JP2713349B2 - Method for measuring human G-CSF - Google Patents

Description

The present invention relates to trace amounts of human G-CSF, especially human GC in plasma.
The present invention relates to a highly sensitive enzyme immunoassay for SF.

G-CSF is one of the hematopoietic factors and has been shown to be present in the culture solution of human bladder cancer cell line 5637 (ATCC HT8-9) (Welt et al .; Proc. Natl. Acad.Sci.U.
SA82, 1526-1530, (1985)). In addition, the DNA sequence encoding this gene has been determined (Japanese Translation of PCT International Publication No. 63-500636), and G-CSF can be produced by genetic recombination.

G-CSF is a factor that acts on bone marrow stem cells and promotes differentiation into neutrophils (Metcalf et al .; Blood, 67 (1), 37-
45, (1986)), phagocytosis against differentiated neutrophils and O ₂ ^-
It has been reported that neutrophil functions such as production are enhanced (Yuo et al., Blood, 70 (2), 404-411 (198
7)). In addition, the effect of early recovery of myelosuppression induced by the administration of chemotherapeutic agents has been demonstrated at the level of animal experiments, and clinical effects are greatly expected (Cohen et al .; Proc. Natl. Acad. Sci., 84,2984-2988 (1987)). G
-Considering the clinical application of CSF, the therapeutic effect and G in the body
-It is necessary to clarify the correlation with the level of CSF,
For this purpose, G-CSF present in the body must be accurately quantified.

On the other hand, it has been reported that G-CSF has an effect of supporting the proliferation of leukemia cells (Berenga et al .; Blood, 69).
(6), 1771-1776 (1987)), and to clarify the relationship between the G-CSF level in the body and the disease state, G-CSF in plasma was also used.
A highly sensitive method for measuring the amount of CSF is likely to be a powerful tool.

However, there is a substance that interferes with the immunological measurement of G-CSF in plasma, and it is thought that this substance binds or conjugates with G-CSF and masks the antigenicity of G-CSF. Therefore, it was not possible to directly measure the amount of G-CSF in plasma. Therefore, the following methods have been used for measuring G-CSF.

That is, the plasma is mixed with an organic solvent to remove lipids and most proteins in the plasma. Next, G-CS is used for the ion exchange resin.
A substance in plasma containing F is adsorbed, G-CSF and an interfering substance are separated by utilizing a difference in affinity with a resin, and only G-CSF is recovered. G-CSF labeled with a radioactive substance
Then, an antibody against G-CSF is added, the intensity of the radioactivity of the resulting G-CSF antibody complex is measured, and the G-CSF amount is determined from the value.

As an alternative to radioimmunoassay (RIA) using radioactive substances, enzyme immunoassay (EIA) is well known. This method involves binding an antibody against the antigen to be measured to a solid phase, and using this method. After washing with an antigen, and then reacting with an enzyme-labeled antibody to measure the amount of the antigen (Ishikawa et al .; Clinical Chemistry, No. 3).
Vol., P. 374 (1974)).

As for the EIA of human G-CSF, a method has been reported in which serum is added to an insolubilized anti-human G-CSF antibody and reacted with peroxidase-labeled Fab ′ (Journal of the Hematological Society of Japan). Vol. 51, No. 2, 390
page).

Problems to be Solved by the Invention The conventional method that combines the above-described extraction operation and RIA,
The extraction operation is complicated, the extraction efficiency is low, there is variation, the number of samples that can be handled at a time is small,
Since a radioactive substance is used, there are drawbacks in that there are many restrictions on the use of a labeled antibody, the treatment of waste liquid, and the like.

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

When considering the clinical application of G-CSF, it is essential to accurately determine the amount of G-CSF in the body, and therefore, G-CSF is simpler, more sensitive, and has less variation.
There is a need for a method for measuring F.

Means for Solving the Problems The present inventors have intensively studied to improve the above-mentioned problems, and as a result, using an antibody digested fragment against human G-CSF, intervening a nonionic surfactant or the like in the reaction system. As a result, the present inventors have found an excellent measuring method that can satisfy the above requirements, and have reached the present invention.

