JPS648307B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a reagent for detecting irregular antibodies comprising a mixed solution of polyethylene glycol obtained by dissolving polyethylene glycol in different states in a phosphate saline buffer. Irregular antibodies are red blood cell antibodies that do not conform to Landsteiner's law, such as anti-D, anti-C, anti-E, anti- ^Dia , anti-S,
Anti- ^Jra , anti- ^Xga , anti-Fy ^b, etc. are known. One of the conventional methods for detecting such irregular antibodies is the albumin Coombs method, and an example of the technique is as follows. Put 1 drop of Selectogen (a suspension of human type O red blood cells containing antigens corresponding to irregular antibodies) and 2 drops of test serum into a test tube, add 2 drops of 22% bovine albumin solution, and incubate at 37°C for 15 minutes. Sensitize for ~30 min. After washing this product three times with physiological saline by centrifugation at 2500 rpm for 5 minutes, discard as much of the supernatant as possible. Next, add 2 drops of Coombs serum and run at 1000 rpm.
Centrifuge for a minute and visually judge the strength of red blood cell agglutination. This albumin-Coombs method requires 22% bovine albumin solution and Coombs serum, and has the disadvantage that Coombs serum is expensive. An object of the present invention is to provide a reagent for detecting irregular antibodies that is inexpensive and has high detection accuracy. The present inventor has researched reagents for detecting irregular antibodies as an alternative to bovine albumin and Coombs serum, and found that easily available and inexpensive grease-like or wax-like polyethylene glycol is equivalent to 22% bovine albumin solution and Coombs serum. Based on this knowledge, we developed a reagent for testing irregular antibodies (Japanese Patent Application No. 165313/1986, referred to as prior invention). As a result of successive research on detection reagents using polyethylene glycol, the present inventor discovered that a mixed solution of polyethylene glycol in different states had superior functionality than the prior invention, and also found that this mixed solution of polyethylene glycol discovered that adding a nonionic surfactant to the erythrocyte aggregation image made it easier to see the agglutination image of red blood cells.
The present invention was completed based on these new findings. The detection reagent according to the present invention is a mixed solution of polyethylene glycol obtained by dissolving polyethylene glycol in different states in a phosphate saline buffer, and examples thereof will be described next. A suitable phosphate saline buffer in the present invention has a phosphate concentration of 1/75 molar, and salt is dissolved therein at a ratio of 0.8%. A nonionic surfactant such as polyoxyethylene sorbitan fatty acid ester is dissolved in this phosphate saline buffer at a ratio of 0.2%. Adding such a surfactant to the polyethylene glycol solvent prevents red blood cells from adhering to the inner wall of the test tube, and centrifugal sedimentation causes the red blood cells to fall to the bottom of the test tube, making it easier to see red blood cell agglutination images. As a result, detection accuracy improves. Polyethylene glycol (PEG) in different states
(abbreviated as PEG) is in the form of grease, wax, flakes, and liquid.
is dissolved in the above phosphate saline buffer to the following concentration: Waxy PEG with an average molecular weight of 6,000 4.5-10.0% Flake-like PEG with an average molecular weight of 20,000 0.5-1.2% Liquid PEG with an average molecular weight of 400 0-7.0% Regarding the method for detecting irregular antibodies using the reagent of the present invention (referred to as the method of the present invention) A specific example of the technique is as follows. PEG #6000, #20000 and #400 were added to the above phosphate saline buffer at concentrations of 6%, 0.9% and 2%, respectively.
Dissolve and add the above surfactant to it.
The reagent of the present invention is prepared by dissolving it at a ratio of 0.2%.
Put 1 drop of selectogen and 2 drops of test serum into a test tube, add 2 drops of the reagent of the present invention, and incubate at 37℃ for 15-30 minutes.
Sensitize for minutes. Use this with physiological saline
Wash twice by centrifuging at 2500 rpm for 5 minutes, and discard the supernatant. Next, add 2 drops of the reagent of the present invention and turn the temperature at 3400 rpm.
Centrifuge for 15 seconds and visually judge the strength of red blood cell agglutination. The reactivity of the conventional albumin Coombs method, the prior invention method, and the present invention method to several irregular antibodies was compared. The results are shown in Table 1. The agglomeration score was calculated according to Dunsford and Bowly's method (4+).
The calculation was divided into six stages from (0) to (0).

【table】

[Table] As can be seen from Table 1, the anti-E used in the test was undetectable by the conventional method and could only be detected by the promelin method, but it was easily detected by the method of the present invention. The method of the present invention was able to detect many irregular antibodies as listed in Table 1 in addition to anti-E. Regarding detection sensitivity, the method of the present invention was more sensitive than the conventional method for anti-E, anti-D, and anti-anti, and the conventional method was more sensitive than the method of the present invention for anti-Jr ^a . For other antibodies, both methods showed the same sensitivity. In addition, the prior invention method uses anti-S, anti-Xg ^a , anti-Jr ^a with low antibody titers.
were undetectable, but the method of the present invention was also able to detect these irregular antibodies. The above shows an example of the reagent according to the present invention and an example of the method for detecting irregular antibodies using the same, and the present invention is not limited thereto, and the polyethylene glycol used may be changed within the gist of the invention. For example, for polyethylene glycol, #6000 can be #4000, #20000
#11000 may be used instead of #400, and #300 may be used instead of #400. In this case, the concentration of each polyethylene glycol in the mixed solution can be changed depending on its average molecular weight. The detection reagent according to the present invention is a mixed solution prepared by dissolving polyethylene glycol in different states in a phosphate saline buffer, and detects irregular antibodies using easily available and inexpensive polyethylene glycol. However, it is extremely useful as a reagent for detecting irregular antibodies because it can achieve results equivalent to or better than the conventional albumin-Coombs method. Furthermore, the addition of a nonionic surfactant to the phosphate buffer has the advantage of further improving detection accuracy.

Claims (1)

[Claims] 1. A mixed solution of polyethylene glycol in different states obtained by dissolving each of grease-like, wax-like, flake-like, and liquid polyethylene glycol in a phosphate-salt buffer. Reagent for detection of irregular antibodies. 2 Grease-like, wax-like, flake-like, and liquid polyethylene glycols were added to a phosphate saline buffer solution at 4.5-10.0% and 0.5-1.2%, respectively.
The reagent for detecting irregular antibodies according to claim 1, wherein a mixed solution is prepared by dissolving the antibodies to a concentration of 0 to 7.0%. 3. The reagent for detecting irregular antibodies according to claim 1 or 2, wherein a nonionic surfactant is added to a mixed solution of polyethylene glycol.