JPS6113156A - Reagent for quantitative analysis of human interferon-alpha and determination method thereof - Google Patents

Abstract

PURPOSE:To measure the concn. of HuIFN (human interferon)-alpha, by reducing an anti-HuIFN-alpha antibody or its pepsine hydrolysate F(ab')2 while bonding the resulting reduced product to beta-D-galactosidase through a dimaleimide compound. CONSTITUTION:An anti-HuIFN-alpha antibody or F(ab')2 being a pepsine hydrolysate thereof is reduced to form a thiol group and a large excess of a dimaleimide compound is reacted with one of said thiol group or that of beta-D-galactosidase and subsequently reacted with the other thiol group, after the unreacted dimaleimide compound was removed, to prepare an enzyme labelled antibody wherein the antibody is bonded to the enzyme. By determining HuIFN-alpha according to a sandwich method using this enzyme labelled antibody, 1I.U./ml of HulFN-alpha can be determined.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a reagent for quantifying human interferon-α in liquids such as serum or culture fluid, and a method for quantifying human interferon-α using the reagent.

Conventionally, the quantification of human interferon-α in liquids was
It is performed in a bioassay, and the minimum detection sensitivity is I1. U
, /lut, but the operation was complicated and the fluctuations in the measured values were large. Recently, quantification has been carried out using radioimmunoassays49, 2 or enzyme immunoassays that utilize antigen-antibody reactions, but both are monoclonal antibodies labeled with radioisotopes or enzymes, and human immunoassays. Assay failure (J, gen, Vi
rol.

62.181-185 (1982) i Medicine History vo
l, 129 Black 8 181-186 (1984))
.

As a result of intensive studies on highly sensitive enzyme immunoassays, the present inventors determined that human interferon-α was quantified by the Sand-Deutsch method using a polyclonal anti-human interferon-α antibody labeled with β-D-galactosidase. By doing so, we discovered a quantitative method with sensitivity comparable to that of bioassays.

Specifically, an enzyme-labeled antibody is produced by reducing anti-human interferon-α antibody or F(ab'), which is a pepsin-decomposed product thereof, and binding β-D-galactosidase via a dimaleimide compound. By quantifying human interferon-α using an antibody by the Sand-Deutsche method, 1), U, /,
This makes it possible to quantify the total amount of human interferon-α.

Interferon is a glycoprotein with a molecular weight of approximately 20,000 that is produced and secreted by animals infected with a virus, and is a substance that is the main cause of so-called virus interference, a phenomenon in which infection with one virus inhibits infection with another virus. It is roughly divided into α type, β type, and β type depending on the difference in antigenicity, and is classified as interferon-α, interferon-β, and interferon-γ, respectively.

In addition, to indicate the animal species of the animal cell that produced interferon, it is expressed as human interferon, mouse interferon, etc.2. Human interferon-α is an α-type interferon produced by human cells.

Recently, it has been revealed from the nucleotide sequence of the human interferon-α gene and the amino acid sequence of human interferon-α that there are more than 10 subtypes of human interferon-α (Naturevol, 286. &10,1)0,19
80).

A method for purifying human interferon-α (hereinafter referred to as HuIFN-α) was carried out by treating human I-IJ lymphoblastoid cells with Sendai Reihess treatment,
A method of inducing and purifying HuIFN-α, a method of introducing the HuIFN-α gene into E. coli using genetic engineering, mass culturing the E. coli, causing HuIFN-α to be produced, and purifying the same are known. Preferably, various subtypes coexist,
This is an easy-to-produce method in which Namalva cells are treated with Sendai virus.

Anti-HuIFN-α antibodies can be obtained by a known method (Introduction to Immunology Experiments Vol. That is, rabbit, mole 5. , Init, subcutaneously of mammals such as rats, mice, goats, etc.
Antiserum can be obtained by administering HuIFN-α intramuscularly, salting out by adding sodium sulfate to the antiserum, dissolving the resulting precipitate in phosphate buffer, and adding DEA.
E + Reulose ion exchange/chromatography] By separating the IgG fraction, anti-HuIFN-
Obtain α antibody.