That is, the present invention relates to an enzyme immunoassay for human G-CSF, which comprises using an antibody digested fragment as a labeled antibody, and using an insolubilized antibody and human G-CSF.
Characterized in that an antigen-antibody reaction is performed 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 IgG has been removed in order to minimize non-specific binding, that is, Fab 'is preferable.

The labeling enzyme used in the present invention may be any enzyme that is commonly used in EIA, but horseradish peroxidase is particularly preferable.

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

As the insolubilized antibody and the labeled antibody of the present invention, it is preferable to use a polyclonal antibody that can bind to a plurality of 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 the measurement, a non-ionic reaction is performed during an antigen-antibody reaction between human G-CSF and an insolubilized antibody. Surfactant, and also sodium ethylenediaminetetraacetate (EDTA) is present in the reaction system.

Nonionic surfactants that can be used in the present invention include:
Polyoxyethylene lauryl alcohol ether, polyoxyethylene monostearate, polyoxyethylene octyl phenyl ether, polyoxyethylene sorbitan trioleate, polyoxyethylene sorbitan monostearate, polyoxyethylene sorbitan monooleate, polyoxyethylene sorbitan monopalmi And polyoxyethylene derivatives such as polyoxyethylene sorbitan monolaurate, and those having a hydrophilic lipophilic balance (HLB) value of 11 to 17 are particularly preferred. A typical example is polyoxyethylene sorbitan monolaurate (Tween 20) having an HLB value of 16.7.

The amount of the nonionic surfactant used varies somewhat depending on the type of the nonionic surfactant used, but is preferably 0.01 to 1% by weight, particularly preferably 0.1 to 1% by weight, based on the reaction system.
However, an excess of the surfactant instead inhibits the binding of the antibody to G-CSF and enhances non-specific color development. Therefore, 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 when EDTA is further present in the antigen-antibody reaction system for the same purpose as the addition of the nonionic surfactant. EDTA is preferably used at a ratio of about 0.5% by weight for this purpose. As a method for allowing a nonionic surfactant and EDTA to be present in the antigen-antibody reaction system of the measurement method of the present invention, these methods are previously used in plasma or serum samples. Or add the substance
Alternatively, they can be separately added before or after the sample is added to the reaction system.

In the measurement method of the present invention, better results can be obtained by setting the antigen-antibody reaction time between human G-CSF and the insolubilized antibody longer than that of ordinary EIA. Therefore, such a reaction is about 4
It is preferably carried out at a temperature of 12 to 24 hours.

In the measurement method of the present invention, except for the points described so far, the means and methods usually used in the conventional conventional EIA
It can be performed in the same manner as the conditions.

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

Example 1 Purification of Antibody Saturated ammonium sulfate solution was gradually added to rabbit anti-human G-CSF serum while stirring, and the crude immunoglobulin fraction was precipitated by 45% saturation, and centrifuged at 2000 rpm for 10 minutes. The precipitate was collected. The precipitate was dissolved in PBS and dialyzed against the same buffer. Next, Protein A
The IgG fraction was purified by a column (MAPSII; BIO RAD).

Example 2 Production of Enzyme-Labeled Antibody Method of Ishikawa et al.
Edition 1982 Medical Shoin).

(1) Preparation of Fab ′ from IgG 100 mg of IgG purified using the method of Example 1 was dialyzed against 0.1 M acetate buffer (pH 4.5) containing 0.1 M sodium chloride, and 4% by weight of pepsin was added. After reaction at 37 ° C. for 24 hours, the digestion was stopped by adjusting the pH to 7.0. Next, gel filtration is performed by HPLC (TSKGel G3000sw) and F
(Ab ') _Two fractions were collected.

(2) Preparation of peroxidase-labeled Fab 'F (ab') ₂ was adjusted to 8 mg / ml, and 2 ml thereof was dialyzed against 0.1 M phosphate buffer pH 6.0, and 1/10 volume of 0.1 M2-mercaptoethylamine-5 mM. EDTA was added, and a reduction reaction was performed at 37 ° C. for 90 minutes. Next, gel was passed through HPLC to obtain Fab '.

