JPS61254861A - Modified human gamma-globulin - Google Patents

Abstract

PURPOSE:To make possible automatic biochemical analysis by adjusting monomer type IgG and polymer type IgG to one to 0.7 or above as modified human gamma-globulin and using the same as an antigen in measuring the RF in the human serum. CONSTITUTION:The human gamma-globulin is dissolved to 1% concn. in 0.02M HEPES buffer soln. (7.2phipH) contg. NaN3 at 0.1% concn. and NaCl at 0.8% concn. and the soln. is treated for 30min at 53-63 deg.C, by which the soln. is thermally modified. The light absorptivity of water blank is first measured by using an automatic biochemical analyzing instrument, then the absorptivity of the specimen serum S and the 1st reagent R1 consisting of NaCl, NaN3, chondoloitin sulfuric acid, TX-100 and polyethylene glycol is measured for 5min; further the 2nd reagent R2 consisting of the modified human gamma-globulin subjected to the above-mentioned thermal modification is added to the soln. and the absorptivity thereof is measured.

Description

DETAILED DESCRIPTION OF THE INVENTION (Industrial Field of Application) The present invention relates to modified r-globulin, particularly to modified human γ-globulin.

R-globulin is a type of biologically produced protein and is used in various immune reactions. The modified human r-globulin according to the present invention can be used to improve reactivity in this immune reaction.

(Prior Art) RP, or rheumatoid 7 acta (Rheu
matoid factor (rheumatoid factor), which is mainly composed of macroglobulin with a molecular weight of approximately 1 million, is a substance presumed to be IgM with a sedimentation constant of 198. acts as an antibody against

Therefore, it has already been put into practical use to diagnose rheumatism, especially rheumatoid arthritis, by causing an immune reaction using human γ-globulin as an antigen and RP as an antibody. RF measurement methods for this purpose include the RA test that uses polystyrene latex particles or bentonite particles and uses an agglutination reaction, and the Wallar-Rose reaction 1 that uses sheep red blood cells and uses an agglutination reaction.
Nine Rolls by simplifying the means in the r-Rose reaction
Although the er modified method and the RAHA test are used, the RA test and the RAHA test are currently mainly used. In these measurement methods, native human r
- Although the RA test using globulin has high sensitivity, the problem is that the positive rate due to non-specific reactions is also high.
On the other hand, the RAHA test has high specificity, but has problems in that it has poor sensitivity and requires a relatively long reaction time.

(Problems to be Solved by the Invention and Means and Effects for Solving the Problems) Therefore, the present inventors have discovered that antigens and antibodies specifically react,
After conducting extensive research to develop an RF measurement method with high sensitivity and short reaction time, we found that using denatured human γ-globulin as an antigen could bring specificity to the reaction. In addition, the present inventors have discovered that the time required for 'PiT can be shortened, and have completed the present invention.

The modified human γ-globulin according to the present invention is characterized in that the ratio of monomeric IgG to polymeric IgG is 1 to 7 or more.

As a preliminary modification method for obtaining the human γ-globulin modified product according to the present invention, any known method such as a thermal denaturation method or a urea denaturation method can be used.

The reason why the ratio of monomeric IgG to polymeric IgG in the human γ-globulin modified product according to the present invention is defined as 1:0.7 or more is that if the content of polymeric IgG is less than this, it will contribute to improving the reactivity. Since the content is low, there is no upper limit to the content of polymeric IgG, and for example, 100% polymeric IgG can be used after being separated.

When performing RF measurement using the denatured human γ-globulin according to the present invention as an antigen, a buffer containing a water-soluble polymeric substance as a reaction accelerator in addition to the denatured human γ-globulin is added to the sample serum. is added, and an antigen-antibody reaction is performed. In this case, polyethylene glycol, polysaccharide or protein can be used as the water-soluble polymer substance,
In this case, the polysaccharides and proteins include dextran, chondroitin sulfate, alginic acid, gum arabic, methylcellulose, heparin, gelatin, alsmin, casein, fibrinogen, lectin, and ConA.

P) IA etc. can be exemplified.

When carrying out RF measurement using the human γ-globulin modified product according to the present invention, the reaction is not specific and the reactivity is high due to the use of the reaction accelerator, so it is recommended to use it instead of the conventional agglutination reaction. As a result, it is possible to measure and judge RF based on turbidity using a biochemical analyzer, eliminating the introduction of errors caused by conventional visual observation judgments. can.

(Usability of human γ-globulin variant I according to the present invention) -1°f Human r-globulin variant according to the present invention! The product can be used as a diagnostic reagent for various immune complex diseases in addition to the above-mentioned RF measurement using immunoturbidimetry, and is expected to improve immunoreactivity.

Furthermore, IgG is used after being labeled with an isotope, etc. when detecting immune complexes during binding and inhibition of immune complexes to cell receptors, and it is also expected that modified products according to the present invention may be used as this IgG. Ru.

