JPH03197865A - Measuring method for phosphatidylcholine - Google Patents

Abstract

PURPOSE:To safely immunologically measure phosphatidylcholine (PC) with a simple operation and high sensitivity in a short period of time by using a monoclonal antibody which specifically recognizes the PC. CONSTITUTION:The PC which is prepd. from the egg yolk, is immobilized on nitrocellulose paper and is homogenated in PBS is directly immunized as an antigen to the spleen of a mouse. The PC formed by coating acid treated Salmonella Minnesota bacteria with the PC is additionally immunized from the vein of the tail part after two weeks. The spleen is extracted from the mouse after 3 days of the final immunization and the antibody forming cells are prepd., by which the hybridoma is prepd. The analysis of the supernatant of the hybridoma culture is then executed and the hybridoma producing the antibody which reacts specifically with the PC is selected. Plural kinds of the monoclonal antibodies which specifically recognize the PC are thus obtd. The independent measurement of the PC in a sample by identifying the same from other phospholipids is, therefore, possible. The measurement of the many specimens with the simple operation and the high sensitivity in a short period of time is thus executed.

Description

DETAILED DESCRIPTION OF THE INVENTION (Industrial Field of Application) The present invention relates to phosphatidylcholine (hereinafter abbreviated as PC)
This invention relates to a method for measuring PC using a monoclonal antibody specific for.

(Prior Art) PC is a phospholipid that is a main component of cell membranes, and plays a very important role in living organisms. The most important role is that involved in cell membrane skeleton formation and membrane permeation, but HDL, which is a substrate of lecithin cholesterol acyltransferase (LCAT),
It plays a role as an activator of blood coagulation factor X, the main lipid component of blood. Furthermore, it plays an important role as a surfactant in body fluids such as bile, intestinal fluid, and plasma.

Clinically, plasma phospholipid concentrations increase due to hepatic parenchymal disorders such as severe hepatitis and decompensated cirrhosis, and plasma phospholipid concentrations decrease in intrahepatic cholestasis and obstructive jaundo. Are known. Of the phospholipids in plasma, about 70 to
Since 80% of these diseases are PC, early diagnosis and follow-up of these diseases are possible by measuring PC in plasma.

Conventional methods for quantifying phospholipids include the wet ashing method and the enzymatic colorimetric method. The wet ashing method is a method in which extracted phospholipids are wet-ashed, inorganic phosphoric acid is quantified, and the amount of phospholipids is measured by conversion. Although it has excellent quantitative properties, it is not used much at present because the operation is complicated and time-consuming. What can be measured with this method is the total amount of phospholipids, and it is not possible to quantify only PC.

The enzymatic colorimetric method is currently the mainstream method for measuring phospholipids, and is a method in which choline-containing phospholipids are decomposed by phospholipase D and the produced choline is quantified by a color reaction. This method allows measurement with a one-step operation and a reaction time of about 5 minutes. However, since choline-containing phospholipids are measured, sphingomyelin, which is contained in large amounts in living organisms, especially plasma, is also included in the measured value, making it impossible to quantify only PC.

Conventionally, fractionation and quantitative methods such as column chromatography, thin-layer chromatography, gas chromatography, and high-performance liquid chromatography have been used to separate and measure PC in phospholipids, but all of these methods are complicated and time-consuming. There were such drawbacks.

(Problems to be Solved by the Invention) The purpose of the present invention is to immunologically immunodefine PC in a safer and more sensitive manner with a simpler operation and in a shorter time than conventional methods by using a monoclonal antibody specific to PC. The objective is to provide a method for measuring

(Means for Solving the Problems) The present inventors have arrived at the present invention as a result of intensive studies regarding the above problems. That is, the present invention is an immunoassay method for PC using a monoclonal antibody that specifically recognizes PC, and the details thereof will be explained below.

Examples of immunological measurement methods include the sandwich method and the competitive method.

In the sandwich method, for example, (1) an immobilized monoclonal antibody specific for PC is combined with (2) a sample containing PC and a labeled antibody of another species specific for PC, and (3) the above-mentioned labeled antibody is added to bind to PC. (2) includes the step of detecting the label of the immobilized or free labeled antibody by the reaction performed in section (2).

In the competitive method, for example, (1) an immobilized monoclonal antibody specific for PC is added to (2) a sample containing PC and labeled PC, and
By binding C to the immobilized antibody (3) by the reaction obtained in section (2) above, the immobilized or free labeled P
The method includes a step of detecting a label of C.

In the method of the present invention, the monoclonal antibodies used can be prepared by methods known per se (G, K).

hler&C, former l5teln, Nature, 2
56, 495. (1975)). Hereinafter, the method for producing a monoclonal antibody of the present invention will be explained in detail.

(A) Preparation of PC PC can be prepared from egg yolk, etc., but may also be prepared from other samples. The method for preparing PC itself is known and is not limited in any way.

(B) Establishment of hybridomas Hybridomas can be prepared by immunizing a suitable animal with the above-mentioned antigen and then extracting lung cells according to a known method.

