JPH05297000A - Method for measuring insulin antibody - Google Patents

Abstract

PURPOSE:To safely, easily, and quantitatively measure the amount of anti-insulin antibodies in human body fluids to such an extent that the measured results can be used as a clear index of insulin dosages based on the combination of a standard sample against which the amount of the anti-insulin antibodies can be accurately calculated and enzyme-labeled protein A which is specifically bonded to the Fc fragment of IgG. CONSTITUTION:A sample to be inspected, such as the human blood serum, etc., and a standard sample containing an insulin antibody derived from an animal (for example, the guinea pig) other than human beings are made to react to insulin arranged in parallel on a solid phase. Then the amount of the insulin antibodies in the sample to be inspected is found by measuring the enzyme activity after making the insulin antibodies bonded to the insulin on the solid phase to react to enzyme-labeled protein A.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for measuring insulin antibody, and more particularly to a method for measuring insulin antibody capable of quantitatively measuring insulin antibody in human body fluid.

[0002]

BACKGROUND OF THE INVENTION Insulin is an important drug commonly used for treating diabetes mellitus, controlling (especially during infection, surgery, pregnancy) and preventing complications (retinopathy, vascular disorder, neuropathy). However, in this use,
Production of (anti) insulin antibodies is often a problem.

That is, when insulin antibody is produced in the body fluid of a human (patient), even if insulin is subsequently administered to the patient, its efficacy is significantly diminished. Therefore, the amount of insulin antibody is the amount of insulin administered to the patient. It is closely related to (when the insulin antibody titer is high, a large dose of insulin is required to obtain the drug effect). Therefore, accurate measurement of this insulin antibody amount has clinically very important significance.

Conventionally, in the measurement of insulin antibody,
A method for measuring an antigen-specific antibody in a body fluid (a so-called immunoassay method), which is generally used for inspection of autoantibodies, has been used as it is.

The conventional measuring methods in this field can be technically classified into two types.

[0006] As a first technique, an antigen labeled with a radioisotope (RI) is reacted with a test sample (test sample) containing an antibody, and the resulting immune complex is precipitated with a precipitant (eg, anti-antibody). A method of precipitating with human immunoglobulin, polyethylene glycol, methanol, ammonium sulfate, etc., and measuring the radioactivity in this precipitate to determine the amount of insulin antibody (clinical adult disease, vol. 10, 249-
255 (1980)); and a specific antibody is immobilized on a solid-phase carrier and the like, and a radioisotope-labeled antigen and a test sample are simultaneously added to the solid-phase carrier to determine the inhibitory activity against the reaction between the solid-phase antibody and the isotope. And measuring the amount of insulin antibody as a relative concentration (diabetes, 30
Volume 549-554 (1987)).

On the other hand, as a second technique, an antigen is immobilized on a solid phase carrier, a test sample containing an antigen-specific antibody is allowed to react with this, and an anti-immunoglobulin antibody labeled with a radioisotope or an enzyme ( There is a method of reacting a second antibody) to measure a specific antibody (JOURNAL OF IMMUNOLOGICA
L METHODS, 76 , 185-194 (1985); EXP.AN
IM. 37 , 67-72 (1988); CLIN. CHEM. 2
7 , 1753-1757 (1981)).

[0008]

However, the first technique using the radioisotope out of the above has problems in terms of safety, equipment, effective period of the reagent, and the like. In addition, as a problem common to the first and second techniques, there is a drawback in that the amount of antibody must be relative in these measurements and it is difficult to be an index for insulin treatment over time.

More specifically, in the conventional method, since human serum itself having an insulin antibody is used as a standard sample, the measurement data is necessarily
"Presence or absence of anti-insulin antibody" in comparison with standard serum
Alternatively, the relative ratio is “how many times the antibody amount in the standard serum (dilution factor)”.

