JPS6113185B2 - - 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 exhibits hemoglobinuria or oliguria due to severe hemolysis, the amount of free hemoglobin or hemoglobin-haptoglobin complex in the plasma or the amount of hemoglobin in the urine is It is important to measure quantities.

Hemoglobin has been measured by absorbance methods such as the cyanmethemoglobin method, benzidine method, and hydrosulfite method, but it has not been possible to separate and quantify free hemoglobin and hemoglobin-haptoglobin complex hemoglobin. , it took some time to obtain the above results.

An object of the present invention is to provide a method and a quantitative measurement kit for rapidly and accurately quantitatively measuring not only free hemoglobin but also hemoglobin in the form of a complex with haptoglobin using simple means.

According to the present invention, (1) A hemoglobin-containing specimen is first brought into contact with immobilized haptoglobin to collect free hemoglobin in the specimen as a complex with haptoglobin, and then brought into contact with an immobilized anti-hemoglobin antibody to collect free hemoglobin in the specimen. A method for quantifying hemoglobin in a hemoglobin-containing sample, characterized by collecting a haptoglobin-hemoglobin complex as a complex with an anti-hemoglobin antibody, and (2) an upper immobilized haptoglobin layer and a lower immobilized anti-hemoglobin antibody layer. Provided is a kit for quantitatively measuring hemoglobin in a specimen, comprising free hemoglobin and hemoglobin present in the form of a complex with haptoglobin.

The origin of the haptoglobin (hereinafter also 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.

Anti-hemoglobin antibodies (hereinafter referred to as anti-Hb) can be purified from anti-human hemoglobin serum obtained by administering and immunizing mammals with human hemoglobin. Known methods include salting out, ethanol fractionation, and chromatography. provided by the method.

The method of immobilizing Hp and anti-Hb on a carrier to make them insoluble is generally known as a method of insolubilizing active proteins such as enzymes, and is described in detail in JP-A-51-105186.

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) In the production of immobilized Hp and immobilized anti-Hb used in the present invention, instead of the enzyme, Hp and anti-Hb
is achieved by the same method except by using respectively.

The carrier used to chemically bind and insolubilize Hp or anti-Hb is a water-insoluble organic or inorganic polymeric material having a group capable of binding to these proteins, such as a carboxyl group or an amino group. For example, polyacrylamide gel [Bio-gel P-300, etc., sold by Bio-gel, USA], agarose [Sepharose 4B, etc., sold by Pharmacia, Sweden], dextran [Sephadex G200, DEAE- DEAE-Sephadex, QAE-Sephadex, etc. (marketed by Pharmacia, Sweden), cellulose (DEAE
-Cellulose, CM-cellulose, AE-cellulose, etc.) which do not directly bind to Hp and anti-Hb (hereinafter referred to as Hp, etc.), and are materials that have been given binding ability by treating them with cyanogen bromide. Used to advantage. A method for treating polysaccharides with cyanogen bromide is described by P. Cuatrecasas, P. and Anfinsen, C.
B.) [Methods in en zymology]
XII, Jacobi ed. (Methods in Enzymology.
XII. ed. 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 with Hp, etc. be able to. Furthermore, inorganic materials such as glass can be treated with aminoalkylsilane to provide amino groups that bind to Hp, etc., and can be used to insolubilize Hp, etc.

Examples of carriers that physically or electrostatically adsorb and insolubilize Hp include diatomaceous earth, activated carbon, and ion exchangers such as DEAE-Sephadex and DEAE-cellulose. or
As a crosslinking agent that insolubilizes Hp etc., a bifunctional compound such as glutaraldehyde can be used, for example.

The matrix used to trap and insolubilize the enzyme is a high molecular weight organic polymer gel or fiber, but these can also be used for Hp etc. by selecting an appropriate means. For example, during monomer polymerization, if Hp is present in the polymerization system, it will be trapped 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 Hp etc. can be used, but simpler carriers are preferred. For example, DEAE-cellulose is bound to Hp etc. by thoroughly washing with distilled water and stirring while keeping the temperature at room temperature, and this bound carrier with Hp etc. is then polymerized with a secondary carrier. obtain.

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

Immobilized Hp and immobilized anti
Hb is set in a suitable device for use as a hemoglobin measurement kit. The device is cylindrical, and a certain amount of immobilized Hp is placed inside the cylinder made of a suitable material.
is set in the upper layer and immobilized anti-Hb in the lower layer.

The particle diameters of the immobilized Hp and immobilized anti-Hb to be set are 40 to 200 microns and are used.

The amount of immobilized Hp and immobilized anti-Hb set in the device is generally 0.5 to 70 cm ³ in volume, and the length of the tube is 2 to 20 cm, preferably 4 to 10 cm, to generally provide a good kit.

For example, fixed in a cylindrical device with an inner diameter of 1.5 cm and a length of 10 cm.
When filled with Hp or immobilized anti-Hb, each approximately 5 to
Combine 100 mg of hemoglobin or hemoglobin-haptoglobin complex.