F (a
b') Using the well-known method of 2, the analogy can be obtained as follows. Anti-HuIFN-α antibody (5-20
Add 1 to 5% by weight of pepsin based on the antibody to an acetate buffer (pH 4 to 5) solution) and incubate at 4 to 40°C for 2 hours.
After allowing the reaction to occur for up to 4 days, and then adjusting the pH to 7 to 8, gel filtration was performed using Sephadex G-150 (manufactured by Pharmacia), and both fractions with a molecular weight of approximately 100,000 were fractionated. F(ab')2, which is a pepsin-digested product of anti-HuIFN-α antibody, is obtained.

In addition, the anti-HuIFN-a antibody or its pepsin-degraded product F(ab')* refers to HuIFN-a as CNBr.
- It is preferable to perform affinity purification using a Hu, IFN-α column bound to Sepharose ■4B (7 columns, manufactured by Remashi Taisho Co., Ltd.), and most preferably, affinity-purified F(ab'). 2.

The binding of anti-HuIFN-α antibody or its pepsin-digested product F(ab')2 and β-ri with lactosidase'
reduces F (ab')2, which is the anti-HuIFN-α antibody or its pepsin-degraded product, and binds to it via a dimaleimide compound. The binding method is enzyme immunoassay f! (-2nd edition-6-α, p.

92-p, l 05, Igaku Shoin). The method is described below.

Examples of dimaleimide compounds used for binding β-ri with lactosidase include N, N'-0-7 enylene dimaleimide 1. N, N/-p-phenylene dimaleimide, N,
N'-oxydimethylene dimaleimide and the like. Preferably it is N, N'-o-7 22 dimaleimide.

The binding method involves reducing F(ab/)2, which is an anti-HuIFN-α antibody or its pepsin-decomposed product, to generate a thiol group, and attaching a large amount to either this thiol group or the thiol group of β-D-galactosidase. After reacting with excess dimaleimide compound, unreacted dimaleimide compound is removed and the antibody and enzyme are bonded by reacting with the other thiol group.

As an example of the above binding method, F(ab')2 solution (5 to 20 rQ
/Ml of phosphate buffer (pH 5,5-6.5 solution) by adding mercaptoethylamine to a final concentration of 0.005-0.1 mol, and then
React at ~40°C for 5 minutes to 1 day, then pH 4.5 to 6.
．． Sephadex G- equilibrated with 0 phosphate buffer
25 (manufactured by Almacia) to remove the low-molecular component mercaptoethylamine, and the eluted protein was filtered with an acetate buffer of N and N'-o-7 enylene dimaleimide. (pH 4,5-6.0)
The 2θ
- IOθ times the number of moles, and after reacting at 4 to 40°C for 5 minutes to 24 hours, unreacted N,N'-o-phenylenedi, which is a low molecular component, was removed by gel filtration in the same manner as above. Maleimide was removed and the resulting protein fraction was divided into 1.
After concentrating to a protein concentration of 0 to 1O/ml, add β-D-galactosidase solution (1.0 to 10mv/ml).
phosphate buffer (pH 6.0-7.0) solution)
is the reduction product Fab' of pepsin degradation product F(ab')2
Adding 10 to 50 mol% of maleimide of pH He
After adjusting the temperature to 0 to 7.0, the reaction mixture is allowed to react at 4° C. for 10 hours to 10 days. After that, Sefa. -S ① Collect the eluted fraction of lactosidase using CL-6B (Fal, manufactured by Cia Corporation).

A similar method can be used when using an anti-HuIFN-α antibody, when using another dimaleimide compound, or when β-ri is reacted with the thiol group of lactosidase and the dimaleimide compound first.

