Method for agglutinating erythrocytes, method for separating erythrocytes, and hemagglutination reagent
11320350 · 2022-05-03
Assignee
Inventors
Cpc classification
G01N1/4044
PHYSICS
International classification
Abstract
Methods of agglutinating and separating erythrocytes, by which erythrocytes can be instantaneously agglutinated into a sufficient size in a blood sample and completely separated from the blood sample; and a hemagglutination reagent are provided. The method of agglutinating erythrocytes according to the present invention includes adding a solution containing a cholic acid-based surfactant and an acid to a blood sample. The method of separating erythrocytes according to the present invention includes separating the erythrocytes agglutinated by the above-described method of the present invention. The hemagglutination reagent according to the present invention contains a cholic acid-based surfactant and an acid.
Claims
1. A method of agglutinating erythrocytes, the method comprising adding a solution containing a cholic acid-based surfactant and an acid to a blood sample, wherein said solution has a pH of from 2.5 to 4.0 and wherein the cholic acid-based surfactant is sodium taurodeoxycholate hydrate.
2. A method of separating erythrocytes from a blood sample, the method comprising separating erythrocytes agglutinated by the method according to claim 1.
3. A hemagglutination reagent, which contains a cholic acid-based surfactant and an acid, wherein said reagent has a pH of from 2.5 to 4.0 and wherein the cholic acid-based surfactant is sodium taurodeoxycholate hydrate.
4. Use of the reagent according to claim 3, as a reagent for agglutinating erythrocytes.
5. The method of claim 1, wherein the method does not employ centrifugation.
6. The method of claim 1, wherein said sample consists of whole blood, optionally diluted with a diluent.
7. The method of claim 6, wherein said diluent consists of a physiological saline solution.
8. The method of claim 1, wherein the concentration of the sodium taurodeoxycholate hydrate in the solution is from 0.1 to 6.0% by weight.
9. The method of claim 3, wherein the concentration of the sodium taurodeoxycholate hydrate in the reagent is from 0.1 to 6.0% by weight.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
(1)
(2)
(3)
(4)
MODE FOR CARRYING OUT THE INVENTION
(5) The method of agglutinating erythrocytes according to the present invention is characterized in that a solution containing a cholic acid-based surfactant and an acid is added to a blood sample.
(6) (Blood Sample)
(7) The blood sample, as used herein, refers to a sample containing erythrocytes, obtained by collecting blood from a human or animal subject. As the blood sample, whole blood collected from subjects may be used as it is or a sample obtained by diluting the collected whole blood with physiologic saline or the like may be used. For ease of operation, whole blood collected from subjects is preferably used as it is. The amount of the blood sample used in the present invention is usually from 10 μl to 50 ml, preferably from 10 μl to 1 ml, more preferably from 10 μl to 100 μl.
(8) (Cholic Acid-Based Surfactant)
(9) The cholic acid-based surfactant, as used herein, refers to a surfactant having a steroid skeleton in the molecule, specifically, a surfactant having a structure of cholic acid or a derivative of cholic acid. The derivative of cholic acid means any compounds derived from cholic acid, specifically including dehydroxylated compounds such as deoxycholic acid and substituted amide compounds such as taurodeoxycholic acid.
(10) The cholic acid-based surfactant, as used herein, is preferably selected from the group consisting of anionic surfactants such as cholic acid, sodium cholate, deoxycholic acid, sodium deoxycholate, sodium taurodeoxycholate, chenodeoxycholic acid, sodium chenodeoxycholate, glycocholic acid, sodium glycocholate, glycodeoxycholic acid, sodium glycodeoxycholate, glycolithocholic acid, sodium glycolithocholate, lithocholic acid, sodium lithocholate, taurocholic acid, sodium taurocholate, tauroursodeoxycholic acid, and sodium tauroursodeoxycholate; nonionic surfactants such as polyoxyethylene cholesteryl ether and N,N-Bis(3-D-gluconamidopropyl)cholamide (BIGCHAPS); amphoteric surfactants such as 3-((3-Cholamidopropyl)dimethylammonium)-1-propanesulfonate (CHAPS); and hydrates thereof. These cholic acid-based surfactants may be used alone or in combination of two or more.
(11) Among them, the cholic acid-based surfactant is preferably anionic cholic acid-based surfactants such as sodium cholate, sodium deoxycholate, sodium taurodeoxycholate and hydrates thereof, particularly preferably sodium taurodeoxycholate and a hydrate thereof, from the viewpoint of rate of agglutination and more complete separation of erythrocytes.