(Production Examples, etc.) - 1°f Next, the present invention will be explained with reference to production examples of the modified human r-globulin according to the present invention and application examples of using the modified products using RF measurement.

Production Example 1 (Thermal denaturation of human r-globulin and reactivity with RF when this denatured globulin is used for RF measurement) Human γ-globulin (“Cohn Fraction ■” manufactured by Sigma) was dissolved in HEPES buffer (pH 7.2) to a concentration of 1%.
Heat denaturation was performed by treating at various temperatures of 3°C (2°C intervals) for 30 minutes.

This was filtered through gel using Sephadex G-200 (manufactured by Pharmacia) equilibrated with 0.1 M phosphate buffer to separate polymeric IgG and monomer i1gG, and the protein concentration of each was measured according to the Lowry method. The results of calculating the ratio between the two were as shown in Table 1 below.

Table 1 Next, each of these modified human γ-globulins was applied to immunoturbidimetry according to the method described in the convenience example below, and the reactivity was investigated by turbidity measurement. The results are shown in the graph of Figure 1. It was a common knowledge. From this graph, the denaturation temperature is 59-6
It can be seen that when the denaturation temperature is between 1°C, there is a sudden change that affects the reactivity, but when the denaturation temperature is set at 61°C or higher, the effect on the reactivity becomes gradual. Considering this result and the results shown in Table 1 above, it can be seen that it is advantageous when the ratio of poly-i-1gG to monomeric IgG is 1:0.7 or more. 100 parts of monomeric IgG and 100 parts of polymeric IgG were collected from the fraction of human r-globulin denatured at temperature (53-63 degrees Celsius, 2 degrees Celsius), and these were used to extract O/1o12/1o14. /10.6/10%8/
A 6-step dilution series of 10 and 10/1o was created, and using these, the 0.D. It was common sense. From this graph, %/-F-sexuality 1gG1
00* (1' degree of 0) human γ-globulin cannot be used for measurement, and as the degree of denaturation increases, a favorable effect appears on reactivity, but in the case of heat denaturation, the denaturation humidity was set at 61 °C. It turns out that the case is the best.

Production Example 2 (Urea denaturation of human γ-globulin and reactivity with RF when used for t-RF measurement of this denatured globulin) Human r-globulin was added to a solution containing 6 M urea and 0.15 M NaC2. (Dissolve Sigma's "Cohn Fraction ■" to a concentration of 1%, and add 6
Dialyzed against M urea for 24 hours at room temperature and further diluted with 0.15
Dialyzed for 72 h at 4 °C against M NaC2 and then further dialyzed at 4 °C against 0.02 M HEPES buffer (p) 17.2) containing 0.1% NaN3 and 0.8% NaCL). The protein was dialyzed for 24 hours, and then separated into cibolimeric IgG and monomeric IgG by gel filtration in the same manner as in Production Example 1.The protein concentration ratio was determined to be 1.26:1. .

Note that the above polymeric IgG versus monomeric 1gG is 1
．． A dilution series of O/10% 2/26 to 1 human r-globulin denatured product, 100% undenatured monomeric IgG and 100% completely denatured polymeric IgG separated by this modification method were used. 10%'/
10s6/10% 6 stages of 8/10 and 10/1o were created and the values were measured as described in the reference example below (0, D, and the results of investigating the effect on reactivity are shown in the graph in Figure 3. From this graph, the polymeric Ig
It can be seen that it is more advantageous to use r-globulin (with a high G content, ie, a high degree of modification), and that it becomes impossible to measure when unmodified material is used.

Example of use (RF using modified human r-globulin according to the present invention)
(For measurement) Equipment used and parameter settings For this RF measurement, a Hitachi model 705 biochemical automatic analyzer was used to save labor. This analysis was done.

Table 2 (B) Reagent a) First reagent (R1) 0.1 M NaC2, 0.1 To NaN3, 0.1% chondroitin sulfate, 0.1 To TX
-100 (surfactant) and 0.02M containing 0.25% polyethylene glycol (molecular weight 4000)
HEPI8 buffer (pE 7.2) b) Second reagent (R2) 0.1 M (0.8%) NaCA and 0.15b
NaN3 and 1.0% human r-globulin (“Cohn Fracton If J” manufactured by Sigma)
- containing 0.02 M HEPES buffer at 61 °C for 3
Heat denatured for 0 minutes (1 g of monomeric clopurin)
G to polymeric IgG ratio of 1 to 0.71) (C) Operation method The measurement procedure is generally as shown in Figure 4. First, the absorbance of a water blank is measured, and then the absorbance of the sample is measured. Serum S and first reagent R1 or both are added and the absorbance is measured continuously over a period of 10 minutes, 31 times at 20 second intervals. Within this measurement period, ie 5 minutes after the addition of the first reagent R1, the second reagent R2 is added. That is, the absorbance measurement within 5 minutes from the addition of the first reagent R1 is performed on the reaction solution of the sample serum S and the first reagent R1, and the absorbance measurement after 5 minutes from the addition of the first reagent R1 is performed on the sample serum S. This is done for the reaction solution of S and the second reagent R2. It should be noted that for each absorbance value measured 31 times after the addition of the first reagent R1, the absorbance value of the water blank is automatically subtracted by nine calculation mechanisms built into the analyzer.