In order to confirm that the anti-HuI FN-α antibody or its pepsin component or decomposition product F (ab')2 and β-D- have been bound by lactosidase using the above method, it is necessary to use anti-HuI FN-α Fluorescein-labeled Ig to the antibody
Just add a small amount of G.

That is, since fluorescein fluorescence was observed in the separated β-D-galactosidase fraction, anti-HuIFN
-α antibody or its pepsin-degraded product F(ab')
It can be confirmed that 2 and β-D-galactosidase were bound together. From the β-D-galactosidase activity of each fraction, the fluorescein fluorescence intensity of the total elution fraction, and the fluorescein fluorescence intensity of both fractions in which β-D-galactosidase enzyme activity was observed, β-D- was found to be 41 molecules of lactosidase. Anti-HuIFN-α antibody or its pepsin-degraded product F (a
b') The average number of molecules of the reduction product Fab' of 2 can be known.

For the purposes of the present invention, β-D-
The reduction product Fab' of anti-HulFN-α antibody or pepsin degradation product F(ab')2 per molecule is 1.0 to 5.
It is preferable that 0 molecules be bonded. More preferably 1.0
~3.0 molecules.

Most preferably, β-ri is an affinity-purified pepsin-digested product F (ab
1.5 to 2.5 molecules of Fab', the reduction product of ')2, bind. .

” The anti-HuIFN-α antibody thus obtained or the pepsin-digested product F(ab')2 and β-ri1 were incubated with serum or culture using a conjugate of lactosidase (hereinafter referred to as labeled antibody). HuIFN-α in liquids such as liquids
The determination is carried out by the well-known Sanderch method.

For example, the Sand Deutsch method applies to polystyrene, nylon,
Glass balls, tubes or silicone strings,
Alternatively, anti-Hu
After the solid phase antibody obtained by binding the IFN-α antibody (hereinafter referred to as surface phase) is reacted with the liquid to be measured, the solid phase is washed. After this, a labeled antibody is added to the same phase to perform a reaction, and the same phase is washed. The enzyme activity on the solid phase is then measured.

For measurement of enzyme activity, a compound that emits fluorescence, visible light, and ultraviolet light due to β-D-galactosidase is used as a substrate. For example, as a substrate, O-nitrophenyl-β-ri is lactopyranoside, 4-methylumbelliferyl-β
-D- is lactopyranoside, etc. Preferably, lactopyranoside is used as the fluorescent 4-methylumbelliferyl-β-ri. The specific measurement is carried out, for example, by the following method. That is, 4-methylumbelliferyl-β-
Riichi is a lactopyranoside solution (final concentration 0.5-10X
10−4 M) 100–200 μl and 25 μl of solid phase
After reacting at ~40 °C for lθ ~60 min, 0.01-I
M. glycine buffer (pH 9,5-1). 0), 1~'
3 ml was added to stop the enzyme reaction, and the fluorescence intensity of the resulting 4-methylumbelliferone was measured at an excitation wavelength of 865 nm.
Measurement is performed at a fluorescence wavelength of 448 nm.

For quantitative determination, a standard solution of HuIFN-α with a known concentration is measured to create a calibration curve, and the Hu I FN-α concentration can be determined from the enzyme activity of the liquid to be measured. According to the method obtained in this way, a sample amount of 20 to 100 .mu.e is used.

It was possible to measure the HuIFN-α concentration of U,/ml, and the correlation with the bioassay was also good.

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

Example I HuIFN-a o) Preparation and Anti-HuI
Preparation of FN-α antibody and enzyme-labeled antibody (1) nu IFN-a (7) 75 Produced Hu
IFN-α was obtained by treating cultured lymphoblastoid cells, Namalva cells, with Sendai virus to induce human interferon, and then purifying the culture solution using a known method (
Nature vol, 287, 408-41) +
1980). That is, human interferon is precipitated from the culture solution with trichloroacetic acid, extracted with acidic ethanol, affinity purified using a polyclonal anti-HuIFN-α antibody column, and then foreign proteins in the culture solution are removed using an anti-bovine serum antibody column. The titer is l
O, 7X106IIJ, to 20X1061. U/+
HuIFN-α solution (0.01 M with 0.1 5M NaC5) in J. lis-glycine buffer (p*7.
2) solution) was obtained.