(12) (Acid)
(13) As the acid used in the present invention, any substance releasing hydrogen ions (H.sup.+) in an aqueous solution may be used. Specific examples of the acid include inorganic acids and organic acids, and salts thereof. Examples of the inorganic acid include hydrochloric acid, hydrobromic acid, sulfuric acid, sulfamic acid, phosphoric acid, and nitric acid. Examples of the organic acid include acetic acid, propionic acid, succinic acid, glycolic acid, stearic acid, lactic acid, malic acid, tartaric acid, citric acid, malonic acid, ascorbic acid, pamoic acid, maleic acid, adipic acid, alginic acid, aspartic acid, hydroxymaleic acid, phenylacetic acid, glutamic acid, benzoic acid, salicylic acid, sulfanilic acid, 2-acetoxybenzoic acid, fumaric acid, benzenesulfonic acid, toluenesulfonic acid, methanesulfonic acid, 2-naphthalenesulfonic acid, ethanedisulfonic acid, oxalic acid, isethionic acid, glucoheptanoic acid, glycerophosphate, hemisulfanic acid, heptanoic acid, hexanoic acid, hydrochloric acid, hydrobromic acid, hydroiodic acid, 2-hydroxyethanesulfonic acid, 2-naphthalenesulfonic acid, pectinic acid, phosphoric acid, sulfuric acid, 3-phenylpropionic acid, picric acid, pivalic acid, thiocyanic acid, p-toluenesulfonic acid, butyric acid, camphoric acid, camphorsulfonic acid, digluconic acid, cyclopentanepropionic acid, disulfuric acid, dodecylsulfuric acid, ethanesulfonic acid, and undecanoic acid. In the present invention, the acids may be used alone or in combination of two or more.
(14) The organic acids are preferably used as the acid used in the present invention from the viewpoint of agglutination speed and more complete separation of erythrocytes. Among them, preferably organic acids selected from the group consisting of acetic acid, tartaric acid, malonic acid, malic acid and citric acid, more preferably polycarboxylic acids, and particularly preferably citric acid are used.
(15) (Hemagglutination Reagent)
(16) In the present invention, erythrocytes are agglutinated by adding a solution containing the cholic acid-based surfactant and the acid described above to a blood sample. Such a solution containing the cholic acid-based surfactant and the acid to be added to a blood sample can be used as a reagent for agglutinating erythrocytes (hemagglutination reagent).
(17) Such a solution containing the cholic acid-based surfactant and the acid (hereinafter referred to as “hemagglutination reagent”) can be prepared by a method comprising adding the cholic acid-based surfactant to an aqueous acid solution obtained by diluting the acid with a physiologic saline or the like. The concentration of the acid in the hemagglutination reagent is usually from 1 mM to 1 M, preferably from 5 mM to 500 mM, more preferably from 10 mM to 200 mM, from the viewpoint of agglutination speed and more complete separation of erythrocytes. The pH of the hemagglutination reagent is preferably from 2.0 to 5.0, more preferably from 2.2 to 4.5, still more preferably from 2.5 to 4.0. The pH of the hemagglutination reagent can be measured with a commercially available pH meter by a glass electrode method. The hemagglutination reagent may contain surfactants (for example, surfactants such as Tween 20 and Emulgen A500), reagents and the like, which do not cause hemolysis, in addition to the acid, the cholic acid-based surfactant, and the physiologic saline.
(18) The concentration of the cholic acid-based surfactant in the hemagglutination reagent is usually from 0.05 to 15.0% by weight, preferably from 0.06 to 13.0% by weight, more preferably from 0.075 to 8.0% by weight, particularly preferably from 0.1 to 6.0% by weight.
(19) The ratio (molar ratio) of the cholic acid-based surfactant and the acid in the hemagglutination reagent is usually from 1:7 to 1:2000, preferably from 1:8 to 1:1667, more preferably from 1:13 to 1:1333, particularly preferably from 1:17 to 1:1000, from the viewpoint of agglutination speed and more complete separation of erythrocytes. The amount of the hemagglutination reagent added to the blood sample to be used (volume ratio) is usually from 1:4 to 1:80, preferably from 1:8 to 1:20, more preferably from 1:10 to 1:13.
(20) (Method of Agglutinating Erythrocytes)
(21) In the present invention, when the hemagglutination reagent described above is directly added to a blood sample at room temperature, erythrocytes are instantaneously agglutinated into a sufficient size. In order to allow the agglutination to be progressed more completely, the blood sample is preferably left to stand for 5 seconds to 30 seconds, preferably for about 5 seconds in the state wherein the blood sample contacts the hemagglutination reagent, and then mixed by upside-down mixing several times. By agglutinating erythrocytes in this manner, an agglutinate having a sufficient size can be obtained.
