KR100530611B1 - A radioimmunoassay for measuring a concentration of macrophage migration inhibitory factor - Google Patents

Abstract

The present invention comprises the steps of: a) attaching a monoclonal antibody against macrophage hepatic inhibitory factor (MIF) to the stationary phase; b) adding at least two standard MIF solutions to bind the anti-MIF monoclonal antibody; c) adding a biotin-bound anti-MIF polyclonal antibody to bind MIF bound to said monoclonal antibody of the stationary phase; d) adding a radioisotope labeled streptavidin to attach to biotin bound to the anti-MIF polyclonal antibody and removing unreacted streptavidin; e) preparing a standard dose response curve by measuring the radioactivity of the streptavidin labeled with the radioisotope attached to the biotin; f) performing steps b) to d) on an unknown concentration of MIF sample instead of a standard MIF solution and measuring radioactivity; And g) substituting the radioactivity obtained in step f) into the standard dose response curve to calculate the concentration of MIF in the sample.

In the case of using the radioimmunoassay for measuring the concentration of MIF according to the present invention, it specifically acts on MIF, so it is possible to accurately measure even the smallest concentration, and conveniently and conveniently measures the concentration of MIF in a biological sample for a large amount of samples. can do.

Description

A radioimmunoassay for measuring a concentration of macrophage migration inhibitory factor

The present invention relates to a radioimmunoassay for measuring the concentration of macrophage migration inhibitory factor (MIF).

Macrophage-derived hypolipidemic factor (MIF) is the first identified T lymphocyte-derived cytokine and is known to play an important role in delayed type hypersensitivity as a substance that inhibits migration from capillaries to macrophages in vitro. .

In 1966, David and Bloom et al. Revealed that in vitro delayed hypersensitivity reactions, substances liberated when lymphoid cells react with antigens mediate hypersensitivity reactions and named them macrophage hypodermic factor (MIF). Bloom BR, Bennett B Mechanism of a reaction in vitro associatied with delayed-type hypersensitivity Science 153: 80-82, 1966; David JR Delayed hypersensitivity in vitro: its mediation by cell-free substances formed by lymphoid cell-antigen interaction Proc Natl Acad Sci USA 56: 72-77, 1966. Nathan et al. Characterized MIF as a factor affecting macrophage function, and Weiser et al cloned human cDNA for MIF from activated T lymphocytes, and MIF consists of about 114 amino acids. It was confirmed that the material has a molecular weight (Nathan CF, Karnovsky ML, David JR. Alterations of macrophage functions by mediators from lymphocytes. J Exp Med 1971 1; 133 (6): 1356-76; Nathan CF, Remold HG, David JR. Characterization of a lymphocyte factor which alters macrophage functions.J Exp Med 1973 1; 137 (2): 275-90; Weiser WY, Temple PA, Witek-Giannotti JS, Remold HG, Clark SC, David JR.Molecular cloning of a cDNA encoding a human macrophage migration inhibitory factor.Proc Natl Acad Sci US A. 1989 86 (19): 7522-6).

In 1993, Bernhagen et al. Revealed that MIF is a pituitary-derived cytokine that increases fatal endotoxemia, and then isolated and purified MIF in rats and humans, and identified the biochemical properties of MIF and the secondary structure of proteins (Bernhagen). J, Calandra T, Mitchell RA, Martin SB, Tracey KJ, Voelter W, Manogue KR, Cerami A, Bucala R. MIF is a pituitary-derived cytokine that potentiates lethal endotoxaemia Nature . 1993 21; 365 (6448): 756-9 Bernhagen J, Mitchell RA, Calandra T, Voelter W, Cerami A, Bucala R. Purification, bioactivity, and secondary structure analysis of mouse and human macrophage migration inhibitory factor (MIF) Biochemistry . 1994 29; 33 (47): 14144- 55).

In 1993, Wistow et al. Found that MIF mRNA expression correlates with the degree of differentiation of the lens cell, suggesting that MIF plays an important role in the differentiation process (Wistow GJ, Shaughnessy MP, Lee DC, Hodin). J, Zelenka PS.A macrophage migration inhibitory factor is expressed in the differentiating cells of the eye lens Proc Natl Acad Sci US A. 1993 15; 90 (4): 1272-5).

In 1995, Calandra et al. Found that MIF inhibits the action of glucocorticoids to mitigate inflammatory and immune responses as modulators in the production of cytokines by glucocorticoids (Calandra T, Bernhagen J, Metz CN, Spiegel LA, Bacher M, Donnelly T, Cerami A, Bucala R. MIF as a glucocorticoid-induced modulator of cytokine production Nature. 1995 7; 377 (6544): 68-71).

