JPH08313530A - Method for measuring total hemoglobin and measuring kit - Google Patents

Abstract

PURPOSE: To measure the total (T-) hemoglobin (Hb) of a specimen by enzyme immunoassay using a standard Hb by forming an Hb-Hp composite by adding an Hb-removed human serum or human haptoglobin (Hp) to the specimen and causing the specimen to react to a free (F-1) Hb contained in the human serum or Hp. CONSTITUTION: An Hb-Hp composite is formed by adding an Hb-removed human serum or Hp to an F-Hb contained in a specimen, such as the blood serum, blood plasma, urine. After an anti-Hb antibody is caused to react to an immobilized solid phase and the solid phase is cleaned, an enzyme-labeled anti-Hb antibody is caused to react to the solid phase. Then an excessive amount of enzyme-labeled anti-Hb antibody is washed away and the the antibody is caused to bring out color by adding an enzyme substrate and color former to the antibody. Thereafter, a reaction stopper is added to the antibody and the coloring intensity is measured with a colorimeter. A standard Hb-Hp composite solution is also measured as a reference and total hemoglobin of the specimen is directly determined from a previously drawn standard curve.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a total hemoglobin measuring method and a total hemoglobin measuring kit, and more specifically to a total hemoglobin that can be easily, quickly and accurately measured by an immunological method used in the field of clinical examination. The present invention relates to a hemoglobin measurement method and a total hemoglobin measurement kit.

[0002]

BACKGROUND OF THE INVENTION Problems to be Solved by the Invention Extracorporeal circulation, burns, incompatible blood transfusions, hemolytic diseases, etc. may cause a large amount of hemolysis, and may cause complications such as renal damage associated with hemolysis. There are many.

Therefore, it is clinically important to measure the total amount of hemoglobin in plasma or serum. Usually, plasma or serum total hemoglobin (hereinafter T-Hb)
Chemical methods such as the cyanmethemoglobin method, the oxyhemoglobin method, the benzidine method, and the modified benzidine method are known for the quantification of.

However, in the cyanmethemoglobin method, T-Hb in blood is added to cyanmethemoglobin by adding potassium ferricyanide and potassium cyanide to give 540
Although the measurement is carried out at a wavelength of nm, the use of the highly poisonous potassium cyanide entails dangerous operations and low measurement sensitivity.

In the oxyhemoglobin method, a sample is diluted with water and converted into oxyhemoglobin, and then the measurement is carried out at a wavelength of 540 nm. When the pH is increased, it is changed to methemoglobin, or when it becomes near neutral, cloudiness occurs. The measurement is lacking in accuracy because of the drawback that it occurs. The benzidine method and the modified benzidine method utilize the peroxidase activity of hemoglobin, and hydrogen peroxide oxidizes benzidine to develop a color. The degree of this color development is quantified, but only Hb is used in this method. However, since methemoglobin and methhemalbumin are also quantified together, and since benzidine itself has carcinogenicity, 3,3′-5,5′-tetramethylbenzidine is used, Condition, accuracy,
There is a problem in sensitivity and specificity, and a more accurate measurement method has been desired.

From the above, the present inventors have long studied the T-Hb measurement by the enzyme immunoassay (ELISA) which is safe in operation and has high specificity and sensitivity. In this measurement method, Hb in plasma or serum is present as a hemoglobin-haptoglobin complex (hereinafter Hb-Hp) and free Hb (hereinafter F-Hb), and therefore, ELI
We faced a serious problem that accurate quantification was not possible due to different reactivity to anti-Hb antibody used in SA method.
This results in inaccurate measurement particularly in a sample of a patient with hypohaptoglobinemia or a sample of a patient with significant hemolysis. (See comparative example)

Therefore, as a result of further diligent research, the present inventors have found that F-Hb in a sample can be obtained by previously adding hemoglobin-removed human serum or human haptoglobin (Hp). Is all Hb
-Hp complex, focusing on trace amounts of T-Hb present in plasma, serum, and urine.
We have found a method to measure accurately, simply and rapidly in the form of Hp complex, and have completed the "total hemoglobin measurement method and kit".