However, human-derived anti-human insulin antibodies are extremely difficult to obtain per se (human insulin antibodies originally present in humans themselves are not easily produced in humans). The amount of anti-insulin antibody in the standard serum varies greatly depending on its origin (individual difference between humans as donors), and the standard serum itself for measuring the amount of antibody in the same patient may differ at each measurement. Therefore, it is difficult to use the measurement data obtained by the conventional method as an index for continuous and continuous insulin administration. Therefore, the actual amount of insulin to be administered to a patient was actually determined by the “experience” and “can” of the doctor involved in this.

On the other hand, the above-mentioned conventional enzyme-labeled antibody (second
Enzyme-linked immunosorbent assay (EIA or ELIS) using antibody
In A), since the measurement data was obtained as optical density or absorbance (OD value), there was a problem peculiar to EIA based on the use of the enzyme-labeled antibody.

That is, it is difficult (actually) to avoid variations in the OD value due to changes over time in the enzyme activity of the enzyme-labeled antibody itself, the method for preparing the enzyme-labeled product, or the reaction yield. Using itself as measurement data has a problem in terms of data accuracy and reproducibility (so-called reproducibility of day difference).

Therefore, the object of the present invention is to eliminate the above-mentioned drawbacks of the conventional method and to quantitatively determine the amount of anti-insulin antibody in human body fluid (or substantially quantify it to the extent that it can be a clear indicator of insulin administration). Purposely) to provide a measurable method for measuring insulin antibody.

[0014]

Means for Solving the Problems As a result of intensive studies, the present inventors have found that a standard sample containing an anti-insulin antibody derived from a non-human animal is used as a standard sample without using a standard human-derived serum in the conventional method. Use; and, without using the second antibody (anti-human immunoglobulin antibody labeled with radioisotope or enzyme) in the conventional method, using the enzyme-labeled protein A is extremely useful for quantifying anti-insulin antibody in human body fluids. I found it effective.

The method for measuring insulin antibody of the present invention is based on the above findings. More specifically, a test sample and a standard sample containing insulin antibody derived from a non-human animal are
It is characterized by reacting with insulin arranged on a solid phase, and then reacting insulin antibody bound to insulin on the solid phase with enzyme-labeled protein A.

In the present invention, an anti-insulin antibody bound to insulin on a solid phase and a reagent to be further bound (having a function corresponding to the second antibody in the conventional method in the present invention) are human or mouse, Protein A that specifically binds to the Fc fragment of IgG (immunoglobulin G) of animals such as rabbits and guinea pigs (hereinafter referred to as "P
A)) is used, the standard sample containing an anti-insulin antibody derived from a non-human animal is used in parallel with the test sample derived from human (sample). It can be reacted (under substantially the same environment).

The amount of anti-insulin antibody in the above-mentioned standard sample derived from an animal can be quantitatively determined by, for example, the Scatchard plot method, as described later. Therefore, according to the present invention, the above PA
And the amount of anti-insulin antibody derived from animal based on the combination of the amount of anti-insulin antibody in human body fluid quantitatively (at least substantially quantitatively to the extent that it can be an objective and clear indicator of insulin dose). ) It becomes possible to measure.

The effects of the present invention as described above can be obtained only based on the combination of the above PA with an animal-derived anti-insulin antibody, and if one of them is lacking, quantitative measurement is not possible. It is possible. That is, for example, even if the second antibody as in the conventional method is simply used instead of the PA, only uncertain data can be obtained rather than in the conventional method.

More specifically, for example, if guinea pig serum (containing a certain anti-insulin antibody) is used as a standard sample, the test sample is usually human body fluid (eg, serum). As the antibody, both an enzyme-labeled anti-guinea pig IgG antibody and an enzyme-labeled anti-human IgG antibody are required. Since these two types of second antibodies generally have completely different properties that affect not only the titer but also the labeling enzyme and other measurement data, it is important to measure the standard sample (guinea pig serum) and human serum "in parallel". Would be completely absent.

Hereinafter, the present invention will be described in detail with reference to the drawings as necessary.

