JPH02287257A - Method for measuring human g-csf - Google Patents

Abstract

PURPOSE: To realize a more convenient measurement with high sensitivity while suppressing fluctuation by using an antibody consuming fraction for human G-CSF and employing a nonionic surfactant and the like in a reaction system. CONSTITUTION: An antibody consuming fraction is used for a labeled antibody in the measurement. An antigen-antibody reaction is caused between an insoluble antibody and human G-CSF under presence of a nonionic surfactant. According to the method, the effect of interferential substances in plasma is eliminated resulting in the enhancement of sensitivity and reliability of measurement.

Description

DETAILED DESCRIPTION OF THE INVENTION Field of Industrial Application The present invention relates to a highly sensitive enzyme-linked immunoassay method for human G-CSF in microscopically opened human G-CSF, particularly in plasma.

Conventional technology G-CSF is one of the hematopoietic factors and is known to exist in the culture solution of human bladder cancer cell line 5637 (ATCCHT8-9) (Welt et al.; Proc.
.

Natl^cad, sci, U, s, A, 82.152
6-1530. (1985)).

In addition, the DNA sequence encoding this gene was determined (Japanese Patent Publication No. 500636/1983), and GCS was produced by genetic recombination.
It is now possible to produce F.

G-CSF is a factor that acts on bone marrow stem cells and promotes their differentiation into neutrophils (Metcalf et al.; Bfood).

67(1), 37-45. (1986)), it has been reported that it also has an effect on differentiated neutrophils to enhance neutrophil functions such as phagocytosis and 02 production (Katao et al.;
Blood, 70(2), 404-411 (198
7)). In addition, the ability to quickly recover from bone marrow suppression induced by the administration of chemotherapeutic agents has been demonstrated in animal experiments, and clinical effects are highly expected (Cohen et al.; Pr0C, Nat1.^cad, sci, , 84.
2984-2988 (1987)).

When considering the clinical application of G-CSF, it is necessary to clarify the correlation between the therapeutic effect and the level of G-CSF in the body. Must.

On the other hand, it has also been reported that G-CSF has the effect of supporting the proliferation of leukemia cells (Berenga et al.; Bfoo
d, 69(6), 1771-177G (1987
)) In order to clarify the relationship between G-CSF levels in the body and pathological conditions, a highly sensitive method for measuring G-CSF in plasma seems to be an effective means.

However, the immunological presence of G-CSF in plasma? There is a substance that interferes with the AII concentration, and this substance is G-CSF.
It is thought that the antigenicity of G-CSF is masked by binding or conjugating with G-CSFf in plasma.
It was not possible to directly measure fi. Therefore, the following methods have been used to measure G-C3F.

That is, plasma is mixed with an organic solvent to remove lipids and most of the proteins in the plasma. Next, add G-CS to the ion exchange resin.
By adsorbing F-containing substances in plasma and separating G-CSF and interfering substances by utilizing the difference in affinity with the resin, G-1cs
Collect only F. G-CSF labeled with a radioactive substance and antibodies against G-C3F are added to this, and the resulting G
The intensity of radioactivity of the -CSF antibody complex is measured, and G-CSFftk is determined from the measured value.

In addition, radioimmunoassay (R
As an alternative method to IA), enzyme immunoassay (ERA)
This is a well-known method, in which an antibody against the antigen to be measured is bound to a solid phase, which is then reacted with the antigen, washed, and then further reacted with an enzyme-labeled antibody to form the antigen 6.
(Ishikawa et al.; Clinical Chemistry, Part 3)
, p. 374 (1974)).

Regarding the ERA of human G-CSF, after adding serum to the insolubilized anti- and l~G-CSF antibodies and allowing them to react,
Sixteen methods characterized by the addition of peroxidase-labeled Fab' have been reported (Journal of the Japanese Society of Hematology, Vol. 51, No. 2, p. 390).

The problem that Akira is trying to solve The conventional method that combines the above-mentioned extraction operation and RIA is
■ The extraction operation is complicated, the extraction efficiency is low, and there are variations; ■ The number of samples that can be handled at a time is small; ■
Since tIi is a sensitive substance, it has drawbacks such as many restrictions on the use of labeled antibodies and treatment of waste fluid.

On the other hand, in the EIA of G-CSF reported so far, sufficient sensitivity was not obtained and variations were large.

When considering the clinical application of G-CSF, it is essential to accurately grasp the amount of G-CSF in the body.
A method for measuring CSF is needed.

Means to Solve the Problems As a result of intensive studies to improve the above problems, the present inventors used antibody digested fragments against human G-C3F and intervened a nonionic surfactant etc. in the reaction system. By doing so, we have discovered an excellent measuring method that can satisfy the above requirements, and have arrived at the present invention.