In this case, after measuring the water blank)
As is clear from the parameter setting values in Section 1, the sample serum (However) 20 μt and the blank solution (R1) which is the first reagent.
350 μt was added to start the actual measurement, and 5 minutes later, the second reagent, denatured human γ-globulin solution (R2
) 50 μt was added, and the measurement was continued for an additional 5 minutes. In addition, AS8 in the equipment parameters in item (6) above
As the setting value is set to END in the AY C0DE section, the average value of the absorbance values measured in the last two measurements (14th and 15th measurements) is also Regarding the measurement of the reaction solution (the solution in which R2 was further added and reacted with S), the average value of the absorbance measured in the last two measurements (the 30th and 31st measurements) was used.

(b) The following dilution series was prepared using the standard serum with a standard curve of 2'm, that is, the serum determined to be negative by the RAHA test, and the serum determined to be 1280 times higher.

0 (undiluted negative serum) 128 (1:9) 256 (2:8) 384 (3 near) 512 (4:6) 640 (5:5) 768 (6:4) 896 (7:3) 1024 (8:2) 1152 (9:1) 1280 (undiluted positive serum) Next, according to the procedure in section 0 above, these serum solutions were used as sample serum (S), 20 μt each, and the first reagent (R1 )3
When absorbance was measured using 50μt and 50μt of the second reagent (R2) and the relationship between absorbance (mABs) and RF value (corresponding to the dilution factor) was plotted, the excessive graph shown in Figure 5 was obtained. Obtained. As is clear from this graph, there is a stable linear relationship between the absorbance value and the RF value, and therefore this graph can be used as a standard calibration curve. The relationship is stored as a calibration curve in the storage mechanism of the automatic analyzer.

(G) Correlation with conventional method Absorbance was measured for serum (70 samples) for which the RF value was undetermined according to the method described in the above section, and the 0, D, and D values were determined. Applying the agglutination method, we diluted the sample serum several times to find out the agglutination limit concentration that would cause it to agglutinate to the same extent as expected, and plotted the correlation between these two methods, and the general graph shown in Figure 6 was obtained. Ta. It is clear from this graph that the two methods have a good correlation, and therefore this measuring method has sufficient practicality.

(Effects of the Invention) The human γ-globulin modified product according to the present invention can improve the specific reactivity when used for immune reactions, and in particular, it can be used as an antigen during RF measurement in human serum. When used, it is possible to perform the measurement with an automatic biochemical analysis device.

Incidentally, the denaturation itself can be carried out relatively easily by a method known in the field of biochemistry.

[Brief explanation of the drawing]

Figure 1 is a graph showing the relationship between denaturation temperature and turbidity when heat-denatured human γ-globulin according to the present invention is reacted with RF. The dilution rate of the modified human r-globulin according to the invention is RF
FIG. 3 is a graph showing the effect on the reactivity of human r-globulin according to the present invention which has been denatured with urea, and is similar to FIG. 2. FIG. - An explanatory diagram illustrating the operation when performing RF measurement using modified globulin, Figure 5 is R of Figure 4.
A graph showing the standard calibration curve for F measurement, Figure 6 is
2 is a graph showing the correlation between the RF measurement method shown schematically in the figure and the conventional latex aggregation method. Patent Applicant: Nissui Pharmaceutical Co., Ltd. Figure 1: Jacket (Declaration of Honor) Procedural Amendment July 5, 1985 Michibe Uga, Director General of the Patent Office1, Indication of the Case Patent Application No. 96098 of 19852, Name of Invention Human Two Globulin Modified Product3, Amendment Representative: Yasuaki Kobayashi 4, Agent: 105 Detailed explanation of the invention in the specification (1) In the 1st page, line 17 of the specification, the word "affected by disease" is amended to read "affected by disease." (2) On page 1, line 19, the word "rheumatoid" is corrected to "rheumatoid." (3) On page 2, lines 11 and 12, “υallar J
Correct the statement as "Ii′υaaler." (4) In the second line of page 8, the words ``interstairs'' are corrected to read ``r spacing''. (5) In the 11th line of page 12, the text "rFracton J" is corrected to (rFraction j).

Claims (1)

[Claims] (1) Monomeric IgG and polymeric IgG are 1:0
．． 7 or more.