(Solid phase) In the present invention, a solid phase used in a usual EIA (also referred to as enzyme immunoassay, ELISA) can be used as the solid phase without particular limitation. As a material for the solid phase, a plastic such as polystyrene is usually preferably used from the viewpoint of physical adsorption of an antigen and / or an antibody (which is considered to be based on a hydrophobic bond).
The shape of the solid phase is not particularly limited, and for example, a microplate shape, a bead (or ball) shape, a ring shape, a tube shape, or a gel shape can be used. For example, a plastic having a plurality of wells Microplates (96-well-microplates, etc.) manufactured by the present invention are preferably used from the viewpoint that a large number of samples (specimens) can be rapidly measured. In this case, the quickness of the measurement can be further improved by combining it with an absorptiometer for a 96-well plate, if necessary.

(Insulin) Next, the insulin to be placed (or coated) on the solid phase will be described. In the present invention, the insulin to be coated on the solid phase can be appropriately determined according to the human condition to be measured (for example, the origin of insulin used for past treatment) (for example, bovine insulin). For measurement of a human-derived sample under treatment using bovine insulin, it is preferable to coat the solid phase with bovine insulin, but it is usually human insulin and / or mammals (in terms of availability). It is preferable to use insulin derived from bovine, pig, etc.). The insulin used in the present invention is 20
IU / mg or more, more preferably 25 IU / mg or more (particularly about 25 to 28 IU / mg) is preferably used. Here, "IU" means "international unit"("BiochemicalDictionary" (2nd edition), page 142 (199).
0) Tokyo Kagaku Doujin).

For example, human insulin is a peptide hormone composed of an A chain consisting of 21 amino acid residues and a B chain consisting of 30 amino acid residues as shown in FIG. The polypeptide chain of this book is between the 7th A chain and the 7th B chain, and between the 20th A chain and 1st B chain.
It is linked by a disulfide bond between the 9th position (the 6th and 11th positions in the A chain, and the 7th and 7th positions between the A and B chains).
The S-S bond is formed by the cysteines at the 20th, 20th and 19th positions). The molecular weight is 5807 and the isoelectric point is 5.3 to 5.4. ("Biochemistry Dictionary" (2nd edition) 1
42 (1990) Tokyo Kagaku Dojin).

On the other hand, in porcine insulin, the 30th B chain of human insulin is changed from Thr to Ala; in bovine insulin, the 8th A chain of human insulin is Thr → Ala, A. The 10th chain is Ile
→ Val, 30th B chain is changed from Thr → Ala.

In the present invention, this insulin is obtained from humans or animals, as well as chemically and / or biochemically synthesized insulin, or obtained by so-called biotechnology such as genetic engineering. Any of the above can be used.

(Animal other than human) When immunizing with insulin to obtain an anti-insulin antibody, a mammal is preferably used as the animal other than human. More specifically, mammals such as guinea pigs (guinea pigs), mice, and rabbits are preferably used, but guinea pigs, mice, or rabbits are preferably used from the viewpoint of ease of handling and price.

From the viewpoint of ease of antibody production, when obtaining the above-mentioned insulin antibody, human insulin is immunized into a (non-human) animal, or animal-derived insulin is immunized into another animal (for example, , And immunize guinea pigs with bovine insulin).

(Standard sample) As a standard sample (standard or reference), a body fluid (usually serum) of the above animal immunized with insulin to produce anti-insulin antibody is used. In the present invention, it is preferable to use, as this insulin antibody, an antibody that has little cross-reactivity with pro-insulin, glucagon, somatostatin and the like.

(Test Sample) As the test sample (or sample), human body fluid such as blood (usually serum) is preferably used.

(Enzyme-labeled protein A) PA (protein A) is a protein having a molecular weight of about 42,000 which accounts for about 5% of the cell wall components of Staphylococcus aureus, and is an immunoglobulin G (IgG) of human, mouse, rabbit or the like. Protein that specifically binds to the Fc fragment of In the present invention, the PA is human, mouse, rabbit, and / or guinea pig Ig per 1 mg of the PA.
G is 7 mg or more, further 8 mg or more (especially 9 mg or more)
It is preferable to use PA having reactivity with (Pharmacia, Separation New
s) April 1979 issue; see "Protein A-anti-antibody reagent" section).

