JPS6114466B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION Hemoglobin has so far been measured using absorbance methods such as the cyanmethemoglobin method, benzidine method, and hydrosulfite method.
The measurement method was complicated and it took time to obtain measurement results.

The present invention consists of a method for simply and quickly quantifying free hemoglobin and hemoglobin-haptoglobin conjugates in a specimen using an immobilized anti-hemoglobin antibody and determining the amount of collected hemoglobin. Further, according to the present invention,
It is also possible to quantify total hemoglobin in the blood.

Anti-hemoglobin antibody (hereinafter referred to as anti-hemoglobin antibody) used in the present invention
Hb) is not particularly limited in its origin, and is used as a method for purifying antibodies from anti-human hemoglobin serum obtained by administering and immunizing mammals with human hemoglobin (Immunochemistry: Asakura Shoten). It can be provided by conventionally known methods such as salt folding method, ethanol fractionation method, and chromatography method.

The carrier for immobilizing anti-Hb is generally known 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 immobilized anti-Hb used in the present invention can be produced by the same method except that the purified anti-Hb described above is used instead of the enzyme.

The carrier used to chemically bind and insolubilize anti-Hb is a water-insoluble organic or inorganic polymeric material having a group capable of binding to anti-Hb, such as a carboxyl group or an amino group. For example, polyacrylamide gel [Bio-gel]
P-300, sold by Biogel, USA], agarose (Sepharose 4B, etc., sold by Pharmacia, Sweden), dextran (Sephadex) G200, DEAE-Sephadex, QAE-Sephadex (QAE) -Sephadex) etc.: released by Pharmacia, Sweden], and cellulose (DEAE-cellulose, CM-cellulose,
Materials that are polysaccharides that do not directly bind to anti-Hb, such as AE-cellulose, and 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. Cuatrecasas, P. and Anfinsen, CB [Methods in Enzymology.
The affinity chromatography method proposed by Jakoby, W.B., pp. 345, (1971) has already been established as a method for separating 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 with anti-Hb binding ability. I can do it. Furthermore, inorganic materials such as glass can be treated with aminoalkylsilane to provide amino groups that bind to anti-Hb, and can be used to insolubilize anti-Hb.

Examples of carriers that physically or electrostatically adsorb anti-Hb to make it insolubil include diatomaceous earth, activated carbon, and ion exchangers such as DEAE-Sephadex and DEAE-cellulose. Again resistance
As a crosslinking agent that insolubilizes Hb, a bifunctional compound such as glutaraldehyde can be used.

The matrix used to confine and insolubilize the enzyme is a high molecular weight organic polymer gel or fiber, and these can also be used for anti-Hb by selecting appropriate means. For example, during monomer polymerization, if anti-Hb is present in the polymerization system, it will become 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.

Although any carrier capable of adsorbing anti-Hb can be used for the polymer-trap method, simpler carriers are preferred. For example, DEAE-cellulose is combined with anti-Hb by thoroughly washing with distilled water and stirring while keeping the temperature at room temperature.
The Hb-binding carrier can then be polymerized with a secondary carrier.

The reaction between the carrier having binding ability obtained as described above and anti-Hb can be carried out at about 3 to 25°C near neutrality (PH 6 to 8).

The immobilized anti-Hb thus obtained is placed in a suitable device for use as a hemoglobin measurement kit. The device has a cylindrical or flat plate shape, and if it is cylindrical, a kit is formed by setting a certain amount of immobilized anti-Hb in the cylinder made of a suitable material. In the case of a flat plate, the immobilized anti-Hb is set on a paper, glass, plastic or other suitable flat plate with a certain thickness.

The particle size of the immobilized anti-Hb to be set is 40 to 200μ
The homogenized product is used.

The amount of immobilized anti-Hb set in the device is generally 0.5 to 70 cm ³ in volume in the case of a cylinder, and the length of the cylinder is 2 to 20 cm, preferably 4 to 10 cm, to generally provide a good kit. In the case of a flat plate, the thickness is preferably 0.5 to 5 mm and the volume is 0.5 to 6 cm ³ . 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 anti-Hb, a converted amount of hemoglobin of about 5 to 100 mg is bound.

For the kit made of the immobilized anti-Hb set in this manner, a calibration curve between the set amount of the immobilized anti-Hb and the equivalent amount of hemoglobin is determined in advance, and a corresponding amount, for example, at a concentration, is placed in the apparatus.

