JPS6113184B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION When hemolysis occurs in a living body, hemoglobin liberated in plasma is combined with haptoglobin and transported to the liver, where it is metabolized and iron is recycled.

However, in the event of a disease or trauma accompanied by a high degree of hemolysis, plasma haptoglobin is exhausted and excess free hemoglobin circulates in the body as a free substance, with a portion being excreted in the urine. Therefore, measuring free hemoglobin in blood and urine has important clinical significance.

On the other hand, when injecting a haptoglobin preparation into a patient who presents with hemoglobinuria or oliguria due to severe hemolysis, the amount of free memoglobin or hemoglobin-haptoglobin complex in the plasma, or the amount of hemoglobin in the urine is measured. It is important to measure quantities.

Until now, hemoglobin has been measured using absorbance methods such as the cyanmethemoglobin method, benzidine method, and hydrosulfite method.
It was not possible to separate and quantify free hemoglobin and hemoglobin in the hemoglobin-haptoglobin complex, and furthermore, it took time to obtain measurement results.

The present invention consists of a method for simply and quickly performing differential quantification by collecting free hemoglobin in a specimen using immobilized haptoglobin and determining the amount of collected hemoglobin.

The origin of the haptoglobin (hereinafter sometimes referred to as Hp) used in the present invention is not particularly limited, as long as it can be recovered in large quantities, such as Hp from mammals.

The carrier for immobilizing Hp is generally used as a method for insolubilizing active proteins such as enzymes.
Detailed in No. 105186.

In other words, conventionally implemented or proposed methods for insolubilizing enzymes include bonding the enzyme to a polymeric polysaccharide activated with cyanogen bromide, or covalently bonding the enzyme to a silicon material. Physical methods such as activated carbon adsorption (US Pat. No. 2,717,852)
Alternatively, an electrostatic method such as adsorption to a basic anion exchanger (Japanese Patent Publication No. 45-6870), or a method of embedding the enzyme in a suitable matrix such as fibrin polymer (Japanese Patent Publication No. 49-41584) The insolubilized haptoglobin used in the present invention can be produced by the same method except that the purified haptoglobin described above is used instead of the enzyme.

The carrier used to chemically bind and insolubilize haptoglobin is a water-insoluble organic or inorganic polymeric material having a group capable of binding to haptoglobin, such as a carboxyl group or an amino group. For example, polyacrylamide gel [Bio-
Gel (Bio-gel) P 300, etc.: Released by Bio-gel, USA], Agarose (Sepharose 4B, etc.: Released by Pharmacia, Sweden), Dextran [Sephadex G200, DEAE-Sephadex] , QAE-Sephadex (marketed by Pharmacia, Sweden), and cellulose (DEAE
Polysaccharides that do not directly bind to haptoglobin, such as cellulose, cellulose (CM-cellulose, AE-cellulose, etc.), which have been given binding ability by treating them with cyanogen bromide, are advantageously used. A method for treating polysaccharides with cyanogen bromide is described by P. Atrecasas, P. and Anfinsen, CB [Methods in Enzymology, ed. by
The affinity chromatography method proposed by Jakoby, WB) pp 345, (1971) has already been established as a method for (insolubilizing) proteins.

Similarly, polyamides that do not have binding groups, such as high molecular weight polymers such as nylon, polyacetal, and polystyrene, can be treated with a suitable treatment agent such as cyanogen bromide to provide groups capable of binding to haptoglobin. can. Furthermore, inorganic materials such as glass can be treated with aminoalkylsilane to provide amino groups that bind to haptoglobin, and can be used to insolubilize haptoglobin.

Examples of carriers that physically or electrostatically adsorb haptoglobin to insolubilize it include diatomaceous earth, activated carbon, DEAE-Sephadex, DEAE-
Examples include ion exchangers such as cellulose. Further, as a crosslinking agent for insolubilizing haptoglobin, a bifunctional compound such as glutaraldehyde can be used.

The matrix used to trap and insolubilize the enzyme is a gel or fiber of a high molecular weight organic polymer, and these can also be used for haptoglobin by selecting an appropriate means. For example, during monomer polymerization, if haptoglobin is present in the polymerization system, it becomes entrapped in the resulting polymer gel.

The conditions for treating the carrier with a treatment agent that provides a protein-binding group are as follows.

In the case of polysaccharide carriers, cyanogen bromide is usually used;
Do this while cooling at pH 10-12.

A methacrylate polymer having a large network structure, which is a high molecular weight polymer, can also be used in the same manner.
Activation with cyanogen bromide at 12 gives good results.

Activation of the inorganic carrier glass is carried out with organosilanes. The conditions are such that organosilane is dissolved in an inert solvent and reacted at room temperature for a sufficient period of time.

For the polymer-trap method, any carrier capable of adsorbing haptoglobin can be used, but simpler carriers are preferred. For example, DEAE-cellulose can be bound to haptoglobin by thorough washing with distilled water and stirring while maintaining the temperature at room temperature, and this haptoglobin-binding carrier can then be polymerized with a secondary carrier.

The reaction between the carrier having binding ability obtained as described above and haptoglobin occurs at around neutrality (PH6~
8) can be carried out at about 3 to 25°C.

The immobilized Hp thus obtained is set in a suitable device for use as a hemoglobin measurement kit. The device has a cylindrical shape or a flat plate shape, and in the case of a cylindrical shape, a certain amount of immobilized Hp is set in a cylinder made of a suitable material to form a kit. In the case of a flat plate, the immobilized Hp is set on a paper, glass, plastic or other suitable flat plate to a certain thickness.