[0008]

The method for measuring T-Hb of the present invention is to measure F-Hb in a sample (eg, serum, plasma, urine, etc.) with Hp.
After reacting with Hb-Hp to form a complex, Hb-Hp complex is formed.
-Hp complex using anti-Hb antibody-immobilized solid phase ELIS
It is characterized in that the measurement is carried out by method A.
In addition, the T-Hb measurement kit of the present invention is a measurement kit used for the measurement of the present method described above, in which hemoglobin that reacts with free Hb present in a sample is removed, or a reagent composed of Hp is used. It means a measurement kit characterized by including.

More specifically, hemoglobin-free serum or Hp is added to F-Hb in a sample to form an Hb-Hp complex, which is then reacted with an anti-Hb antibody-immobilized solid phase.
Further, after washing the solid phase, the enzyme-labeled anti-Hb antibody is reacted to remove excess enzyme-labeled anti-Hb antibody by washing, and then an enzyme substrate and a coloring agent are added to develop color.

After adding the reaction terminator, the color development intensity is measured with a colorimeter. As a control, the solution of the standard Hb-Hp complex is also measured in the same manner and directly quantified from the standard curve prepared in advance.

The hemoglobin-depleted human serum used in the present invention is a hemoglobin-haptoglobin complex or free hemoglobin present in human serum.
For example, it is human serum containing free Hp that has been removed beforehand by affinity chromatography using an anti-human Hb antibody. On the other hand, haptoglobin is not particularly limited, but the present measurement is possible as long as it is a purified product of Hp that is commercially available as a purified product from Sigma.

As the anti-Hb antibody, either a polyclonal antibody or a monoclonal antibody can be used. Usually, the polyclonal antibody is commercially available from Institute of Medical Biology, and the monoclonal antibody is commercially available from MEDIX BIOTECH Co., Ltd. and the like, which are easily available. The monoclonal antibody can also be easily obtained by a usual method of cell fusion of mouse splenocytes immunized with human Hb and mouse bone marrow cells. Furthermore, in order to increase the purity of the antibody, Hb-
Affinity chromatography with Sepharose can be appropriately used.

Next, although there are various methods for measuring T-Hb in the present invention, the sandwich method of the generally used ELISA method is preferable. As the solid phase material for immobilizing the anti-Hb antibody, any of the solid phase materials conventionally used in the ELISA method and the like can be used, but in the measurement of a large number of samples, for example, a microplate,
Magnetic beads and plastic beads are preferable. In addition, there are various enzymes as enzyme-labeled substances that can be used in the present invention. Among them, peroxidase,
Alkaline phosphatase, β-galactosidase and the like are particularly preferable.

There are several methods for labeling an antibody with an enzyme, but the most important conditions imposed on each are 1) no reduction in enzyme and antibody activity. 2) The bond is stable and can be stored for a long time. 3) The procedure is as simple as possible and the labeled antibody can be easily obtained. The enzyme labeling methods that have been developed to satisfy these conditions can be roughly classified into two.
One of them is a method of chemically linking an enzyme and an antibody, which is the glutaraldehyde method (S. Avrameas,
Immunochemistry , 6 , 43, 196
9) and the periodate oxidation method (P.K. Nakane, A .;
Kawaoi, J .; Histochem . Cytoch
em . , 22 , 1084, 1974) correspond to this. The other is a method of binding an enzyme and a first antibody using an antibody or the like as an intermediary. In this method, the avidin-biotin method (SM Hsu, L. Raine, H. Franc) is used.
ger, Am . J. Clin . Pathol . , 75 ,
734-738, 1981) is known.

In the present invention, any labeling method can be used for measurement. As one aspect of preparation of the labeled antibody used in the present invention, a method of obtaining an antibody labeled with peroxidase is known. Specifically, all free amino groups of peroxidase are blocked with 1-fluoro-2,4-dinitrobenzene (FDNB), and then the adjacent hydroxyl group of the dinitrophenylated peroxidase sugar moiety is cleaved with periodic acid to form an aldehyde group. Reborn. Unreacted periodate is decomposed by adding ethylene glycol, and the reaction is stopped.

Next, 0.01M sodium carbonate buffer (p
After dialysis with H9.5), the newly formed aldehyde group is reacted with the amino group of the antibody to form a Schiff base.
The Schiff base reaction product is then reduced with sodium borohydride to chemically stabilize it. After reduction, excess sodium borohydride is removed by dialysis to obtain the desired enzyme-labeled antibody. Further, it is preferable to purify the obtained labeled antibody by gel filtration chromatography before use.