In the present invention, for example, PC prepared from egg yolk is immobilized on nitrocellulose paper, homogenized in PBS, and used as an antigen to directly immunize the lungs of mice (BALB/C) (Experimental Medicine Vol. 6, pp. 915-919). Two weeks later, the PC was coated with acid-treated Salmonella minnesota and boosted through the leg vein. Three days after the final immunization, the lungs were extracted from the mice and antibodies were produced. Cells can be prepared. The prepared tile cells were transformed into myeloma cell line P3-X6 using conventional cell fusion technology.
Hybridomas can be prepared by performing cell fusion in 3-Ag653 and DMEM medium in the presence of 50% polyethylene glycol.

(C) Selection of hybridomas The target hybridomas can be obtained by, for example, solid-phase enzyme immunotherapy.
Standard method (Math, Enzymol, 70.419
-439. (1980)), hybridoma culture supernatants are analyzed, hybridomas producing antibodies that specifically react with PC are selected, and the limiting dilution method is performed twice.

(D) Recovery and Purification of Antibodies According to conventional methods, the hybridomas are cultured in a growth medium, or the hybridomas are intraperitoneally administered to experimental animals (mice, rats, etc.) in which these cells can proliferate and cultured in vivo. By this, antibodies can be produced. Antibodies can be recovered from the culture supernatant when hybridomas are grown in a growth medium, or from the ascites of experimental animals when grown within the peritoneal cavity of the animal. The culture supernatant or ascites can be roughly purified by ammonium sulfate precipitation fractionation, and then purified by ion exchange chromatography.

The purity of the antibody can be confirmed by the formation of two bands, one heavy chain and one light chain, in 12% 5DS-polyacrylamide electrophoresis under mercaptoethanol reduction.

By the above method, multiple types of monoclonal antibodies that specifically recognize PC were obtained. The binding constants of each antibody were in the range of 107-1011 M-1. Using these monoclonal antibodies, quantitatively detect PC11) J
Immunological +1 by sandwich method or competitive method that can be determined
11 rules became possible.

In addition, it specifically recognizes PC, phosphatidylserine (PS), phosphatidylethanolamine (PE)
, phosphatidylic acid (PA), calciolipin (CL
), phosphatidylinositol (PI), or phosphatidylglycerol (PC) to obtain a monoclonal antibody that does not show cross-reactivity with PS, PE, PA, CL, It is sufficient to select one that does not cross-react with PI and PG.

A known method can be used to immobilize the antibody or PC used in the method of the present invention on a solid phase. Examples of the solid phase include polystyrene, polyethylene, polyvinyl chloride, polycarbonate, sepharose particles, latex,
Agarose, cellulose, polymethacrylate, etc. are used.

Furthermore, the method of labeling the antibody or PC and the method of detecting the same are not limited at all, and labeling and detection can be performed by known methods. When using an enzyme as a label, examples of the labeling substance include peroxidase,
Enzymes such as β-D-galactosidase, alkaline phosphatase, urease, catalase, and β-glucuronidase are used. As a radioactive substance, 3H, +251
For example, fluorescamine, fluorella centiocyanate, tetrarhodamine isothiocyanate, etc. are bound to a monoclonal antibody using a conventional method. However, the labeling substance should not be limited to the above-mentioned substances.

In addition to the above-mentioned substances, known reagents used in the measurement include substrates, solubilizers, buffers, detergents, reaction terminators, and the like.

(Effects of the Invention) As is clear from the above explanation, according to the present invention, (1)
It is possible to distinguish PC in a sample from other phospholipids and measure it alone, and (2) it is possible to determine ILJ of a large number of samples with high sensitivity and in a short time with simpler operations compared to conventional methods.

(Example) Examples of the present invention will be described below to explain the effects of the present invention in detail. Note that the present invention is not limited to the following examples.

Example 1 Egg yolk-derived PC (manufactured by Funakoshi Pharmaceutical Co., Ltd., manufacturer code A-31) was used as an antigen for initial immunization in mice (BALB
/C) and further boosted using PC.

First, PC was immobilized on nitrocellulose paper, homogenized in PBS, and used as an antigen for mice (BAL).
The lungs of B/C) were directly immunized, and two weeks later, the PC coated with acid-treated Salmonella minnesota was boosted through the original vein. Three days after the final immunization, the lungs were removed from the mice and antibody-producing cells were prepared.

Using conventional cell fusion technology, the lung cells prepared above were
Miniroma cell line Pa-X83-Ag853 and DMEM
Hybridomas were prepared by cell fusion in a medium in the presence of 50% polyethylene glycol.

The target hybridoma is selected by analyzing the hybridoma culture supernatant using solid-phase enzyme immunoassay, and then
A hybridoma producing an antibody that reacts only with the above was selected, and the hybridoma was established by performing limiting dilution twice. The obtained hybridoma cells were cultured in a 96-well microtiter plate according to a conventional method, and one week after the start of culture, the antibody titer and specificity of the culture supernatant were evaluated by solid-phase enzyme immunoassay. made a selection.