The enzyme-labeled PA used in the present invention is the above-mentioned PA.
Is obtained by labeling with an enzyme usable in the EIA method. As the enzyme used for labeling here, alkaline phosphatase, β-galactosidase, glucose oxidase, peroxidase and the like are preferably used, but as described below, a simple and highly sensitive enzyme activity measurement can be performed using a substrate and a colorimetric method. From a possible point of view, it is preferable to use a peroxidase such as horseradish peroxidase (HRP).

As a method for labeling PA with the above-mentioned enzyme, a known method (for example, glutaraldehyde method) can be used and is not particularly limited.

(Skim milk) In the insulin antibody measuring method of the present invention, a skim milk addition buffer is preferably used for diluting a test sample (specimen). When the skim milk-added buffer solution is used as described above, according to the study by the present inventors, a non-specific binding factor in a sample (which was one of the causes of the decrease in specificity in the conventional EIA sandwich method)
It is possible to suppress the influence of. "Skim milk" here is so-called skim milk powder, which is obtained as a powder by removing water from skim milk, which is the part with the smaller fat content obtained when milk is subjected to a cream separator. Say.

The above-mentioned skim milk also has a function as a so-called blocking agent that blocks the non-insulin-attached portion of the solid phase. As such a blocking agent, BSA (bovine serum albumin) and the like have been conventionally used, but according to the studies by the present inventors, some test samples have anti-BSA antibody. , The antibody may bind to the blocking agent and contribute to the non-specific reaction. Even in such a case, the skim milk can be preferably used.

As the buffer used together with the skim milk, a general buffer such as a phosphate buffer (PBS) can be used without particular limitation. The amount of skim milk added to the buffer solution is 5 to 5 based on the weight of the buffer solution.
It is preferably about 11% (more preferably about 8 to 11%, particularly about 9 to 11%).

(EIA Assay Method) Next, a preferred embodiment of the anti-insulin antibody assay method of the present invention will be described.

In the anti-insulin antibody measuring method described below, materials or reagents such as insulin, anti-insulin antibody, standard sample and test sample (specimen) are appropriately buffered solutions such as phosphate buffer (PBS). It may be diluted with. In addition, in the following measurement, it is preferable to add a degrading enzyme inhibitor (eg, aprotin) that inhibits a degrading enzyme derived from water pancreas to a test sample in order to improve accuracy of data.

Insulin (eg, bovine insulin) was diluted with a buffer solution to about 10 to 100 μg / mL, and then diluted on a solid phase (eg, 96-well microplate) for each well (hereinafter, the amount used was Add 50-200 μL / well and write at low temperature (eg 4
(° C) overnight (10-20 hours) (1-2 at room temperature)
Let stand for about a hour) to coat the solid phase with insulin.
After collecting the excess insulin solution, the solid phase is washed with the buffer several times (for example, about 2 to 4 times).

Thereafter, if necessary, a blocking solution (eg, gelatin-containing buffer solution) 100 to 200 μL
Is injected into the well and left at 25 ° C. for about 1 to 2 hours to block the non-insulin-adhering portion of the solid phase surface in order to lower the background and improve the measurement accuracy and reproducibility. preferable.

Next, a standard sample containing anti-insulin antibody (standard; derived from animals other than humans) is diluted with a buffer solution so that the amount of antibody in the diluted solution is about 0 to 20 ng / mL (normally, a calibration curve is used). For preparation, make several points within this range) Standard solution 50
~ 200 μL, and test sample (eg human serum)
50 to 100 times diluted with a buffer solution
About 200 μL is injected into each well,
The plate is allowed to stand overnight at 4 ° C (about 1-2 hours at 25 ° C) to react the insulin on the plate with the anti-insulin antibody in the standard solution and the sample solution (first
reaction).