As the color-developing substrate and color-developing agent, hydrogen peroxide and 3,3'-5, in the case of peroxidase, are used.
5'-Tetramethylbenzidine or 3,3'-diaminobenzidine is used, while in the case of alkaline phosphatase the bromochloroindole phosphate nitro-tetrazolium drug substance is used. Further, the reaction conditions of the color former may be the same as those of the usual enzyme reaction, and the temperature is not particularly specified, but it is most preferably carried out near room temperature.

An Hb-Hp complex is used as the Hb standard product, and this complex can be easily prepared by adding an excess of the Hp purified product (manufactured by Sigma) to the Hb purified product and reacting for several hours. . Further, instead of the Hp purified product, Hb-depleted normal human serum obtained by previously treating normal human serum with an anti-human Hb antibody-affinity column was used.
An Hb standard product (Hb-Hp complex) can also be prepared by adding a purified product (manufactured by Sigma). Comparative examples are shown below, but this comparative example shows the reason that accurate quantification cannot be obtained by the usual assay method because the reactivity of human Hb-Hp complex and F-Hb to anti-human Hb antibody is different. It is a thing.

[Comparative Example] Human Hb-human Hp complex and F-Hb anti-human Hb antibody
On measurement due to difference in reactivity to <human Hb-human Hp complex and human Hb, human Hp
Preparation of Solution >> twice-crystallized human Hb (manufactured by Sigma) 160
0.1% bovine serum albumin (hereinafter BSA) -phosphate buffered saline (hereinafter PBS) so as to be ng / ml
Diluted with. Next, Hp (manufactured by Sigma) is 800 ng / m
The mixture was diluted with 0.1% BSA-PBS to 1 l, and 1 ml of each was mixed in equal amounts and incubated at 37 ° C. for 1 hour, and then a human Hb-human Hp complex was prepared. on the other hand,
To examine the reactivity of human Hb and human Hp, double-crystallized human Hb was adjusted to a concentration of 80 ng / ml, and Hp was adjusted to a concentration of 400 ng / ml with 0.1% BSA-P.
Prepared by adding each BS.

<< Preparation of anti-human Hb antibody-immobilized microplate >> Polyclonal antibody (No. 703, 307) obtained by immunizing rabbits in house, Monoclonal antibody (No. 6) prepared in house and Japan 5 of Biotest's monoclonal antibody (SU110, SU112)
The seed was used as the test subject. 2 μg in PBS beforehand
200 μl of each antibody diluted to 100 μl / ml was added to each well of a 96-well microplate (manufactured by Coaster) and reacted at 4 ° C. overnight. After the reaction, the liquid in each well was removed, 250 μl of PBS was added to each well, and after stirring, the liquid was removed. After repeating this operation 3 times, 1% BSA-PBS was added as a masking reagent and incubated at 37 ° C. for 1 hour. After masking, the solution in each well was removed, and the stock solution (0.1% BS containing 0.1% NaN ₃ was added).
200 μl was added to each well.

<< Human Hb-Human Hp Complex and Human H
b measurement> For the measurement of human Hb, human Hb-human Hp complex and human Hp, the ELISA method (sandwich method) was used. That is, after removing the stock solution of the anti-Hb antibody-immobilized-96-well microplate prepared above, 200 μl of the sample was added to each well, and standard Hb (manufactured by Sigma) 0 to
200 μl of 160 ng / ml was similarly added to each well. After stirring for 1 minute with a microplate mixer, 37
The reaction was carried out at ℃ for 1 hour. After removing the liquid in each well, wash solution (0.1% BSA-PBS) is added to each well at 250 μm.
The solution was removed by adding 1 l each and stirring. This operation was repeated 3 times, 200 μl of peroxidase-labeled anti-Hb antibody solution was added to each well, and the mixture was similarly reacted at 37 ° C. for 1 hour. After the reaction, the liquid was removed, and the same washing operation as above was performed. Next, a substrate solution (hydrogen peroxide + o -phenylenediamine) was added and reacted at room temperature for 15 minutes. The reaction was stopped with 50 μl of 4N sulfuric acid and measured with a colorimeter for microplate (492 nm / 630 nm, Sjeia Auto Reader Sanko Junyaku Co., Ltd.). The human Hb value was calculated from a standard curve prepared in advance using standard Hb (manufactured by Sigma).