Then, in 1996, Bacher et al. Confirmed that MIF plays a very important role in the activity of T lymphocytes, and in 1998, Calandra et al. Examined the immune system cells produced by exotoxins secreted by Gram-positive bacteria. It was identified as an important factor mediating activity (Bacher M, Metz CN, Calandra T, Mayer K, Chesney J, Lohoff M, Gemsa D, Donnelly T, Bucala R. An essential regulatory role for macrophage migration inhibitory factor in T-cell activation.Proc Natl Acad Sci USA .1996 23; 93 (15): 7849-54; Calandra T, Spiegel LA, Metz CN, Bucala R. Macrophage migration inhibitory factor is a critical mediator of the activation of immune cells by exotoxins of Gram-positive bacteria Proc Natl Acad Sci US A. 1998 15; 95 (19): 11383-8).

Recently, MIF is a Gram-negative lipopolysaccharide (LPS) and a Gram-positive toxin. Toxic Shock Syndrome Toxin-1 (TSST-1) and Streptococcal pyrogenic exotoxin A It is important for the host response by A), and it is reported that the use of immunoneutralization method which suppresses this action of MIF and deletion of gene of MIF can prevent endotoxin and toxic shock by Staphylococcus aureus. (Calandra T, Spiegel LA, Metz CN, Bucala R. Macrophage migration inhibitory factor is a critical mediator of the activation of immune cells by exotoxins of Gram-positive bacteria Proc Natl Acad Sci US A. 1998 15; 95 (19 ): 11383-8; Calandra T, Echtenacher B, Roy DL, Pugin J, Metz CN, Hultner L, Heumann D, Mannel D, Bucala R, Glauser MP.Protection from septic shock by neutralization of macrophage migration inhibitory factor Nat Med. 2000 6 (2): 164-70). In addition, the use of anti-MIF antibodies has been shown to mitigate the side effects and inflammation of glomerulonephritis and arthritis, and rejection of xenologous tissue (allograft), and to investigate the important effects of MIF in sepsis. Studying the fact that the substance TNF-α causes fatal peritonitis in rat animal models knocked out and the increased survival rate by treating anti-MIF antibodies has been shown to lead to prevention and symptom relief of sepsis. .

In addition, a 1999 study by Mitchell et al. Showed that MIF increases the activity of MAP kinases activated by mitogens and phospholipase A2 in the cytoplasm, acting as a substance that regulates cell proliferation and ultimately fibroblasts. (Mitchell RA, Metz CN, Peng T, Bucala R. Sustained mitogen-activated protein kinase (MAPK) and cytoplasmic phospholipase A2 activation by macrophage migration inhibitory factor (MIF). cell proliferation and glucocorticoid action J Biol Chem. 1999 18; 274 (25): 18100-6).

In recent years, the cloning of human MIF's cDNA from activated T lymphocytes has accelerated the study of a number of unknown functions, leading to MIF being a hormone derived from the anterior lobe of the hypothalamus, increasing fatal endotoemia, inflammatory response and immunity by glucocorticoids. Inhibiting the inhibitory action of the response, and is known to play an important role in the activation of T lymphocytes according to the stimulation and differentiation of antigens, MIF is not only mediate the immune response but also acts on the proliferation and differentiation of cells.

As a research method for MIF performing various functions as described above, Burmeister et al. In 1987 produced a monoclonal antibody to human MIF and characterized its immunohistochemical staining method in which immune diseases and diseases are affected by MIF. (Burmeister G, Tarcsay L, Sorg C. Generation and characterization of a monoclonal antibody (1C5) to human migration inhibitory factor (MIF) Immunobiology. 1986 171 (4-5): 461- 74) In addition, in 2000, Yuka Mizue et al studied the one-step sandwich ELISA method to measure the concentration of MIF and measured the blood concentration of MIF, and revealed that MIF is significantly increased in autoimmune diseases. In addition, MIF has been reported to be careful when measuring MIF concentration (Mizue Y, Nishihira J, Miyazaki T, Fujiwara S, Chida M, Nakamura K, Kikuchi K, Mukai M. Quanti). tation of macrophage migration inhibitory factor (MIF) using the one-step sandwich enzyme immunosorbent assay: elevated serum MIF concentrations in patients with autoimmune diseases and identification of MIF in erythrocytes Int J Mol Med. 2000 5 (4): 397-403).