After removing the excess sample solution and standard solution, each well is washed with a buffer solution about 3 to 5 times.

Then, an enzyme (eg, horseradish peroxidase (HRP)) labeled antibody PA buffer solution containing about 100 to 200 ng / mL of protein A (PA) is injected into each well at about 50 to 200 μL, and the temperature is 25 ° C.
After allowing to stand for 1 to 3 hours, the anti-insulin antibody (Fc fragment) is reacted with PA (second reaction).

After removing the excess PA solution and washing with a buffer solution about 3 to 5 times, the activity of the above enzyme is measured to determine the amount of anti-insulin antibody in the standard and the sample.

More specifically, the above-mentioned enzyme activity is measured by
It can be performed as follows. For example, a substrate corresponding to the above-mentioned enzyme (when the enzyme is HRP, for example, ortho-phenylenediamine (OPD) is 0.3 to 0.6 mg / m 2
Inject 50 to 200 μL in each well at a concentration of about L, and
After reacting the substrate with the enzyme for about 10 minutes to 1 hour at 5 ° C. and confirming that the wells of the positive standard have sufficiently developed color, the reaction is stopped (for example, using an absorptiometer for 96-well plate). Measure the absorbance of the wells.

[0045]

EXAMPLES The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these examples. Example 1 Bovine insulin (Sigma, I-3500) was added to 10 m.
M carbonate buffer solution _{(Na 2 CO 3 -NaHCO 3,} pH
Dilute to 50 μg / mL with 9.9), 96 wells (well)
-Adding 100 μL per well to an immunoplate (Coster Microplate) and incubating at 4 ° C. for 20 hours. Then, the supernatant is discarded, and 0.15M NaCl is added.
1 and 0.05% Tween-20 in PBS for washing (10 mM phosphate buffer, pH 6.8) three times.

Next, a blocking agent (Bl manufactured by Dainippon Pharmaceutical)
A blocking solution consisting of 0.01 M-PBS (phosphate buffer solution) containing 25% of occ Ace) was added to each well in an amount of 100 μL and left at 25 ° C. for 2 hours to perform a blocking operation.

After removing the excess blocking solution, 100 μL of the standard solution and 100 μL each of the test human serum diluted solution were injected into each well and reacted at 4 ° C. overnight (first reaction).

Here, the standard solution was guinea pig serum containing anti-insulin antibody (Linco Research,
No. 4011) is diluted with PBS (0.1M PBS)
(PH 6.8), 10% skim milk (Difco Laborato
ries 0032-02), 0.1% Tween-20 (Hanai Chemical) and aprotin (Basel, 25000 KIU / m
L) 500 KIU / mL is included) and the antibody amount is 0 to 20 n
6 points were used that were diluted to g / mL. As the test human serum, human serum diluted 50 to 100 times with the above-mentioned dilution PBS was used.

After removing the standard solution and the sample solution, each well was washed four times with the above-mentioned washing PBS. Then, diluted PBS for PA (protein A) (0.
1MPBS, 0.1% Tween-20, 10% Bl
HRP (horseradish peroxidase) -labeled PA (Capp) containing 100 to 200 ng / mL diluted with occ Ace and aprotin 500 KIU / mL)
100 μL) was added to each well and left at 25 ° C. for 2 hours (second reaction).

After removing the excess HRP-PA solution and washing three times with the above-mentioned washing PBS, 100 μL of the substrate solution was added to each well and left at 25 ° C. for 20 minutes (coloring reaction). Here, as the substrate solution, 30 mg of OPD
(Orthophenylenediamine, Sigma) 0.01M
Dissolve in 50 mL of PBS (pH 5.0), 30% H ₂
The one to which 25 μL of O ₂ was added was used.

The above color development reaction was carried out using 100 μL / well of 5
After stopping with N-sulfuric acid, an absorbance meter (immunoreader,
It was measured with a Dinatec MR5000) and a calibration curve was determined from the standard. Using this, the amount of anti-insulin antibody in human serum was determined. An example of the calibration curve for measuring the anti-insulin antibody obtained here is shown in FIG.