(2) Antibody production method The nu I FN-α solution was mixed with an equal amount of 70 India Complete Adjuvant to prepare an emulsion, which was administered to rabbits. 2 x 1061 per dose. U,
to 10 x 10' 1. U, (D Hu I
FN-tx was administered to the heel, subcutaneously, and intramuscularly, and a total of 18 doses were administered at intervals of 2 to 4 weeks. Blood was collected from the bony artery 10 days after the final administration. The obtained blood was stored at room temperature for 6
After leaving it for a while, a o o o r.

Antiserum was collected by centrifugation at p and m. 6.82 ml of sodium sulfate was added to 85 g of antiserum for salting out, and the resulting precipitate was diluted with sodium phosphate buffer (0,
017.5M, pH 5.3, hereinafter abbreviated as buffer ■)
The supernatant was removed by dialysis against the same buffer, and the supernatant was transferred to a DE-52 [phase] (Whatman) column (I
Ion-exchange chromatography was carried out using X 40 cm (equilibrated with buffer solution), and a fraction absorbing light at a wavelength of 280 nm was collected and designated as an anti-HuIFN-α IgG fraction. Both obtained fractions were concentrated by ultrafiltration using Dia 70 membrane ■ (manufactured by Amicon), and the IgG concentration was 20".
12.8 ml of a solution with a concentration of 9/ml was obtained. Anti-HuIFN-a
IgG solution 0, IM sodium acetate buffer (1) H4,
After dialysis against 5), add 7.7 ~ pepsin to 37°C.
, and kept warm for 25 hours. After adjusting the pH of the pepsin-treated IgG solution to 7.9 using IN sodium hydroxide solution, gel filtration (column equilibration) was performed using a Sephadex G-150 (manufactured by Pharmacia) column (1.5 x 40 cnI). and 0.1 M sodium borate buffer (1)H8,0) was used for elution), and the molecular weight was approximately i.

Both aliquots were separated and anti-Hu I FN-a F (a
b') 2150■ was obtained. Obtained anti-HuIFN-αF
(ab')2 in 0.1M sodium phosphate buffer (pH
HuIFN-α was dialyzed against CNB r-
Affinity purification was performed using a HuIFN-α column coupled to Sepharose 4B (manufactured by Famicon). Anti-Hu adsorbed on the column
0.2 M glycine-HO2 [r solution (pH 2,3) was used for elution of IFN-αF(ab')2(7), and the eluted F(ab')2 was 0.2 M glycine-HO2[r solution (pH 2,3). I N sodium hydroxide φ
The pH was immediately adjusted to 7.0 with a solution of 100% alcohol.・After dialysis against 0.1M sodium phosphate buffer (pH 7.0), affinity purified anti-Hu
IFN-aF (ab')25. I got l my (8
1yI labeled antibody synthesis method Affinity purification anti-Hu I FN-aF (ab'4
After dialysis of ■ against O,lM sodium phosphate buffer pH 6.0 containing 1mM ethylenediaminetetraacetic acid (hereinafter abbreviated as buffer ■), this affinity-purified anti-H
'uIFN-a F(ab')24''!/480
Fluorescein-labeled F (ab'
)z 0. I Q / 1001t (l buffer ■, 0
．． 1M mercaptoethylamine/100μe buffer■,
After mixing 320 μe of buffer solution, the mixture was kept at 87° C. for 90 minutes to reduce F(ab')2. Gel filtration was performed using a Sephadex ■ G-25 (manufactured by Pharmacia) column (l x 30 crn) equilibrated with buffer solution ■, and the protein fraction eluted first was approximately 8.87 IQ, 8.1 r.
xl is N,N'-o-phenylenedimengreimide saturated,
The mixture was directly introduced into 2.6 gJ of 0.1 M sodium acetate buffer (pH 5,0) containing 1 mM ethylenediaminetetraacetic acid and kept at 30° C. for 20 minutes. A Sephadex G-25 (manufactured by Pharmacia) column (l) equilibrated with 0.02 MIII' acid sodium buffer (pH 5,0)
Gel filtration is performed using X 40 an), and the first eluted fraction with absorption at a wavelength of 280 nm is collected, concentrated by ultrafiltration using a diaflow membrane (manufactured by Amicon), and reduced to one molecule. Fab' with 0.52 molecules of maleimide group introduced into Fab'2.4'F/1.28 wl
I got it. The definition of the maleimide group in Fab' was carried out using a known method (Immunology Experiment Procedure X1.3497~
8520.1982).