Radioimmunoassay is a method that can measure the concentration of trace substances in the range of nanogram (ng) and picogram (pg) per milliliter (ml), which is difficult to measure by other common methods. It is used not only for the measurement of antigen-antibody and allergen in blood, but also for the increase of tumor markers in malignant tumors, and for all substances that have excellent sensitivity, specificity, diversity, and convenience, and which can make antibodies. It is widely used because of the merit that it can measure and process many samples at once.

The present invention is to provide a radioimmunoassay that can accurately measure the concentration of MIF to a small amount, specifically acts on MIF, and can conveniently and conveniently measure the concentration of MIF in a biological sample for a large amount of samples. Accordingly, an object of the present invention is to provide a radioimmunoassay that can measure the concentration of MIF in a biological sample.

The present invention to achieve the above technical problem,

a) attaching the monoclonal antibody to the macrophage hepatic inhibitory factor (MIF) to the stationary phase;

b) adding at least two standard MIF solutions to bind the anti-MIF monoclonal antibody;

c) adding a biotin-bound anti-MIF polyclonal antibody to bind MIF bound to said monoclonal antibody of the stationary phase;

d) adding a radioisotope labeled streptavidin to attach to biotin bound to the anti-MIF polyclonal antibody and removing unreacted streptavidin;

e) preparing a standard dose response curve by measuring the radioactivity of the streptavidin labeled with the radioisotope attached to the biotin;

f) performing steps b) to d) on an unknown concentration of MIF sample instead of a standard MIF solution and measuring radioactivity; And

g) calculating the concentration of MIF in the sample by substituting the radioactivity obtained in step f) into the standard dose response curve.

EMBODIMENT OF THE INVENTION Hereinafter, this invention is demonstrated in detail.

The present invention relates to a radioimmunoassay for measuring the concentration of MIF in a biological sample. The method for measuring the concentration of MIF according to the present invention is largely prepared by preparing a standard dose response curve using a standard recombinant MIF solution and preparing a standard dose response curve. Concentration measurement is carried out in two steps called the measurement of the concentration of the sample to be measured. In addition, the standard dose response curve is prepared by immobilizing monoclonal antibodies to MIF, binding monoclonal antibodies to standard MIF antigens, binding polyclonal antibodies with biotin to the antigens, and labeling with the biotin and radioisotope. Attached to the streptavidin, and the preparation of a standard dose response curve by measuring the radioactivity of the streptavidin labeled with radioisotope attached to the biotin.

Immobilization of the anti-MIF monoclonal antibody is immobilized by immobilizing the antibody with a buffer solution in a well plate such as a 96 well plate for 18 to 24 hours, and blocking the wells to which the monoclonal antibody is not immobilized. Unfixed antibody is removed by washing several times with a mild detergent such as Tween-20. On the other hand, the MIF is preferably, but not limited to, recombinant human MIF.

After immobilizing the monoclonal antibody on the stationary phase, an antigen-antibody reaction is induced by addition of a known concentration of standard MIF antigen solution, and the unreacted antigen is removed by washing several times with a mild detergent such as Tween-20. do.

After the antigen-antibody reaction between the monoclonal antibody and the recombinant MIF antigen, the antigen-antibody reaction is induced again between the antigen and the polytin antibody to which the biotin is bound, and the unreacted polyclonal antibody is a mild detergent such as Tween-20. Remove several times with mild detergent.

Biotin-bound polyclonal antibodies bound to the antigen are detected using radioisotopes labeled streptavidin, preferably ¹²⁵ I-streptavidin.

Labeled polyclonal antibodies are isolated and purified by known methods such as ultrafiltration and centrifugation, and the radioactivity of the concentration of each standard recombinant MIF antigen solution is measured, for example, using a gamma scintillation counter. Dose response curves can be prepared.

Next, after the standard dose response curve is prepared, binding to a stationary monoclonal antibody, binding of a biotin-bound polyclonal antibody, attachment of a label, and radioactivity measurement are performed on the target sample to which the concentration of MIF is measured. The concentration of MIF in the target sample can be determined by performing the calculation and substituting the measured radiation value into the standard dose response curve.

The radioimmunoassay for measuring the concentration of MIF according to the present invention is applicable to various body fluid components including collected blood, saliva, urine, spinal fluid, lymph and cell disruption.

Hereinafter, the present invention will be described in more detail with reference to Examples and drawings. However, the following examples are provided only to make the present invention easier to understand, and the present invention is not limited to the following examples.