Solid-phase enzyme immunoassay uses various phospholipids, such as PS, PA, PE, and PC, in microtiter plates.

CL, PI, and PC were coated at 0.5 nmol/well and blocked with 3% BSA (bovine serum albumin) for 2 hours at room temperature. lomM Tris-HCI
, 150mM NaCl, pt (after washing with 7,4,
Culture supernatant was added at soμI/well and left at room temperature for 2 hours. After washing, biotin-labeled antibody (manufactured by ZYMED, 8
2-6440 Old otln-Gtx Mouse1gG+
IgA+IgM (M+L)) and wasabi peroxidase/avidin D (manufactured by Funakoshi Pharmaceutical Co., Ltd., A-200)
4, VEC41012004) and selected wells with positive antibody titer to obtain antibodies showing reactivity only to PC (hereinafter these antibodies will be referred to as antibodies A, B, and C).

Example 2 The following experiment was conducted for the purpose of investigating the cross-reactivity of Antibody A with PC-like substances.

First, a microtiter plate was prepared with 0.5 nmol of P.
After coating with C and blocking with 3% BSA, It
) nM Tris-IC1,150 (IIM NaCI
(pH 7,4).

Next, 50% of the culture supernatant of the hybridoma producing antibody A was
PC1choline-C1, phosphorylcholine, glycerophosphocholine, and lyso-PC1 for μm, respectively.
Alternatively, sphingomyelin was added and left to stand at room temperature for 2 hours, and then the cross-reactivity between Antibody A and the above-mentioned PC-like substance was investigated in the same manner as in Example 1. As a result, it was found that antibody A specifically reacts only with PC.

Example 3 PC was measured by the sandwich method.

(A) Immobilization of anti-PC antibody: 0.1 M sodium carbonate buffer (p
200 µm of a solution of 3 ug/ml mouse-derived anti-PC antibody (antibody B) dissolved in H9,6) was added and incubated overnight at 4 °C. Next, the solution in each well was removed and washed three times with a solution containing 0.04% Tween-20 in PBS (hereinafter referred to as PBS-T).
300 μl of PBS-T solution containing % BSA was added to each well, and blocking treatment was performed at 4° C. overnight.

(B) Preparation of horseradish peroxidase (HRP) labeled antibody: 0.3M sodium bicarbonate buffer (pH
1% 1 in HRP solution (5B/nl) dissolved in 8,1)
0.11 of an ethanol solution of -fluoro-2,4-dinitrobenzene was added, and the mixture was reacted at room temperature for 1 hour. 1.01 of 0.08M sodium periodate was added to the solution and reacted for 30 minutes. Remove unreacted sodium periodate to 0
．． After adding and removing 18M ethylene glycol 1.01, 0.01M sodium carbonate buffer (pH 9,5)
Dialyzed with Next, 5 mg of mouse-derived anti-PC monoclonal antibody (antibody C) was added and reacted for 5 to 6 hours. 5 mg of sodium borohydride was added and left at 4°C overnight. After this, 1011 containing 0.85% sodium chloride was added to remove unreacted sodium borohydride.
4°C against M sodium phosphate buffer (pH 7,4)
The mixture was dialyzed overnight with stirring. The above reaction product was purified by high performance liquid chromatography using TSK Gel G3000SW (manufactured by Tosoh Corporation) to obtain an HRP-labeled antibody.

(C) Quantification of PC in samples 2. Return the microtiter plate prepared by the method described in (A) of this example to room temperature, wash it with PBS-T solution, and add a standard sample containing PC to each well. 20 μm was added to each. Next, the HRP-labeled antibody obtained in Example (B) was diluted with PBS-T solution, and 200 μl was added to each well. After incubating for 3 hours at room temperature, remove the solution and replace with PBS.
- Washed three times with T solution. 1.2% 2.2-azinody(3-ethylbenzthiarizoline sulfate)-diammonium salt (ABTS) and 0.01% hydrogen peroxide (H2O
2) 0, 1M citrate buffer (pH 4,1) containing
Add 20cjμl of substrate solution consisting of
After enzymatic reaction for 30 minutes at room temperature, 100 μl of 20 (leM oxalic acid solution) was added to stop the enzyme reaction.
5 nm and a reference wavelength of 492 nm using an automatic microtiter plate reader (manufactured by Tosoh Corporation, MPR-A).
4). The results are shown in Table 1.

Table 1 As is clear from the results, the PC in the sample is between 1 and 200.
It was confirmed that it could be quantified in the ng/ml range.

Claims (4)

[Claims] (1) A method for measuring phosphatidylcholine, which comprises using a monoclonal antibody that specifically recognizes phosphatidylcholine. (2) It specifically recognizes phosphatidylcholine and shows no cross-reactivity with phosphatidylserine, phosphatidylethanolamine, phosphatidylic acid, cardiolipin, phosphatidylinositol, or phosphatidylglycerol. A method for measuring phosphatidylcholine according to claim (1) using a monoclonal antibody. (3) The method according to claim 1 or 2, wherein the measurement is performed by a sandwich method. (4) The method according to claim 1 or 2, which is measured by a competitive method.