β-D-galactosidase (hereinafter abbreviated as β-D-Gal) was adjusted to 8.75 mg/I/(0.1
Dissolved in M sodium phosphate buffer (pH 6,5), F
The molar ratio of β-D-Gal to ab'+z is +fflte4
Four types of mixed solutions of Fab/-maleimide solution and β-D-Gal solution were prepared so that the ratio was 0/192°, 28/192, 14/192.7/192. However, the pH of the mixed solution is O,
I M sodium phosphate buffer (pH 7,57)
) Adjusted to H6.5. After reacting at 4°C for 90 to 140 hours, the reaction solution was
Sepharose ■CL- equilibrated with 0.01 MIJy acid sodium buffer pH 7.0 (hereinafter abbreviated as buffer ■) containing Cl, 1 mM Mg C1, 0.1% BsA and 0.1% NaN. 6B (77,,v,
The gel was applied to a column (1.5 x 40 cm) (manufactured by + Co., Ltd.). Buffer ■ was used as the eluate, and the fractions were 1.8. Fluorescence intensity β-D-G of fluorescein for each surface
The enzymatic activity of al was measured. The fluorescence of fluorescein was measured at an excitation wavelength of 499 nm and a fluorescence wavelength of 510 nm. The number of Fab' bound to the β-D-Gal1 molecule was calculated using the following formula.

Here, A is the fluorescein fluorescence intensity of both fractions in which the enzymatic activity of β-D-Gal is observed, B is the fluorescein fluorescence intensity of the entire elution fraction, and α is the β-D-Gal relative to the l'ab' used. It is the molar ratio of D-'Gal.

α is 40/192.28/192.14/192°7
/192, respectively, β-D-Gal1
For the molecule, Fab' is 2.2°2.5, -8.9
,'8. An enzyme-labeled antibody bound to 8 molecules was obtained.

Example 2 Quantification method and calibration curve of Hu I FN-α A calibration curve was prepared using the Hu I FN-α quantification reagent of the present invention obtained in Example 1 as follows, and the quantifiable range was examined. That is, test tube (10w x 72 +u
), 100 µe of buffer solution, and the CPE cytodegeneration-inhibiting dye uptake method conducted at the National Institute of Health (Proteins, Nucleic Acids, Enzymes, Separate Volume No. 25, p.

859-p, 862.19'81, Kyoritsu Shuppan), the titer was measured by a bioassay, and after mixing with 50 μe of standard Hu I FN-α diluted with buffer solution, O, l f/'1 concentration of anti-Hu I FN
-a 0.25M sodium phosphate buffer for IgG (
Immobilized anti-HuIFN-αI obtained by placing a polystyrene ball with a diameter of 8.2 mm in a pH 7,5) solution and allowing it to stand at 4°C for one week to physically adsorb IgG to the polystyrene ball.
gG (hereinafter abbreviated as solid phase) was added, incubated at 87°C for 6 hours with shaking, and then allowed to stand overnight at 4°C (first reaction).