2 mg of horseradish peroxidase was dissolved in 0.3 ml of 0.1 M phosphate buffer (pH 7.0), and 30 μl of a solution of 4 (N-maleimidoethyl) cyclohexane-1-carboxylic acid N-hydroxysuccinimide ester in N, N-dimethylformamide was added. Was added. After reacting at 30 ° C for 60 minutes, HP
Gel permeation by LC gave peroxidase-maleimide.

Next, Fab '2 mg and peroxidase-maleimide 1.8 m
g in 0.1 ml phosphate buffer (pH 6.0) -2.5mM EDTA 1ml at 30 ℃
For 1 hour. 6 μm of 50 mM N-ethylmaleimide
1 and gel filtration by HPLC to remove unreacted substances to obtain peroxidase-labeled-Fab '.

Example 3 Preparation of anti-human G-CSF antibody-insoluble microtiter plate The anti-human G-CSF antibody (IgG) obtained in Example 1 was used at 50 mM.
Dilute to a concentration of 300 μg / ml with carbonate buffer pH 9.2.
50 μl was dispensed into each well and left at room temperature for 2 hours. After removing the reaction solution and washing three times with PBS, the PBS 25 containing 5% BSA is removed.
0 ml was dispensed into each well, allowed to stand at room temperature for 1 to 2 hours, and after removing the reaction solution, the well was washed three times with PBS.

Example 4 Measurement of human G-CSF using peroxidase-labeled-Fab '(1) Measurement in PBS containing 1% BSA Human G-CSF was diluted with PBS containing 1% BSA (1% BSA-PBS) Example 3 using a standard solution (buffer-based standard solution)
Was dispensed at 50 μl per well into each of the microtiter plates prepared in the above. After standing at 4 ° C. for 12 hours, the reaction solution was removed, and the plate was washed five times with PBS. The peroxidase-labeled-Fab 'prepared in Example 2 was diluted with 1% BSA-PBS, and dispensed to each well at 50 μl. After leaving at room temperature for 2 hours, the solution was discarded, and the plate was washed five times with PBS. 100 μl of 0.1% citrate buffer (pH 5.2) containing 0.4 mg / ml O-phenylenediamine-0.006% aqueous hydrogen peroxide was dispensed into each well, and then 10 μl at room temperature.
Allowed to react for minutes. The reaction was stopped by adding 50 μl of 1N HCl, and the absorbance at 492 nm was measured using 600 nm as a control.

The absorbance at 492 nm was plotted on the vertical axis, and the concentration of human G-CSF was plotted on the horizontal axis, and a standard curve was created using a semilogarithmic graph (first curve).
See figure).

(2) Measurement in healthy human plasma Standard solution (healthy human plasma standard solution) prepared by serial dilution of human G-CSF to human healthy plasma prepared by adding 0.5% by weight of EDTA was performed. It measured by the method of Example 4 (1). The calibration curve obtained is shown in FIG. However, the measurement results were low in sensitivity and large in variation. Also, this calibration curve does not match the calibration curve obtained with the buffer-based standard solution for the interfering substance, and is higher than the calibration curve in the low concentration region of human G-CSF,
The value was low in the high concentration region of human G-CSF. This was thought to be because human G-CSF was stably present in plasma, but the binding between human G-CSF and the antibody was partially inhibited by interfering substances in plasma.

(3) Measurement in plasma of healthy humans to which non-ionic surfactants were added. Conditions for adding various non-ionic surfactants to plasma to minimize the influence of interfering substances in plasma as much as possible. A study was conducted. Tween 20 (POLY SCIENCE WARRINGTON, PA) as a non-ionic surfactant
Tween 85 (polyoxyethisane sorbitan trioleate, HLB = 11.0, manufactured by Junsei Chemical Co., Ltd.), Brij
-35 (polyoxyethylene lauryl alcohol ether, HLB = 16.9, manufactured by Nacalai Tesque, Inc.) at 10% (W
/ V) and measured in the same manner as in Example 4 (1). The results are shown in FIG. It can be seen that the addition of a non-ionic surfactant to the plasma gives a good agreement with the calibration curve of the buffer standard solution. In addition, as a study of the amount of the nonionic surfactant to be added, various concentrations of Tween 20 were added to the plasma volume.
10% (W / V) was added, and measured by the method of Example 4 (1). As the concentration of Tween 20 in the plasma was increased, the measurement results gradually approached the calibration curve of the buffer-based standard solution, and the best results were obtained with the addition of 0.1 to 1% Tween 20 (No. 4). Tween 20 greater than 1% by weight
Is not preferred because it causes a non-specific reaction or inhibits binding to anti-human G-CSF antibody. Also, the optimal concentration of the nonionic surfactant was different for each nonionic surfactant.