The kit consisting of immobilized Hp and immobilized anti-Hb set in this way is prepared in advance by a calibration curve between the set amount of immobilized Hp, the amount of free hemoglobin, the set amount of immobilized anti-Hb, and the amount of hemoglobin-haptoglobin complex. is determined and a corresponding quantity is attached to the device, for example as a concentration.

Measurement of hemoglobin amount using a fixed kit
The amount of free hemoglobin and the amount of hemoglobin-haptoglobin complex in the sample are determined from the calibration curve by measuring the length or volume of the part that is adsorbed to Hp and immobilized anti-Hb and colored red. The sample injected into the kit first binds to the immobilized Hp and free hemoglobin in the upper layer, and the hemoglobin-haptoglobin complex binds to the immobilized anti-Hb in the lower layer.

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. Approximately 5 minutes to 20 minutes is sufficient. After sufficient penetration, wash with an aqueous solution of at least twice the volume of the kit. The cleaning solution is
There is no particular limitation 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
_Na2HPO4・NaOH (PH10~11), 1~5M _NaBr ,
Alternatively, contact with an aqueous solution of 1 to 6 M MgCl ₂ (PH 3 to 5) releases the binding between Hp and hemoglobin and the binding between anti-Hb and the hemoglobin-haptoglobin complex, thereby releasing hemoglobin and the hemoglobin-haptoglobin complex. 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 can measure the amount of free hemoglobin and hemoglobin-
This is a reagent that can easily and quickly separate and quantify the amount of haptoglobin complex.

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. Sepharose 4B was fixed using the cyan method, and this was filled into the lower 10 cm of a syringe cylinder with an inner diameter of 1.5 cm and a length of 20 cm.A circularly cut paper was placed on the top of the filling so as not to disturb the top surface of the filling, and then pooled human plasma was added. Hp obtained by further purification (JP-A-50-77516)
Immobilization on Sepharose 4B using cyanogen bromide method
A kit was prepared by filling an upper layer of 10 cm with Hp and placing a circularly cut piece of paper on top of the filling so as not to disturb the top surface of the filling and moistening it with physiological saline. The calibration curves of free hemoglobin and hemoglobin-haptoglobin complex of this kit are linear as shown in FIG.

Example 2 The bottom of a transparent plastic tube with an inner diameter of 1.5 cm and a length of 10 cm was filtered with a glass filter (600 mesh).
After filling this with immobilized Hp and immobilized anti-Hb prepared in the same manner as in Example 1 to a height of 7 cm, a glass filter of the same type was fixed on the top surface of the contents, and a small amount of physiological A total of 200 devices were made, 100 each, filled with saline and sealed at both ends of the column with rubber caps. Of these, 10 devices were selected in which an immobilized Hp column was set in the upper layer and an immobilized anti-Hb column was set in the lower layer, and standard hemoglobin and standard hemoglobin-haptoglobin complex were dissolved in hemoglobin-free serum at 1 mg/ml each. 2 ml of the containing solution was passed through. The length of the red colored part is highest in the upper part.
The length was 1.85cm, the minimum was 1.70cm, and the average was 1.75cm. In the lower part, the maximum height was 1.83cm, the minimum was 1.68cm, and the average was 1.72cm.

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 and anti-Hb obtained by immunizing horses with human hemoglobin were added to the porous glass. Fixed.

A device was prepared by filling a plastic tube with these in the same manner as in Example 2. Of these
When 10 pieces were selected and 2ml of a solution containing 1mg/ml of each of standard hemoglobin and standard hemoglobin-haptoglobin complex dissolved in hemoglobin-free serum was passed through, the length of the red colored part was found to be in the upper layer. Maximum 2.63cm, minimum 2.47cm, average
2.56 cm, maximum 2.58 cm in the lower part, minimum 2.45 cm, average
It was 2.53cm.

[Brief explanation of drawings]

FIG. 1 is a calibration curve of hemoglobin in free hemoglobin and hemoglobin-haptoglobin complex obtained in Example 1 using the method and kit according to the invention.

Claims (1)

[Scope of Claims] 1. A hemoglobin-containing specimen is first brought into contact with immobilized haptoglobin to collect free hemoglobin in the specimen as a complex with immobilized haptoglobin, and then brought into contact with an immobilized anti-hemoglobin antibody to collect the free hemoglobin in the specimen. After collecting the haptoglobin-hemoglobin complex inside as a complex with anti-hemoglobin antibody, the length of each adsorbed part colored red,
Alternatively, a method for quantifying hemoglobin in a hemoglobin-containing specimen, which comprises quantifying free hemoglobin and haptoglobin-hemoglobin complex by measuring volume. 2. A reagent for quantitatively measuring hemoglobin in a specimen containing free hemoglobin and hemoglobin existing in the form of a complex with haptoglobin, comprising an upper layer of immobilized haptoglobin and a lower layer of immobilized anti-hemoglobin antibody layer.