After removing the reaction solution by suction using an aspirator, transfer the solid phase to buffer solution■
Washed twice with 1 ml. After washing, remove the buffer solution using an aspirator, and add 100 μ# of buffer solution to the test tube.
Add 50 μe of the four types of labeled antibodies prepared in Example 1 having 2000 μ units of enzyme activity and a solid phase, and heat at 87°C.
It was incubated with shaking for 5 hours (second reaction).

The reaction solution was removed by suction using an aspirator, and the solid phase was transferred to buffer solution 0.
Washed twice with 1 ml. The buffer solution after washing was removed by suction using an aspirator. The solid phase was transferred to a new test tube, 100 μe of buffer solution (1) was added, and the tube was incubated at 37° C. for 5 minutes. 0.8 mM 4-methylumbelliferyl-β-D- with 50 μe of lactopyranoside;
After carrying out the enzyme reaction at 37°C for 710 minutes, 2.5g+
/'s 0. IM melysin buffer (OH 10.4) was added to stop the enzyme reaction.

The fluorescence intensity of the reaction product 4-methylumbelliferone was measured at an excitation wavelength of 8fi5 nm and a fluorescence wavelength of 4.48 nm. As a standard for fluorescence intensity, the fluorescence intensity of lXl0-7M4-methylumbelliferone dissolved in 0.1 M glycine buffer was set as 100. A calibration curve was created by taking the HuIFN-α concentration on the horizontal axis and the fluorescence intensity on the vertical axis. β-D-
For one molecule of Gal, Fab' is 2.2,
In the calibration curve of the enzyme-labeled antibody bound to the 2.5, 8.8, and 8.9 molecules, the HuIFN-α concentration was
The HuIFN-α concentrations exhibiting a fluorescence intensity that is twice the fluorescence intensity when gl are 1). U,/ml.

21, U, Al, 4.1. U7 41. My U shoulder was warm. In addition, in the case of any enzyme-labeled antibody, Hu I
FN-α concentration is 25601. The calibration curve was an upward-sloping curve up to U, /y. For one β-D-Gae molecule, F
FIG. 1 shows a calibration curve using an enzyme-labeled antibody to which 2.2 molecules of ab' were bound. In addition, in Fig. 1, HuI
FN-α concentration is 401. The part below U, /d is shown in FIG. 2 in an enlarged manner.

Example 3 Correlation with Bioara Strawberry measurement values Example 1 (
Regarding 160 Hu IFN-α final products obtained by adding human plasma albumin as a stabilizer to the Hu IFN-α solution obtained as in 1), the Hu IFN-α solution was determined by bioassay.
The quantitative value of IFN-α was compared with the quantitative value of HuIFN-α using the HuIFN-α quantitative reagent according to the present invention. The bioassay was performed as described in Example 2. Quantification using the HuIFN-α quantification reagent according to the present invention was carried out according to Example 2. That is, in the first reaction, standard Hu I FN− diluted with buffer solution ① was used to create a calibration curve.
HuIF diluted with buffer ■ as α50μe1 sample
50μ4 of N-α final product was used. The operations after the first reaction were the same as in Example 2. Using a calibration curve created from the fluorescence intensity of the sample with the concentration of standard HuIFN-α on the horizontal axis and the fluorescence intensity on the vertical axis, the HuIFN-α concentration in the sample is determined, and then the value is multiplied by the dilution factor to determine the HuIFN-α concentration.
This was taken as the HuIFN-α concentration in the final product. H using bioassay and reagent for HuIFN-α quantification according to the present invention
The relationship between the measured values of Hu IFN-α concentration in the final product of Hu IFN-α is shown in FIG. The regression equation is y=1.0
7x-0,1)6, correlation coefficient is 0.93, number of samples is 16
A good correlation was observed.