Example 1. Immobilization of Monoclonal Antibodies

Immobilization of monoclonal antibody against anti-MIF was performed using 10 mM phosphate buffer solution, 100 μl of antibody concentration was added to a 96-well plate at 200 ng per well, and left at room temperature for 24 hours. In order to remove the immobilized monoclonal antibody, 200 μl of a phosphate buffer solution (pH 7.4) added with 0.05% Tween-20 was added per well, and left standing for 5 minutes, and washed three times.

Phosphate buffer (pH 7.4) with 1% bovine serum albumin, 5% sucrose, and 0.05% sodium azide added per well to block the portion of the remaining wells bound to the anti-MIF antibody 200 µl was added and allowed to stand at room temperature for 2 hours. In order to remove the unimmobilized substances, 200 μl of a phosphate buffer solution (pH 7.4) added with 0.05% Tween-20 was added per well, and the mixture was washed for 5 minutes and repeated three times. After the immobilization and blocking of the antibody plate was stored in a drying table with calcium chloride fixed at a temperature of 4 ℃ was used for the experiment.

Example 2. Preparation of Standard Solutions

A standard solution was prepared to create a standard dose response curve. For diluting the standard solution, a 20 mM Tris buffered saline (pH 7.4) containing 0.1% bovine serum albumin and 0.05% Tween-20 was used, and diluted from 100 ng / ml to 0 ng / ml in two-fold dilutions. . The concentration of each standard solution was 100ng / ml, 50ng / ml, 25ng / ml, 12.5ng / ml, 6.25ng / ml, 3.125ng / ml, 1.56ng / ml, and 0ng / ml. 10 μl of standard solution was added to each well, and 100 μl was added using 20 mM Tris buffer solution (pH 7.4) containing 90 μl of 0.1% bovine serum albumin and 0.05% Tween-20. At room temperature, the dancer's rpm was reacted for 2 hours at × 100 / min. In order to remove unreacted MIF, 200 μl of phosphate buffer solution (pH 7.4) added with 0.05% Tween-20 was added per well, washed for 5 minutes, and repeated three times.

Example 3 Binding of Polyclonal Antibodies to MIF

In order to identify macrophage hepatic inhibitors bound to monoclonal antibodies against immobilized anti-MIF, a polyclonal antibody against biotin-bound anti-MIF was used as a confirming antibody. Biotin-bound anti-MIF polyclonal antibody was diluted to 20 ng per well using 20 mM Tris buffer solution (pH 7.4) containing 0.1% bovine serum albumin and 0.05% Tween-20, and then 100 μl of this was added. At room temperature, the dancer's rpm was reacted for 2 hours at × 100 / min. To remove unreacted biotin-bound anti-MIF polyclonal antibody, 200 μl of a phosphate buffer solution (pH 7.4) added with 0.05% Tween-20 was added per well and left to stand for 5 minutes, followed by washing three times. Was implemented.

Example 4 Preparation of Labeling Substance

In order to detect a biotin-bound anti-MIF polyclonal antibody, the radioisotope iodine-125 ( ¹²⁵ I) was labeled with streptavidin. The method of labeling ¹²⁵ I to streptavidin is as follows.

The streptavidin was diluted to a 1 mg / ml solution using 50 mM phosphate solution (pH 7.5), 20 μg of which was placed in a 1.5 ml Eppendorf tube, and the radioactive isotope Na dissolved in 0.1 N sodium hydroxide solution was added. ¹²⁵ I was added 1 mCi. Chloramine-T as an oxidizing agent was dissolved in a 50 mM phosphate solution (pH 7.5) to 2.5 mg / ml, and 250 µg / 100 ul was added thereto so that a magnetic stirrer was added at room temperature. The reaction was carried out for a minute. In order to neutralize the oxidizer, sodium metabisulfite was dissolved in 50 mM phosphate solution (pH 7.5) to 2.5 mg / ml, and 500 µg / 200 µl was added to terminate the reaction.

The identification of the labeling reaction of ¹²⁵ I was evaluated using the instant thin layer chromatography (ITLC) method. The stationary phase was ITLC-silica gel (ITLC-sg) and acetone was used as the mobile phase. A relative frequency (R _f ) of 0 means ¹²⁵ I labeled streptavidin, and a value of R _f of 1 means ¹²⁵ I of no reaction. Cover yield was evaluated using a gamma ray scanner, shown in FIG.