The particle diameter of the immobilized Hp to be set is 40 to 200μ and is used.

The amount of immobilized Hp set in the apparatus is generally 0.5 to 70 cm ³ in volume in the case of a cylindrical kit, and the length of the cylinder is 2 to 20 cm, preferably 4 to 10 cm, to provide a generally good kit. In the case of a flat plate, the thickness is 0.5 to 5 mm.
0.5~ ^6cm3 is good. As a guideline for the set amount, when a cylindrical device with an inner diameter of 1.5 cm and a length of 10 cm is filled with immobilized Hp, about 5 to 100 mg of hemoglobin will be bound.

The kit consisting of the immobilized Hp set in this manner is placed in the apparatus by determining in advance a calibration curve between the set amount of immobilized Hp and the amount of hemoglobin, and setting the corresponding amount, for example, concentration.

Measurement of hemoglobin amount using a fixed kit
The amount of free hemoglobin in the sample is determined from the calibration curve by measuring the length or volume of the part that is adsorbed to Hp and colored red.

The reaction conditions between the sample and the kit are not particularly limited, but include a low salt concentration (0.05 to 8) with a pH of 5 to 8.
After wetting with an aqueous solution of 0.3M), the sample is added and bonded by development. The time required is approximately 5 minutes to 20 minutes. After sufficient penetration, wash with an aqueous solution of at least twice the volume of the kit. The cleaning liquid is not particularly limited as long as it is an aqueous solution with a low salt concentration of pH 5 to 8.

To regenerate the kit, after the measurement is completed,
~5M guanidine/HCl (PH4~6), 0.05~0.5M
Glycine/HCl (PH2~3), 0.05~0.5M
It is brought into contact with an aqueous solution of Na ₂ HPO ₄ .NaOH (PH 10-11), 1-5 M NaBr or 1-6 M MgCl ₂ (PH 3-5) to break the bond between Hp and hemoglobin and liberate hemoglobin. Thereafter, it can be used again after washing with an aqueous solution of pH 5 to 8 with a low salt concentration.

The kit is stored in a dry state or in a low salt concentration aqueous solution with a pH of 5 to 8 containing a preservative (eg, 0.1% NaN ₃ ). Temperature is low, preferably 10℃
Below are the conditions for not freezing. When stored in an aqueous solution, it can be used for about 2 years without loss of activity.

For measurement, if the sample is serum, plasma, urine, cerebrospinal fluid, etc., it can be used as is without any pretreatment.

The hemoglobin measurement kit thus provided is a reagent that can easily and quickly separate and quantify the amount of free hemoglobin.

Examples are shown below, but the present invention is not limited thereto in any way.

Example 1 Hp obtained by purification from pooled human plasma was fixed on Sepharose 4B using the cyanogen bromide method, and this was fixed on Sepharose 4B with an inner diameter of 1.5 cm.
A syringe with a length of 10 cm was filled with the mixture, and a paper cut into a circle was placed on the top of the filling so that the top surface of the filling would not be disturbed, and a kit was made by moistening it with physiological saline. The free hemoglobin calibration curve of this kit is linear as shown in FIG.

Example 2 The bottom surfaces of two types of transparent plastic tubes, one with an inner diameter of 1.5 cm and a length of 10 cm and an inner diameter of 1 cm and a length of 10 cm, were fixed with a glass filter (600 mesh), and the immobilized Hp prepared in the same manner as in Example 1 was applied to these tubes. After filling each column to a height of 7 cm, a glass filter of the same type was fixed on top of the contents, a small amount of physiological saline was filled on top of the column, and both ends of the column were sealed with rubber caps, 100 each for a total of 400 units. I created one. Of these, 10 devices were selected in which columns with an inner diameter of 1.5 cm were filled with immobilized Hp, and 2 ml of a 1 mg/ml solution of standard hemoglobin dissolved in hemoglobin-free serum was passed through them. The maximum length of the red colored part is 1.85 cm, the minimum length is 1.70 cm, and the average length is 1.75 cm.
It was cm.

Example 3 Porous glass (pore diameter approximately 80μ, glass diameter 100μ)
After activating Hp with gamma-aminopropyl-triethoxysilane, Hp obtained by separately purifying horse plasma as a raw material was immobilized on this porous glass. A device was prepared by filling a plastic tube with this in the same manner as in Example 2. When 10 of these were selected and 2 ml of a 1 mg/ml solution of standard hemoglobin dissolved in hemoglobin-free serum was passed through them, the length of the red colored part was 2.63 cm at the highest, 2.47 cm at the lowest, and 2.56 cm on average. It was hot.

Example 4 Hp immobilized on Sepharose by the cyanogen bromide method was uniformly set on a slide graph plate (3 x 3 cm) to a thickness of 1 mm to prepare a hemoglobin measurement kit.

Example 5 2 mm of Hp immobilized on Cepharose by the cyanogen bromide method was placed on a filter paper (2 x 10 cm).
A kit for hemoglobin measurement was prepared.

[Brief explanation of the drawing]

FIG. 1 shows a calibration curve for measuring free hemoglobin using the method and kit of the present invention, obtained in Example 1.

Claims (1)

[Claims] 1. A reagent for measuring free hemoglobin comprising immobilized haptoglobin. 2. The reagent for measuring free hemoglobin according to claim 1, which comprises immobilized haptoglobin set in a cylindrical device. 3. The reagent for measuring free hemoglobin according to claim 1, which comprises immobilized haptoglobin set in a flat plate shape.