., J. Biochem.
92 (1982) 1413) Next, the measuring reagent of the present invention is a reagent used for carrying out the measuring method of the present invention, and achieves the same purpose as in the measuring method. That is, in the set for measuring apolipoprotein B in blood by an immunological measuring method, the measuring reagent of the present invention includes dry paper and a surfactant for preparing a measuring sample as essential components. It is a feature. Here, the surfactant may be provided as a solution containing a surfactant, for example, phosphate, NaCl, polyethylene glycol p-isooctylphenyl ether are dissolved in 0.01M, 0.15M and 0.1% concentrations, respectively. It can be mentioned that it is provided as a solution whose pH is adjusted to 7.3.

The action of the present invention makes it possible to sensitively measure apolipoprotein B from an extremely small amount of blood collected by collecting blood with dry paper and eluting it into a solution containing a surfactant.

(4) Example Example 1 An anti-apolipoprotein B antibody (rabbit IgG) was dissolved in 0.1 M carbonate buffer (pH 8.5) to a concentration of 10 μg / ml,
100 μl of this was put into a 96-well microtiter plate and left at 4 ° C. overnight. Remove each well by aspiration, wash 3 times with 0.15M NaCl (pH7.5) wash solution, and add 1% bovine serum albumin solution (0.01M phosphate buffer, 0.15M).
Add 250 μl of M NaCl, pH 7.5 and incubate at 37 ° C. for 3 hours. Remove the solution by suction and wash 3 times with the above washing solution to make a measurement well.

Separately, a peroxidase-labeled anti-apolipoprotein B antibody (rabbit Fab) is prepared and combined with the above-mentioned measuring well, and DBS having a diameter of 3 mm and polyethylene glycol p-isooctylphenyl ether are attached thereto to give the measuring reagent of the present invention.

Example 2 The measurement is carried out as follows using the measurement reagent described in Example 1.

Blood is collected from newborns using 3 mm diameter DBS. Phosphate NaC
l, polyethylene glycol p-isooctyl phenyl ether were dissolved in 0.01M, 0.15M and 0.1% concentration, respectively.
The collected blood DBS is immersed in 200 μl of the eluate whose H is adjusted to 7.3 and left overnight at 4 ° C. The obtained solution is dissolved in phosphate and NaCl at concentrations of 0.01M and 0.15M, respectively, and diluted 100 times with a solution whose pH is adjusted to 7.3 to obtain a measurement sample.

Add 100 μl of the measurement sample to each well and stir gently at 4 ° C overnight. The wells are removed by suction and washed 4 times with a washing solution of 0.15M NaCl (pH 7.5).

Peroxidase-labeled anti-apolipoprotein B antibody (rabbit Fab), phosphate, NaCl, and normal rabbit serum were dissolved at concentrations of 1 μg / ml, 0.01M, 0.15M, and 20%, respectively, and the pH was adjusted to 7.4.
100 μl of the prepared solution was added to each well and incubated at 37 ° C. for 3 hours. Aspirate and remove each well, and add 0.15N
Wash 4 times with aCl (pH 7.5). 100 μl of substrate solution containing 2,2′-azino-bis (3-ethyl-benzothiazoline-6-sulfonic acid) and hydrogen peroxide was added to each well, and after 30 minutes at room temperature, 2 mM NaN ₃ solution was added to react. Stop. After this, the absorbance value at 405 nm is measured.

(5) Effects of the invention Experimental example 1 A large number of samples were prepared by collecting blood from a newborn baby with DBS,
After a predetermined number of days, (a) the method described in Example 2 and (b)
Blood apolipoprotein B was measured in the same manner as in Example 2 except that polyethylene glycol p-isooctylphenyl ether was not used.

The results are shown in Fig. 1. The horizontal axis of the figure represents the number of days after blood collection, and the vertical axis represents the recovery rate (%) when the measured value of the sample immediately after blood collection is 100%. In the figure, the solid ● line shows the result by the method (a), and the dotted line ○ shows the result by the method (b). It is known from FIG. 1 that the measured values by the method of the present invention are stable.

Experimental Example 2 Paper blood and serum were collected from 55 persons,
For paper blood, blood apolipoprotein B was measured in the same manner as described in Example 2, and for serum, phosphate and NaCl were dissolved in 0.01 M and 0.15 M concentrations, respectively, and the pH was adjusted to 7.3 to 80,000 times. Adipoprotein B in blood was measured in the same manner as described in Example 2 by diluting the sample.

The results are shown in Figure 2. The horizontal axis of the figure is the measured value from serum (mg
/ dl), the vertical axis represents the measured value (u / dl) from paper blood, and each point indicates the correspondence between the two measured values for each person.

As a standard, 0.25ng / 100μl-4.0ng / 10
Apolipoprotein B solutions of various concentrations in 0 μl were used, and in the case of paper blood, 100 times the measured value was expressed as u / dl. From FIG. 2, it is known that the measured value of the method of the present invention correlates well with the measured value from a large amount of collected serum even though the amount of collected blood is extremely small.

[Brief description of drawings]

FIG. 1 is a graph showing the relationship between the number of days elapsed after blood collection and the recovery rate (%). FIG. 2 is a graph showing the correlation between the measurement value from serum and the measurement value from paper blood.

Claims (6)

[Claims] 1. A method for measuring apolipoprotein B in blood by an immunological measurement method, which comprises collecting said apolipoprotein B in blood as dried filter paper blood and eluting with a solution containing a surfactant when preparing a measurement sample. A method for measuring apolipoprotein B in blood, which comprises: 2. The surfactant is polyethylene glycol p.
-The method for measuring blood apolipoprotein B according to claim 1, which is isooctyl phenyl ether. 3. The method for measuring blood apolipoprotein B according to claim 1 or 2, wherein the immunological measurement method is an ELISA method. 4. A reagent for measuring blood apolipoprotein B by an immunological measuring method, which is provided with a dry filter paper and a surfactant for preparing a measurement sample, for measuring blood apolipoprotein B. reagent. 5. The surfactant is polyethylene glycol p.
A reagent for measuring blood apolipoprotein B according to claim 4, which is isooctyl phenyl ether. 6. The reagent for measuring apolipoprotein B in blood according to claim 4 or 5, wherein the immunological measuring method is an ELISA method.