That is, the present invention relates to an enzyme immunoassay method for human G-CSF, which method uses an antibody-digested fragment as a labeled antibody, and an insoluble antibody and human G-CS.
It is characterized in that the antigen-antibody reaction with F is carried out in the presence of a nonionic surfactant.

As the digested fragment of the labeled antibody used in the present invention, an antibody digested fragment from which the FC portion of gG is removed, ie, Fab', is preferable in order to minimize non-specific binding.

Further, the labeling enzyme used in the present invention may be any enzyme as long as it is commonly used in EIA, but horseradish peroxidase is particularly preferred.

Fab' and labeled antibodies can be prepared by conventional means in the art.

As the insolubilized antibody and labeled antibody of the present invention, it is preferable to use a polyclonal antibody that can bind to multiple sites and is considered to have high affinity for the antigen.

One feature of the measurement method of the present invention is that in order to eliminate the influence of interfering substances in plasma and increase the sensitivity and reliability of measurement,
In the antigen-antibody reaction between human G-C3F and an insolubilized antibody, a nonionic surfactant and further ethylenediaminetetratetraacetic acid mono-lithium (EDTA) are present in the reaction system.

Nonionic surfactants that can be used in the present invention include polyoxyethylene lauryl alcohol ether, polyoxyethylene monostearate, polyoxyethylene octylphenyl ether, polyoxyethylene sorbitan 1-lioleate, and polyoxyethylene sorbitan. Examples include polyoxyethylene derivatives such as monooleate, polyoxyethylene sorbitan monooleate, polyoxyethylene sorbitan monooleate, and polyoxyethylene sorbitan monolaure-1. Particularly preferred are those having an HLB) value of 11 to 17. A typical example is polyoxyethylene sorbitan monolaurate (Tween), which has an HLB value of 16.7.
20).

The amount of the nonionic surfactant to be used varies somewhat depending on the type of nonionic surfactant used, but it is preferably 0.01 to 1% by weight, particularly 0.1 to 1% by weight, based on the reaction system. However, excess surfactant will actually inhibit the binding of antibodies to G,-CSF and will also intensify non-specific color development, so it is desirable to use a nonionic surfactant at 1% by weight or less. .

In the measurement method of the present invention, an excellent effect can be obtained by adding EDTA to the antigen-antibody reaction system for the same purpose as the addition of a nonionic surfactant. EDTA
For this purpose, about 0, r, q♀% of a nonionic surfactant and ED are added to the antigen-antibody reaction system of the measurement method of the present invention.
Methods for making TA present include adding these substances to plasma or serum samples in advance, or adding them separately.
It can also be added before or after adding the sample to the reaction system.

Furthermore, in the measurement method of the present invention, good results can be obtained when the antigen-antibody reaction time between human G-CSF and insolubilized antibody is made longer than in normal EIA. Therefore, such reactions are preferably carried out at about 4°C for 12 to 24 hours.

In the measurement method of the present invention, except for the points mentioned above, the methods and methods commonly used in conventional conventional EIA are used.
This can be done under the same conditions.

Example EXAMPLES Hereinafter, the present invention will be described in detail with reference to Examples.

Purification of rabbit anti-human G-CSF serum with saturated ammonium sulfate solution was gradually added to the rabbit anti-human G-CSF serum to achieve 45% saturation to precipitate the crude immunoglobulin fraction.
m, centrifugation was performed for 10 minutes and the precipitate was collected.

The precipitate was dissolved in PBS and dialyzed against the same buffer. Next, Protein^ column (HAPSIr:
The IoG fraction was purified by BIOItAD).

Example 2 Production of enzyme-labeled antibodies This was carried out by the method of Ishikawa et al. (Eiji Ishikawa et al., "Enzyme immunoassay method 1, 2nd edition, Igaku Shoin, 1982)".

(1) Preparation 1 of Fab' from IgG 1g0100cm purified using the method of Example 1 was 0.
O, IMM drawing solution containing 1M sodium chloride (pH 4,
Dialyzed against 5), added 4% by weight of pepsin, and heated at 37°C.
After reacting for 24 hours, digestion was stopped by adjusting the pH to 7.0. Next, HPLC (TSKGet G3
000sw) to collect two portions of F(ab').

(2) Preparation of peroxidase-labeled Fab' F (a
b'),,8! Itg/xi! and that 2m is 0.
Dialyzed against 1M Phosphorus IIIWlfi solution p116.0 and 1/10th volume of 0.1H2-mercaptoethylamine-5n
HEDTA was added and photoreacted for 90 minutes at 37°C. Next, Fab' was obtained by gel filtration using HPLC.