<< Human Hb-Human Hp Complex, Human F-H
b and reactivity with various anti-Hb antibodies against human Hp >>
Table 1 shows the Hb value for human Hb-human Hp complex, human F-Hb and human Hp using each anti-Hb antibody and the relative activity when each F-Hb value is 100% in parentheses. It was The reactivity of F-Hb against each anti-human Hb antibody was in the range of 78 to 84 ng / ml, which was close to the theoretical value of 80 ng / ml by using any antibody. On the other hand, in the human Hb-human Hp complex, 37
Measurement values were obtained in the range of ˜229 ng / ml (46 to 286%), and it was found that the sensitivity varies depending on the type of antibody. However, Hp did not react with any of the antibodies.

As shown in Table 1 above, it was found that the reactivity with human F-Hb and human Hb-human Hp complex differs depending on the type of anti-Hb antibody. Therefore, when measuring total hemoglobin, Preliminary addition of hemoglobin-depleted human serum or Hp to convert all Hb to Hb-Hp complex as described in the following examples enabled accurate quantification. One mode of the embodiment will be shown below, but the invention is not limited to the embodiment.

[0024]

[Table 1]

[Example 1] Quantitative properties of Hb solution treated with Hp << Preparation of normal human serum for Hb removal >> 10 g of CNBr-activated Sepharose 4B (Pharmacia) was swollen with 1 mM hydrochloric acid water, and then 0.5 M chloride was added. 0.1 including sodium
200 ml of M sodium hydrogen carbonate buffer (pH 8.3)
After equilibration with, 30 mg of purified goat anti-Hb antibody was added. After stirring at room temperature for 2 hours, 500 ml of the above buffer solution was washed, and 100 ml of the above buffer solution containing 1% BSA was used to mask the remaining active groups. After stirring at room temperature for 2 hours, it was washed with the above buffer solution to prepare anti-Hb antibody-Sepharose 4B. Next, 30 ml of anti-Hb antibody-Sepharose 4B was centrifuged at 2,000 rpm for 15 minutes, 15 ml of non-hemolyzed normal human serum was added to the precipitate, and the mixture was shaken at room temperature for 3 hours for adsorption. After adsorption, centrifugation was performed at 2,000 rpm for 15 minutes to obtain a supernatant. Furthermore, 3,000 rp
Centrifugation was performed again for 20 minutes to obtain a supernatant, and Hb-free normal human serum was prepared. For confirmation, the previously prepared Hb-depleted normal human serum was subjected to ELI using anti-human Hb antibody.
Hb was not detected at all when measured by the SA method.

<< Preparation of standard Hb-Hp complex >> Double-crystal human Hb (manufactured by Sigma) was measured by the cyanmethemoglobin method, and it was prepared with distilled water so as to have a concentration of 10 mg / ml. This solution was further adjusted to 320 ng / ml with a volume of 0.
Dilution was performed with 1% BSA-PBS. On the other hand, normal human serum from which Hb had been removed was diluted 10 ³ times with PBS. Human Hb diluted solution prepared above (320 ng / m
l) 50 ml of Hb-removed normal human serum diluted solution (Hp1.
50 μl (corresponding to 4 μg / ml) was added and reacted at 37 ° C. for 1 hour. This reaction solution was dispensed in 1 ml portions into 15 ml vials and freeze-dried to obtain a standard Hb-Hp complex.

<< Preparation of Two Kinds of Hp-treated Diluting Solution >> The human Hb diluting solution (320 ng / ml) prepared above was added to 0.1
% BSA-PBS for additional 160 ng / ml,
It was diluted to 80 ng / ml and used as solutions A, B, and C, respectively. A solution (1.4 μg / ml) prepared from commercially available purified Hp (manufactured by Sigma) was added to 0.5 ml of each Hb solution, and a Hb-removed normal human serum diluted solution (Hp: 1.4 μg / ml) was separately prepared. 0.5 ml each was added to each Hb solution to prepare two types of Hp-treated diluted solutions.