Figure 2 is a radiochromator of the ¹²⁵ I- streptavidin-labeled reaction solution, radiolabeled ¹²⁵ I as a radioisotope in streptavidin, and 2 μl of the labeling reaction solution was added to ITLC-sg and developed with acetone. Figure shows the radioactivity measured by a gamma ray scanner. The label yield of the reaction was found to be 88%.

Ultrafiltration was performed to separate and purify labeled ¹²⁵ I-streptavidin (SA). The labeled reaction solution was placed in Centricon YM-10 (Centricon YM-10) having a pore size of 10,000 molecular weight, and centrifuged at 2,500 rpm for 20 minutes to remove unreacted ¹²⁵ I. ^In order to obtain ¹²⁵ I-SA, the final material was separated and purified by centrifugation at × 2500 rpm for 10 minutes. The radiochromatogram of ¹²⁵ I-SA purified separately is shown in FIG. 3.

3 is a radiotracer ¹²⁵ I- streptavidin as radiation chromatogram after separation and purification of avidin to evaluate the radiochemical purity of the ¹²⁵ I- streptavidin was separated purified by ultrafiltration method Purification pure ¹²⁵ I- streptavidin 2 The μl was added to the ITLC-sg and developed with acetone, followed by radioactivity measurement with a gamma-ray scanner. The radiochemical purity of the purely purified ¹²⁵ I-SA was found to be 99%.

Example 5 Evaluation of Stability of Labeling Substances

In order to evaluate whether the purified ¹²⁵ I-streptavidin is a radioactive tracer that can be used for radioimmunoassay, it was taken at a certain amount of 60 days to evaluate the stability. Evaluation of stability was developed using ITLC-sg as the stationary phase and acetone as the mobile phase and evaluated using a gamma ray scanner, and the results are shown in FIG. 4.

Figure 4 evaluated the stability up to 60 days while storing the purified purified ¹²⁵ I- streptavidin at 4 ℃ and room temperature to evaluate the stability of ¹²⁵ I- streptavidin as a radiotracer. The horizontal axis represents the elapsed time in days, and the vertical axis uses ITLC-sg as the stationary phase of radioactivity bound to streptavidin and acetone as the mobile phase, and then deploys 2 μl of the sample stored at each temperature. Evaluation was made using a gamma ray scanner. At 4 ℃, it showed 95% stability up to 30 days and showed 93% stability at 60 days, and it was confirmed that it could be used stably up to 30 days while storing at 4 ℃ as a radiotracer.

Example 6 Combination of Polyclonal Antibodies and Labeling Substances

To detect biotin-bound anti-MIF polyclonal antibody, 25ng of ¹²⁵ I-streptavidin was added per well, and 100 μl of each was added thereto, followed by reaction for 2 hours using the dancer's rpm at 100 × / min at room temperature. I was. In order to remove unreacted ¹²⁵ I-streptavidin, 200 μl of a phosphate buffer solution (pH 7.4) added with 0.05% Tween-20 was added per well and left to stand for 5 minutes, and washed three times.

Example 7 Radioactivity (cpm) Measurement and Preparation of Standard Dose Response Curve

In order to measure the radioactivity of the bound ¹²⁵ I-streptavidin, each well was measured with a gamma counter, and a standard dose response curve was prepared with the horizontal axis as the concentration of MIF and the vertical axis as the binding radioactivity. Indicated.

Figure 5 shows the standard dose response curve in the radioimmunoassay for measuring the MIF concentration, the correlation coefficient was 0.999 as the amount of radioactivity bound as the concentration of MIF increases. Therefore, it was confirmed that this can be used as a standard dose response curve for measuring the blood MIF concentration.

Experimental Example 1. Measurement of MIF concentration in the sample

In order to confirm the accuracy of the radioimmunoassay for measuring MIF concentration, four samples of different concentrations were measured 9 times or 10 times on the same plate, and the coefficient of variation (CV) was measured. For the samples, the accuracy of the samples was measured by measuring the coefficient of variation (CV) between the values measured on different plates, and the results are shown in Table 1.

Check the accuracy of radioimmunoassay for measuring MIF concentration Sample measure ± standard deviation (ng / ml) Coefficient of variation (CV) (%) ABCD between same plates 9999 61.5 ± 7.068.3 ± 3.026.2 ± 1.316.2 ± 0.1 5.54.44.80.5 EF between different plates 22 64.9 ± 4.815.4 ± 1.2 7.47.6

Table 1 shows the coefficient of variation (CV)% of the values measured by repeating the number of sample measurements 9 times in the same plate to check the accuracy of the radioimmunoassay for measuring the MIF concentration. In addition, the coefficient of variation of the measured values between the different plates was calculated. The method of calculating the coefficient of variation is as follows.