Example 2 The amount of antibody in the standard (commercially available anti-insulin antibody) used in Example 1 was determined from the Scatchard plot using iodine-labeled insulin ( ¹²⁵ I-insulin) as follows. That is, the anti-human insulin guinea pig serum described above was used as the antibody (Ab), and the antigen (Ag)
As ¹²⁵ I-insulin (9314 cpm / 100
μL / tube 12.6 pg) was used.

Ab100 μL and Ag100 described above
μL of buffer (0.01 M PB, pH 6.8) 300
After overnight reaction at 4 ° C. in μL, bound (Bound) and unbound (Free) were separated (so-called B / F separation). Under the above conditions, the antibody was kept constant and the reactivity when the amount of ¹²⁵ I-insulin added was changed.
asked for ot.

When the Ag concentration (Hot) is [A], the Ab concentration is [Z], and the Ag · Ab complex concentration is [B], the following equation holds with the binding constant K. Representing [F] as Ag (Hot) free that does not combine (AB), the above equation can be rewritten as: K = [B] / [F] ([Z] − [B]) [B] / [F] = − k [B] + K [Z] where [B] / [F] is the Y axis, If [B] is taken as the X-axis, a linear expression having a slope of −K is obtained.

If all of the added antibody [Z] is used for binding, [Z] = [B] and [B] / [F] = 0
Becomes That is, in the above linear expression, when y = 0, that is, the X-axis intercept is the concentration of the added antibody.

A Scatchard plot obtained by such a method is shown in FIG. Here, when y = 0, that is, the X-axis intercept indicates 7.1 × 10 ⁴ cpm / tube,
This can be converted into antibody concentration (however, Ag
And Ab react with 1: 1).

When the concentration of 7.1 × 10 ⁴ cpm is converted, The molecular weight of insulin is 5800, and the molecular weight of antibody IgG is 160000.
Since mL is used, the antibody concentration is That is, the amount of antibody is about 400 μg. Regarding the method of obtaining such an antibody amount, reference literature (Tadamitsu Sudo et al., Method for measuring antibody binding constant-K value and antibody concentration, immunoexperimental operation method, 2691 to 2706 p. (1970 to 1980), edited by Japan Immunological Society) ) Can be referred to.

The EIA (or ELIS of Example 1)
A) In measurement, porcine, bovine and human are used as insulin coated on the microplate.
As shown in FIG. 4 and FIG. 5, the antibody (anti-
Responsiveness to human ginea pigs was almost unchanged in all three.

Example 3 Using the samples (A, B, and C) prepared by diluting an insulin antibody-positive sample (human serum) with a buffer solution at different dilution ratios, the measurement results of the measurement system of Example 1 are shown in FIG. Show.
As is clear from FIG. 6, the linear relationship passing through the origin was obtained for all the measured samples, and the accuracy of the above measurement system was proved. In this measurement system, there is a day difference reproducibility expressed as a systematic error, and a coefficient of variation of 1.9-
It was 8.4%.

Example 4 By the measuring method of Example 1, a healthy person (Normal, 94
Example), antinuclear antibody positive patients (ADNA (+), 25 cases),
Rheumatoid factor positive patients (RAHA (+), 27 cases),
FIG. 7 shows the results of measuring the amount of anti-insulin antibody in each of the specimens of diabetic patients (DM, 198 cases).
In FIG. 7, in DM (diabetic patients), insulin (+) indicates a sample of a patient undergoing insulin treatment, and insulin (−) indicates a sample of a patient who had only diet therapy.

As is clear from FIG. 7, in the measurement results of the assay method of the present invention, healthy subjects, antinuclear antibody-positive patients, and rheumatoid factor-positive patients all showed antibody-negative,
Only diabetic patients were antibody positive. Therefore, the anti-insulin antibody measurement method of the present invention was found to be useful as an index for insulin treatment of diabetic autoantibodies and insulin antibody-positive patients.