<< Preparation of Hb measurement method and total hemoglobin standard curve >> The Hb measurement method was carried out according to the comparative example, using the monoclonal antibody 6 of Table 1 as the immobilized antibody and the standard Hb-prepared previously as the standard product. The Hp complex was used. Further, the quantitative properties of the two types of Hp-treated diluted solutions were compared with theoretical values. The Hb standard curve shows the standard Hb-Hp complex as Hb.
The measurement was carried out at 6 stages of 5 to 160 ng / ml as equivalent amounts, and the results were plotted by plotting the abscissa as the Hb equivalent of the standard Hb-Hp complex and the ordinate as the absorbance value of each concentration. (See Figure 3)

<< Purification Hp Treatment and Quantitativeness with Hb-Depleted Normal Human Serum >> Table 2 shows the measurement results of the purified Hp (commercially available product) and Hb-depleted normal human serum treated with each Hb solution. The quantitative value as the Hb concentration for both of the two types of Hp-treated diluted solutions was within the range of 95 to 103% of the theoretical value, and highly accurate quantitativeness was recognized.

[0030]

[Table 2]

[0031]

Example 2 Serum Total Hemoglobulin in Patients with Hypohaptoglobinemia
Quantitative analysis of bottle << Dilution method >> 1 ml of Hp low hemolytic serum (specimen No. 77) from a hypohaptoglobinemia patient was supplemented with 0.1% BSA-PB.
It was diluted 10 ³ times with S to obtain a diluted sample. Next, an equal volume of the Hb-removed normal human serum diluted solution (Hp: 1.4 μg / ml) described in Example 1 was mixed with this diluted solution and reacted at 37 ° C. for 1 hour, and this reaction solution was used as Hp-treated hemolyzed serum. did.
0.1% BSA-PB was added to the hemolyzed serum of this patient (without Hp treatment) and the Hp-treated hemolyzed serum of this patient, respectively.
S was diluted with 2, 4, 8 and 16-fold and used for the experiment.

<< Hb measuring method >> The total Hb was measured using the monoclonal antibody 6 shown in Table 1 as the immobilized antibody in the diluted solution diluted in each of the above steps according to the comparative example. The standard Hb-Hp complex prepared in Example 1 was used as the standard product.

<Quantitativeness> The horizontal axis represents the dilution ratio and the vertical axis represents the Hb value. Hb measurement values of hemolyzed serum (Hp-untreated) and Hp-treated hemolyzed serum and standard Hb as a control were obtained.
The results of plotting the Hb measurement values of the -Hp complex are shown in Fig. 1. All three cases show linearity, but hemolytic serum (untreated)
Is significantly different from the standard Hb-Hp complex in the slope of the line. On the other hand, since the Hp-treated hemolyzed serum has the same slope as that of the standard Hb-Hp complex, this method enables highly accurate quantification.

[0034]

Example 3 Sephacryl S-200HR column chromatography
Of Hb-Hp complex formation of Hp-treated sample by Ruffy
Confirmation << Method for preparing Hb-Hp complex of Hp-treated sample >> Example 2
1 ml of Hp-removed normal human serum (Hp: 1.4 mg / ml) was added to 1 ml of Hp low hemolytic serum (sample No. 77) used for
1 was added thereto and reacted at 37 ° C. for 1 hour to prepare a Hp-treated sample. As a control, Hp low-value hemolyzed serum (Sample No. 77) 1
1 ml of 0.1% BSA-PBS was added to ml, and the same operation was performed to obtain an untreated sample.

<< Confirmation of Hb-Hp complex >> 1 ml of the Hp-treated sample was previously equilibrated with 0.1 M phosphate buffer (pH 7.2) containing 0.15 M sodium chloride.
Phacryl S-200HR column (φ1.8 cm
(× 69 cm, manufactured by Pharmacia) and gel filtration was performed. ELIS for each elution fraction according to Example 1
Human Hb content was measured using Method A. As shown in FIG. 2, the Hp-treated sample was detected as a single peak in the Hb-Hp complex fraction. On the other hand, the control untreated sample is Hb-H.
It was detected as two peaks of the p complex fraction and the Hb fraction. From the above, it was found that F-Hb in the sample was formed as an Hb-Hp complex by the Hp treatment. One embodiment of the total human hemoglobin quantification kit is shown below, but the present invention is not limited to these examples.