(4) Measurement in multiple healthy human plasma 1% by weight Tween 20-0.5% by weight T
Human G was added to various plasma samples to which ween 20 and EDTA were added.
When a standard solution prepared by serial dilution with the addition of -CSF was measured, it was found that the standard solution was in good agreement with the calibration curve of the buffer standard solution (see FIG. 5).

(5) Measurement in plasma of leukemia patient In a plasma sample of leukemia patient, 1% by weight Tween 20-0.5
Table 1 shows the results obtained by adding Tween 20 and EDTA so as to obtain the weight% EDTA, and measuring by the method of Example 4 (1). The G-CSF amount in the plasma was determined from a calibration curve in a buffer standard solution.

[Brief description of the drawings]

FIG. 1 shows a calibration curve of a standard solution (buffer standard solution) prepared by serially diluting human G-CSF with 1% BSA-PBS. FIG. 2 shows a calibration curve for a standard solution (normal human plasma standard solution) prepared by adding human G-CSF to healthy human plasma and serially diluting it. In the figure, ○ indicates a buffer-based standard solution, and the other symbols indicate healthy plasma standard solutions. FIG. 3 shows a calibration curve when a nonionic surfactant is added to a normal human plasma standard solution. The meaning of each symbol in the figure is as follows. ○: Buffer standard solution □: Normal human plasma standard solution containing 1% Tween 20 ■: Healthy human plasma standard solution containing 1% Tween 85 ◇: Healthy human plasma standard solution containing 0.01% Brij-35 ●: Healthy Human Plasma Standard Solution FIG. 4 shows the amount of Tween 20 added to a healthy human plasma standard solution and its calibration curve. The meaning of each symbol in the figure is as follows. ○: Buffer standard solution ●: Healthy human plasma standard solution □: Healthy human plasma standard solution containing 1% Tween 20 ■: Healthy human plasma standard solution containing 0.1% Tween 20 ◇: 0.01% Tween 20 Fig. 5 shows a normal human plasma standard solution containing a plurality of healthy human plasma samples at 1% by weight Tween 20-0.
The calibration curve when adjusted to be 5% by weight EDTA is shown. In the figure, ○ indicates a buffer standard solution, and other symbols indicate Twee
1 shows a normal human plasma standard solution containing n20.

 ──────────────────────────────────────────────────続 き Continuation of the front page (56) References JP-A-63-315954 (JP, A) JP-A-62-130698 (JP, A) JP-A-58-187862 (JP, A) JP-A-62 71861 (JP, A)

Claims (7)

(57) [Claims] 1. A method for measuring human G-CSF in blood by enzyme immunoassay, wherein an antibody-digested fragment is used as a labeled antibody, and an HLB value of an antigen-antibody reaction between the insolubilized antibody and human G-CSF is determined. 11 to 17 nonionic surfactant 0.01 to 1% by weight
The method according to claim 1, wherein the measurement is performed in the presence of 2. The method according to claim 1, wherein said antigen-antibody reaction further comprises sodium ethylenediaminetetraacetate. (3) sodium ethylenediaminetetraacetate having a concentration of
3. The method according to claim 2, wherein it is present in a proportion of 5% by weight. 4. The method according to claim 1, wherein said surfactant is present in the range of 0.1 to 1% by weight. 5. The method according to claim 1, wherein the surfactant is polyoxyethylene sorbitan monolaurate. 6. The method according to claim 1, wherein the antigen-antibody reaction between the insolubilized antibody and human G-CSF is performed at about 4 ° C. for 12 to 24 hours. 7. The method according to claim 1, wherein a polyclonal antibody is used as the insolubilized antibody and the labeled antibody.
The measurement method according to any one of the above.