Dissolve horseradish peroxidase 2q in 0.1M phosphate buffer (all 7.0>0.3ai!
30 parts of a solution of (N-maleimidoethyl)cyclohexane-1-carboxylic acid N-hydroxysuccinimide ester in N-dimethylformamide was added. 6 at 30℃
After reacting for 0 minutes, the reaction mixture was subjected to gel E filtration using HPLC to obtain peroxidase-maleimide.

Next, 2 mg of Fab' and 1.8 m5 of peroxidase-maleimide were added to 0.1 M phosphate buffer (all 6.0).
The reaction was carried out at 30° C. for 1 hour in a 2.5 m HED chamber.

By adding 6 g of 50 mM toethylmaleimide and gel filtration using HPLC, unreacted substances were removed to obtain peroxidase-labeled Fab'.

Example 3 Production of anti-human G-CSF antibody insolubilized microtiter plate The anti-human G-C3F antibody (IgG) obtained in Example 1 was mixed with 50 mH carbonate buffer pl+9.2 at 300 m2/7! The solution was diluted as desired, and 50 doses of the solution were dispensed into each well, and left at room temperature for 2 hours. After removing the reaction solution and washing three times with PBS, 250 d of PBs containing 5% BSA was dispensed into each well, left at room temperature for 1 to 2 hours, and after removing the reaction solution, PBs
Washed with S three times.

History - Measurement of human G-CSF using fUPJ4° peroxidase standard "J-Fab' (1) Measurement in PBS containing 1% BSA PBS containing 1% BS8 (1%BS△- human G-
Standard solution prepared by diluting CS and F (buffer-based standard solution)
50μ of each well was dispensed into the microtiter plate prepared in Example 3. After standing at 4°C for 12 hours, the reaction solution was removed and washed 5 times with PBS. The peroxidase-labeled Fab' prepared in Example 2 was diluted with 1% BSA-PBS, and 50 ρ was dispensed into each well.

2 [1i-1? ! After leaving it for a while, discard the solution and add PBS.
Washed 5 times with 0.4Rg/id O-phenylenediamine 0.1% citric acid buffer pH 5.2 containing 6% hydrogen peroxide solution was dispensed into each well at 100 pi and allowed to react at room temperature for 10 minutes. INIIIJ) 50/
Example 4 Human G- using peroxidase labeled Fab'
Measurement of C3F (1) Measurement in PBS containing 1% F3SΔ 1% BSA
human G in PBS ('1%BSA-PBS) containing
'Standard solution prepared by diluting CSF (buffer-based standard solution)
50 components were dispensed into each well of the microtiter plate prepared in Example 3. After standing at 4°C for 12 hours, the reaction solution was removed and washed 5 times with PBS. BelΔoxidase-labeled Fab′ prepared in Example 2 was added to 1
It was diluted with %BSΔPBS and 50 components were dispensed into each well.

Leave for 2 hours at room temperature [L solution was discarded and washed 5 times with PBS.

0.4m9/ld O-phenylenediamine 0.006
0.1% citrate buffer containing % hydrogen peroxide p115
．． After pouring 100 components of 2 into each well, the mixture was allowed to react at room temperature for 10 minutes. The reaction was stopped by adding 150 ρ of IN HCl, and the absorbance at 492r+m was measured using eoonm as a control.

A standard curve was created using a semi-logarithmic graph, with the absorbance of 492n111 on the vertical axis and the concentration of human G-C3F on the horizontal axis (see Figure 1).

(2) Measurement in u normal human plasma Standard g prepared by adding human G-CSF and serial dilution to rainbow normal human plasma prepared by adding 0.5% by weight of EDT△
<Healthy human plasma standard solution) was measured by the method of Example 4 (1). The obtained test m-line is shown in FIG. However, this measurement method had low sensitivity and large variations. Furthermore, this calibration curve does not match the calibration curve obtained using the buffer-based standard solution due to interference substances, and is higher than the calibration curve 1 in the low Q degree region of human G-CSF.

In the high concentration region of human G-C3F, low levels were observed.

Although human G-CSF exists stably in plasma, interfering substances in IfI plasma cause human G-C3 to
This was thought to be because the binding between F and the antibody was partially inhibited.

(3) Measurement in human plasma with non-ionic surfactants added Various non-ionic surfactants were added to plasma to determine conditions to minimize the effects of interfering substances in plasma. We have considered this. Add Tween 20 (POLY 5CIE) as a nonionic surfactant to healthy human plasma standard solution.
NCE W^IIRINGTON, PA company tJ)
, Twecn 85 (polyoxyethylene sorbitan trioleate, I L B = 11.0,
(manufactured by Junsei Kagaku iI1), Br1j-35 (poly(diethylene lauryl alcohol ether, 1-ILB=1
6.9, Nacalai Tesque (manufactured by Kikusha) at 10% (V/V
) was added and measured in the same manner as in Example 4 (1).