To measure the equivalent amount of hemoglobin using a kit, free hemoglobin and hemoglobin-haptoglobin conjugate (or total blood hemoglobin) are adsorbed onto immobilized anti-Hb, and the length or area of the red colored part is measured. Determine the converted hemoglobin amount in the sample from the calibration curve.

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 a low-temperature concentrated aqueous solution with a pH of 5 to 8.

To regenerate the kit, recycle the kit after measurement is completed.
~4M guanidine HCl (PH4~6), 0.05~0.5M
Glycine/HCl (PH2~3), 0.05~0.5M
Contact with an aqueous solution of Na ₂ HPO ₄・NaOH (PH 10-11), 1-5 M NaBr or 2-8 M MgCl ₂ (PH 3-5) to release the bond between the immobilized anti-Hb and hemoglobin or hemoglobin-haptoglobin conjugate. release. Thereafter, it can be used again after washing with an aqueous solution of pH 5 to 8 with a low salt concentration.

Kits are stored in a dry state or in a low salt aqueous solution containing a preservative (for example, 0.1% NaN ₃ ) with a pH of 5 to 8. Temperature is low salt 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. In the case of blood, dilute it 10 to 100 times with distilled water and use it after sufficient hemolysis.

The hemoglobin measurement kit thus provided is a reagent that can easily and quickly quantify the amount of free hemoglobin, the amount of hemoglobin-haptoglobin conjugate, and the amount of total hemoglobin in blood.

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

Example 1 Anti-Hb obtained by purifying anti-human hemoglobin serum obtained by immunizing rabbits with human hemoglobin by Cohn's ethanol fractionation method (J.Am.Chem.Soc., 72 465 (1950)) was bromated. Cephalose 4B by cyan method
(Sepharose4B) and fix this to a length of 1.5 cm in inner diameter.
A 10 cm syringe was filled with the mixture, and a paper cut into a circle was placed on top of the filling so as not to disturb the top surface of the filling, and then moistened with physiological saline to prepare a kit. The calibration curve of the converted hemoglobin amount of this kit is linear as shown in FIG. Note that there is no difference between the calibration curves for hemoglobin and the hemoglobin-haptoglobin conjugate.

Example 2 The bottoms 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 immobilized tubes prepared in the same manner as in Example 1 were attached to these. Hb
After each column was filled 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 filter, and both the top and bottom ends of the column were sealed with rubber caps.
Created 400 pieces. Of these, 10 devices were selected in which columns with an inner diameter of 1.5 cm were filled with immobilized anti-Hb, 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 was 1.80 cm, the minimum length was 1.65 cm, and the average length was 1.70 cm.

Example 3 Porous glass (pore diameter approximately 80μ, glass diameter 100μ)
After activating the Hb mesh with γ-aminopropyl-triethoxysilane, anti-Hb obtained by purifying it in the same manner as in Example 1 was immobilized on the porous glass.

In the same manner as in Example 2, a device was prepared in which a plastic tube was filled with this. 10 of these
A standard hemoglobin-haptoglobin conjugate was dissolved in hemoglobin-free serum.
When 2 ml of mg/ml solution was passed through, the maximum length of the red colored part was 2.68 cm, the minimum length was 2.51 cm, and the average length was
It was 2.54cm.

Example 4 Ten of the devices obtained in Example 3 were selected, and when 1 ml of normal human blood diluted 50 times with distilled water and sufficiently hemolysed was passed through the blood, the length of the red colored part was determined. The maximum height is 3.96cm, the minimum is 3.68cm, and the average is 3.79cm.
It was cm.

Example 5 Anti-Hb immobilized on Cepharose by the cyanogen bromide method on a glass slide plate (3 x 3 cm)
were uniformly set to a thickness of 1 mm to make a hemoglobin measurement kit.

Example 6 Anti-Hb immobilized on Cepharose by the cyanogen bromide method was placed on a filter paper (2 x 10 cm).
A kit for measuring hemoglobin was prepared by uniformly setting a thickness of mm.

[Brief explanation of the drawing]

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

Claims (1)

[Scope of Claims] 1. A hemoglobin measurement reagent comprising an immobilized anti-hemoglobin antibody for quantifying free hemoglobin, hemoglobin-haptoglobin complex, or total blood hemoglobin. 2. The hemoglobin measurement reagent according to claim 1, which comprises an immobilized anti-hemoglobin antibody set in a cylindrical device. 3. The hemoglobin measurement reagent according to claim 1, which comprises an immobilized anti-hemoglobin antibody set in a plate shape.