[0036]

Example 4 Construction of Total Human Hemoglobin Assay Kit The configuration of the kit comprises the components shown in (1) to (11) below. (1) Human Hp (Hb-removed human normal serum, lyophilized product) (2) Anti-human Hb antibody-immobilized plate (anti-human Hb antibody-binding microplate, sodium azide-containing phosphate buffered saline) (3) Buffer solution (phosphate buffered saline containing 0.1% bovine serum albumin) (4) Enzyme-labeled antibody (peroxidase-labeled anti-human Hb
Antibody, lyophilized product) (5) Enzyme-labeled antibody solution (0.1% bovine serum albumin-containing phosphate buffered saline) (6) OPD (containing 16 mg of o-phenylenediamine,
(Phosphate buffer, lyophilized product) (7) OPD solution (purified water) (8) Standard human Hb-Hp complex (phosphate saline,
Lyophilized product (9) Hydrogen peroxide solution (0.6% hydrogen peroxide solution) (10) Washing stock solution (5-fold concentration solution) (0.5% bovine serum albumin-containing phosphate buffered saline) (11) ) Reaction stop solution (4N sulfuric acid)

B. The measurement method was implemented by the following operating procedures (1) to (13). (1) Human Hp was dissolved in a buffer solution. (2) Test serum (or plasma) was diluted 10 ³ with a buffer solution. (3) Mix 0.5 ml each of human Hp solution and diluted serum,
The reaction was carried out at 37 ° C for 1 hour. (4) Dilute the standard human Hb-Hp complex with a buffer solution and
A standard solution of ˜200 ng / ml was prepared. (5) Human Hp is added to each well of the anti-human Hb-immobilized plate
Solution of treated serum and standard human Hb-Hp complex 200
μl was added. (6) The mixture was stirred for 1 minute with a microplate mixer, covered, and left at 37 ° C. for 1 hour.

(7) After incubation, the plate was turned upside down and the solution remaining in the wells was discarded at once. About 200 μl of the washing solution was added to all wells, stirred for 30 seconds with a microplate mixer, and then discarded at once. This washing was repeated 3 times, and finally the plate was tapped on a paper towel to completely remove the washing solution from the wells. (8) Except for the blank well, 200 μl of the enzyme-labeled antibody solution was rapidly added to each well in the same order, and the mixture was stirred for 30 seconds with a microplate mixer, and left still at 37 ° C. for 1 hour. (9) Washing was performed in the same manner as (7). (10) Add 200 μl of substrate solution (hydrogen peroxide + o-phenylenediamine) to all wells in the same order and at the same time intervals, stir for 30 seconds with a microplate mixer, and keep at room temperature (15-25 ° C.) for 15 minutes while protected from light. did. (11) 50 μl of the reaction stop solution was added to all wells in the same order and at the same time intervals. (12) A blank well was used as a control and measured with a microplate colorimeter (492 nm / 630 nm). (13) A standard curve (Fig. 3) was prepared from the absorption value of the solution of the standard human Hb-Hp complex, and the total human Hb was calculated.

[Brief description of drawings]

FIG. 1 Standard curve of standard Hb-Hp complex, Hp-treated hemolyzed serum and hemolyzed serum (no Hp treatment).

FIG. 2 Confirmation of Hb-Hp complex formation in Hp-treated sample by Sephacryl S-200 HR column chromatography.

FIG. 3: Total hemoglobin standard curve

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Takeshi Maeda 1-5-3 Nihombashi Muromachi, Chuo-ku, Tokyo Wakamoto Pharmaceutical Co., Ltd.

Claims (3)

[Claims] 1. A human hemoglobin-depleted human serum or human haptoglobin is added to a sample, reacted with free hemoglobin present in the sample to form a human hemoglobin-human haptoglobin complex, and then enzyme immunization using a hemoglobin standard product. A method for measuring total hemoglobin, which comprises measuring by a measuring method. 2. The method according to claim 1, wherein the standard hemoglobin standard used for the measurement of total hemoglobin is a standard human hemoglobin-human haptoglobin complex. 3. A kit for measuring total hemoglobin using the measuring method according to claim 1, which comprises a hemoglobin-depleted human serum that reacts with free hemoglobin present in a sample, or a reagent consisting of human haptoglobin. Characteristic measurement kit.