Coefficient of variation (%) = standard deviation / mean × 100

In the same plate, the coefficient of variation did not exceed 5.5% and the coefficient of variation between different plates did not exceed 7.6%.

In addition, the degree of recovery of the measured values was evaluated by adding MIF solutions having different concentrations to the serum of humans having a constant concentration of MIF, and the results are shown in Table 2.

Reproducibility test of radioimmunoassay for measuring MIF concentration Sample Name Blood MIF (ng / ml) Standard MIF Solution Added (ng / ml) Metric (ng / ml) Reproduced value (ng / ml) Reproducibility (%) Sample 26 ^* 0.365 6.25 7.38 7.02 112 0.365 12.5 11.86 11.5 92

* "Sample 26" refers to a sample of patients randomly selected for testing among patients in the hospital.

Table 2 adds the MIF standard solution prepared at a known concentration to a sample with a certain amount of MIF to evaluate the reproducibility of the radioimmunoassay for measuring the MIF concentration, and evaluates how much the MIF concentration is added. . The mean value of reproducibility was found to be 102%.

In addition, the change of the measured value according to the dilution concentration is shown in FIG. 6 in order to evaluate the degree of change and the accuracy of the measured value in the case of serial dilution of 2 times. Figure 6 shows the evaluation of the change in the value and the accuracy of the measured value by diluting the sample with a concentration of 90ng / ml MIF in multiples in order to measure the change in the measurement value according to the sample dilution in the radioimmunoassay for measuring the MIF concentration It was. The correlation between the measured values was 0.97, indicating the accuracy of the measured values.

Finally, the serum concentrations of MIF according to males and females were evaluated by performing radioimmunoassay to measure MIF concentration in 15 males and 25 females as normal serum.

Sample object male female Blood MIF Concentration (Mean ± Standard Deviation) 1.34 ± 1.47 0.17 ± 0.18

Table 3 shows the measurement of the concentration of MIF in the blood of normal subjects by radioimmunoassay for the measurement of the concentration of MIF, 5 of the 15 men detected blood, 8 of the 25 were detected in women. The mean blood concentration of males was 1.34 ± 1.47 ng / ml, and the mean blood concentration of females was 0.17 ± 0.18 ng / ml, which was 7.9 times higher than that of males.

In the case of using the radioimmunoassay and the concentration measurement kit for measuring the concentration of MIF according to the present invention, since it specifically acts on MIF, it is possible to accurately measure even a small amount of concentration. Inner concentration can be measured.

1 is a schematic diagram of a radioimmunoassay for measuring the concentration of MIF according to the present invention.

Figure 2 is a radiochromatogram of the ¹²⁵ I-streptavidin labeled reaction solution as a radiotracer.

3 is a radiochromatogram after separation and purification of ¹²⁵ I-streptavidin as a radiotracer.

4 is a graph showing the stability of ¹²⁵ I-streptavidin as a radiotracer.

5 is a standard dose response curve in radioimmunoassay according to one embodiment of the invention.

Figure 6 is a graph showing the change of the measurement value according to the sample dilution in the radioimmunoassay according to an embodiment of the present invention.

Claims (4)

a) attaching the monoclonal antibody to the macrophage hepatic inhibitory factor (MIF) to the stationary phase; b) adding at least two standard MIF solutions to bind the anti-MIF monoclonal antibody; c) adding a biotin-bound anti-MIF polyclonal antibody to bind MIF bound to said monoclonal antibody of the stationary phase; d) adding ¹²⁵ I-labeled streptavidin to attach to biotin bound to the anti-MIF polyclonal antibody and removing unreacted streptavidin; e) preparing a standard dose response curve by measuring the radioactivity of ¹²⁵ I-labeled streptavidin attached to biotin; f) performing steps b) to d) on an unknown concentration of MIF sample instead of a standard MIF solution and measuring radioactivity; And g) calculating the concentration of MIF in the sample by substituting the radioactivity obtained in step f) into the standard dose response curve. Method for measuring the concentration of MIF in a sample comprising a. The method of claim 1, wherein the MIF is recombinant human MIF. delete The method of claim 1, wherein the sample is selected from the group consisting of collected blood, saliva, urine, spinal fluid, lymph and cell disruption.