The binding is shown in Figure 3. It can be seen that when a nonionic surfactant is added to plasma, the results match well with the calibration curve for the buffer-based standard solution. In addition, as a study on the addition of nonionic surfactants, various concentrations of Twcen 20 were added at 10% (V/V) to the plasma volume.
). As the concentration of Twecn 20 in plasma increases, the measurement results gradually approach the standard curve of 0.1-1% Twecn 20 in the buffer-based standard solution.
The best results were obtained under the addition condition of n 20 (4th
(see figure). Addition of more than 1% by weight of Dingwecn 20 was not preferred because it caused a non-specific reaction or inhibited binding to the anti-Human IG-CSF antibody. Furthermore, the optimal concentration of the nonionic surfactant was different for each nonionic surfactant.

(41?! Measurement in plasma of 2 healthy people 1 car number %Twe
en 20-0.51fft% [r to be DTA
Ween 2083 and EDTA-added various Shiragiku samples were added with human G-C3F and serially diluted to create a standard solution, which was measured. (See Figure 5).

(5) Measurement in leukemia plasma Samples of 1υ% Tween 20-
Tween 2 to be 0.5ff[%E DTA
Table 1 shows the results obtained by adding 0 and EDTA and measuring according to the method of Example 4 (1). In addition, these G-CS in plasma
Fffi was determined from a calibration curve using a buffer-based standard solution.

table CML: Chronic myeloid leukemia ALL: Acute lymphocytic leukemia CLL: chronic lymphocytic leukemia

[Brief explanation of the drawing]

FIG. 1 shows a calibration curve for a standard solution (buffer-based standard solution) prepared by serially diluting human G-C3F with 1% BSA-PBS. FIG. 2 shows a calibration curve for a standard solution (healthy person plasma standard solution) prepared by adding human G-CSF to healthy person's plasma and serially diluting it. In the figure, O indicates the buffer base standard beta, and other symbols indicate healthy human blood + 5 standard solutions. Figure 3 shows healthy human plasma vp, 111. A calibration curve is shown when a nonionic surfactant is added to the solution. The meaning of each symbol in the diagram is as follows. O: Buffer system standard solution port = Healthy human blood 51 standard solution containing 1% Tween 20■
: Healthy human blood + standard solution containing 1% Tween 85◇
: Healthy person plasma standard solution containing o, oi% Br1j-35. : Healthy person plasma standard solution.
The addition port and its calibration curve are shown. The meaning of each symbol in the diagram is as below. 0:! 1iii liquid system 4! IQ-liquid: Healthy human plasma standard solution Inlet: Healthy human plasma standard solution containing 1x Tween 20■
:0. Healthy human plasma standard solution containing IX Tween 20 ◇: Healthy human plasma standard solution containing 0.01% Tween 20
The test ω line is shown when adjusted to be n20-0.5 type O%EDT^. In the figure, O is gmi standard solution, other symbols are Tw
A healthy human plasma standard solution containing een 20 is shown.

Claims (9)

[Claims] (1) An enzyme immunoassay method for human G-CSF, in which an antibody digested fragment is used as a labeled antibody, and an antigen-antibody reaction between the insolubilized antibody and human G-CSF is performed in the presence of a nonionic surfactant. The measuring method characterized in that: (2) The measuring method according to claim 1, characterized in that disodium ethylenediaminetetraacetate is further present during the antigen-antibody reaction. (3) The measuring method according to claim 1 or 2, wherein the surfactant is present in a range of 0.01 to 1% by weight. (4) The measuring method according to claim 3, wherein the surfactant is present in a range of 0.1 to 1% by weight. (5) The measuring method according to any one of claims 1 to 4, wherein the surfactant has an HLB value of 11 to 17. (6) The measuring method according to claim 4, wherein the surfactant is Tween 20. (7) Ethylenediaminetetraacetic acid disodium is about 0.5
The measuring method according to any one of claims 1 to 6, characterized in that it is present in a proportion of % by weight. (8) The measuring method according to any one of claims 1 to 7, characterized in that the antigen-antibody reaction between the insolubilized antibody and human G-CSF is carried out at about 4°C for 12 to 24 hours. (9) The measuring method according to any one of claims 1 to 8, characterized in that a polyclonal antibody is used as the insolubilized antibody and the labeled antibody.