Example 4 HuIF in serum of Mayusa administered with HuIFN-α
N-α Quantification Human plasma albumin was further added as a stabilizer to the HuIFN-α solution obtained as in Example 1 (1).
Sera collected over time from patients who had been administered the FN-α final product were used as samples, and the concentration of HuIFN-α in the serum was determined using a bioassay and a reagent for quantifying HuIFN-α according to the present invention. H in serum by bioassay
The uIFN-α concentration was determined by the method described in Example 2. Quantification of HuIFN-α in serum using the reagent for quantifying HuIFN-α according to the present invention was carried out according to Example 2. However, in order to avoid the undesirable influence of serum on HuIFN-α quantitative determination of the present invention,
0.01 M sodium phosphate buffer pH containing in
7,0 (hereinafter abbreviated as buffer solution ■), and standard IFN
Inactivated rabbit serum was added to HuIFN-α.
was quantified.

That is, to create a Hu I FN-α standard curve, the first reaction was carried out using standard Hu I FN-α diluted with buffer ■50μ4, immobilized sea cucumber serum 50μe, buffer ■50μe and the solid phase. Also, HuIFN- in the sample
For α quantification, sample serum 50μ4, buffer ■100μ
The first reaction was carried out using l and a solid phase. The operations after the first reaction were the same as in Example 2.

The quantitative value of HuIFN-α in serum determined using the reagent for quantifying HuIFN-α according to the present invention was compared with the quantitative value of bioassay. The correlation coefficient is 0.96 (number of samples is 2)
4), a good correlation was observed. Furthermore, Hu I
The quantitative value of HuIFN-a in serum collected over time after administration of FN-α was 101. FIG. 4 shows the results of quantifying a sample with a volume of 7 ml or less using the reagent for quantifying Hu I FN-α according to the present invention. Hu
Clear HuIF with a peak 6 hours after IFN-α administration
A change in N-α concentration was observed.

[Brief explanation of drawings]

Figure 1 shows β-D-galactosidase-labeled anti-HuI using N,N/-0-phenylene dimaleimide as a chemical binder.
The calibration curve of 1HuIFN-α when FN-αFab' is used is shown. The horizontal axis is the HuIFN-α concentration (1, U,
/d), and the vertical axis represents the fluorescence intensity when the fluorescence intensity of lo-7M 4-methylumbelliferone is set to 100. Figure 2 shows that the Hu I FN-α concentration in Figure 1 is 0 to 40.
This is an enlarged view of the 1, U,/ml portion, and the horizontal and vertical axes are the same as in Figure 1. Figure 3 shows the HuIF in the Hu I FNN two-stage final product.
The relationship between the quantitative value by bioassay and the quantitative value using the HuIFN-α quantitative reagent according to the present invention in N-α quantitative determination is shown. The horizontal axis is the quantitative value (I
X 106I, U, /zl), and the vertical axis represents the quantitative value using the Hu I FN-α quantitative reagent according to the present invention. Figure 4 shows HuIFN-α in serum after administration of HuIFN-α.
It represents the change in α concentration over time. The horizontal axis represents the time (hours) after administration of HuIFN-α, and the vertical axis represents the HuIFN-α concentration (1, U
, /#Il).・40 80 160 640 2560 (
1, U, / scrap l)

Claims (4)

[Claims] (1) Human interferon characterized by reducing an anti-human interferon-α antibody or its pepsin-degraded product F(ab')_2 and then binding the reduced product to β-D-galactosidase via a dimaleimide compound. -α quantitative reagent. (2) The reagent according to claim 1, wherein the dimaleimide compound is N,N'-o-phenylene dimaleimide. (3) Claim 1, wherein the anti-human interferon-α antibody or its pepsin-degraded product F(ab')_2 is an affinity-purified anti-human interferon-α antibody or its pepsin-degraded product F(ab')_2. Reagents listed in section. (4) For quantification of human interferon-α by reducing anti-human interferon-α antibody or its pepsin-degraded product F(ab')_2 and then binding the reduced product to β-D-galactosidase via a dimaleimide compound. A method for measuring the human interferon-α concentration in a sample by a sandwich method using a reagent.