Example 5 (Additional Recovery Test ) A standard insulin antibody having different concentrations (5 points each) was added to three types of serum (human serum, samples D, E, and F) The insulin antibody amount (recovery rate) was determined by the measurement system of No. 1. The results are shown in Fig. 8. In this test, the spike recovery was 96.9% on average (average slope = 0.696).

Example 6 (Skim milk and / or Tween
Effect of addition of −20) In the measurement system of Example 1, skim milk (SM) and
The concentration of Tween-20 was variously changed and the fluctuation of the insulin antibody measurement value was examined. As a result, skim milk (S
M) and Tween-20 when Tween-20 was added together
With respect to the concentration of 1, the nonspecific reaction was small at the concentration of 0.1 to 0.5%, and the amount of insulin antibody was measured in the same manner. Considering the "easiness of peeling" of solid-phase insulin, Tween
The concentration of -20 was most preferably 0.1%. on the other hand,
For SM, the non-specific reaction was effectively suppressed in the concentration range of 5 to 10%.

[0064]

As described above, according to the method for measuring insulin antibody of the present invention, a standard sample (standard) derived from an animal other than human and capable of accurately calculating the amount of anti-insulin antibody contained therein. And the enzyme-labeled protein A that specifically binds to the Fc fragment of IgG, the amount of anti-insulin antibody in human body fluid can be measured safely and simply and quantitatively to the extent that it can be a clear indicator of insulin administration. It is possible to measure.

[Brief description of drawings]

FIG. 1 is a diagram showing the amino acid sequence of human insulin.

FIG. 2 is a graph showing an example of a calibration curve for anti-insulin antibody measurement obtained in Example 1.

FIG. 3 is a graph showing the results of Scatchard analysis. The Y-axis shows the amount of specific antibody-label-bound complex / free label, and the X-axis shows the amount of specific antibody-label-bound complex (cpm).

FIG. 4 shows porcine insulin as insulin to be coated on a solid phase in the method for measuring insulin antibody of the present invention.
It is a graph which shows the comparison of the measurement result when using bovine insulin or human insulin.

FIG. 5 shows porcine insulin as insulin to be coated on the solid phase in the method for measuring insulin antibody of the present invention,
It is a bar graph which shows a comparison of the measurement results when using bovine insulin or human insulin.

FIG. 6 is a graph showing the results of a dilution test of human test serum carried out in Example 3.

FIG. 7 is a plot showing the results of measuring anti-insulin antibody in the sera of healthy subjects, antinuclear antibody-positive patients, rheumatoid factor-positive patients, and diabetic patients.

FIG. 8 is a graph showing the results of an addition recovery test of Example 5.

Claims (9)

[Claims] 1. A test sample and a standard sample containing an insulin antibody derived from a non-human animal are reacted with insulin arranged on a solid phase, and then the insulin antibody bound to the insulin on the solid phase and an enzyme-labeled protein. A method for measuring an insulin antibody, which comprises reacting with A. 2. The method for measuring insulin antibody according to claim 1, wherein the non-human animal is a mammal. 3. The method for measuring insulin antibody according to claim 2, wherein the mammal is a guinea pig or a mouse. 4. The method for measuring insulin antibody according to claim 1, wherein the insulin antibody derived from the animal is an insulin antibody obtained by immunizing the animal with insulin from another animal. 5. The method for measuring insulin antibody according to claim 1, wherein the animal-derived insulin antibody is an insulin antibody obtained by immunizing the animal with human insulin. 6. The method for measuring an insulin antibody according to claim 1, wherein the insulin arranged on the solid phase is human insulin. 7. The method for measuring an insulin antibody according to claim 1, wherein the insulin arranged on the solid phase is animal-derived insulin. 8. The method for measuring insulin antibody according to claim 1, wherein a test sample diluted with a skim milk addition buffer is used. 9. The method for measuring an insulin antibody according to claim 1, wherein the enzyme is peroxidase.