(22) Since the erythrocytes agglutinated using the method and the reagent of the present invention have a sufficient size, they can be easily and conveniently separated from the blood sample. Examples of the method of separating the agglutinated erythrocytes include a method in which the supernatant is recovered after the agglutinated erythrocytes are precipitated, and a method of separating them by a filtration with a filter paper. Since the serum component and the plasma component from which erythrocytes have been separated off in this manner are substantially free of erythrocytes, they can be used as analysis samples for biochemical tests.
EXAMPLES
(23) The present invention will now be described in more detail based on Examples. However, the present invention is not limited to these Examples.
Examples 1 to 4 and Comparative Examples 1-1 to 4-2
(24) Hemagglutination Depending on the Presence of Cholic Acid-Based Surfactant and Salt and pH Range
(25) To 71 mL of distilled water, 10 mL of 10 w/v % sodium taurodeoxycholate hydrate (TDOC), 10 mL of 1 M citric acid, and 9 mL of 10 w/v % sodium chloride were added to prepare a solution containing 100 mM citric acid, 1% TDOC, and 0.9% sodium chloride as final concentrations (hemagglutination reagent). A solution was also prepared in the similar manner except that TDOC and/or sodium chloride were not added (hemagglutination reagent), and effects of the presence and absence of the cholic acid-based surfactant and the salt and the pH range on agglutination of erythrocytes were compared.
(26) After adding 60 μL of a whole blood sample to 400 μL of the hemagglutination reagent, the rate of agglutination of erythrocytes was visually evaluated. The rate of agglutination was evaluated as +++, ++, +, or ± in order of rapidity based on the criteria described below. Hemolysis without agglutination was evaluated as −.
(27) +++: erythrocytes were instantaneously agglutinated into a sufficient size and the agglutinated erythrocytes were precipitated.
(28) ++: erythrocytes started to agglutinate after leaving to stand for a few seconds, and agglutinated erythrocytes were gradually precipitated.
(29) +: erythrocytes were agglutinated, but the agglutinates were very small and the agglutination was incomplete.
(30) ±: only very few erythrocytes were agglutinated.
(31) −: erythrocytes were hemolyzed without being agglutinated
(32) The results are shown in Table 1 below.
(33) The pH of the hemagglutination reagent was measured using a desktop pH meter (HORIBA) (similarly in the following Examples and Comparative Examples).
(34) TABLE-US-00001 TABLE 1 tube Rate of Example No. No. pH NaCl TDOC agglutination FIG. Comparative 1 2.5 x x − FIG. 1 Example 1-1 Comparative 2 ∘ x + Example 1-2 Example 1 3 ∘ ∘ +++ Comparative 4 3.0 x x − FIG. 2 Example 2-1 Comparative 5 ∘ x + Example 2-2 Example 2 6 ∘ ∘ +++ Comparative 7 4.0 x x − FIG. 3 Example 3-1 Comparative 8 ∘ x + Example 3-2 Example 3 9 ∘ ∘ +++ Comparative 16 2.5 x ∘ +++ FIG. 4 Example 4-1 Comparative 17 3.0 x ∘ +++ Example 4-2 Example 4 18 4.0 x ∘ +++
(35) As can be seen from the results in Table 1, when the hemagglutination reagent contained the cholic acid-based surfactant and the acid, the rate of agglutination was evaluated as “+++” within a pH range of 2.5 to 5.0 regardless of whether NaCl exists or not, and excellent agglutination effect was observed. On the other hand, when the hemagglutination reagent did not contain the cholic acid-based surfactant and contained only the acid, or only the acid and the salt, the rate of agglutination was evaluated as “−” or “+”, which means that hemolysis occurred or that erythrocytes were agglutinated but the agglutinates were very small and the agglutination was incomplete. The results show that the presence of the cholic acid-based surfactant has a great effect on agglutination of erythrocytes.
Examples 5 to 7
(36) Hemagglutination Depending on Cholic Acid-Based Surfactant and Acid
(37) Various cholic acid-based surfactants (1% by weight) and citric acid (100 mM) were added to a physiologic saline to prepare hemagglutination reagents (pH3.0) and their abilities to agglutinate erythrocytes were compared.
(38) After adding 60 μL of a whole blood sample to 400 μL of the hemagglutination reagent, the rate of agglutination of erythrocytes was evaluated. The rate of agglutination was evaluated as +++, ++, +, or ± in order of rapidity based on the criteria described below. Hemolysis without agglutination was evaluated as −. The following criteria were the same as those in Examples 1 to 4 and Comparative Examples 1-1 to 4-2.
(39) +++: Erythrocytes were instantaneously agglutinated into a sufficient size and the agglutinated erythrocytes were precipitated.
(40) ++: Erythrocytes started to agglutinate after leaving to stand for a few seconds, and agglutinated erythrocytes were gradually precipitated.
(41) +: Erythrocytes were agglutinated, but the agglutinates were very small and the agglutination was incomplete.
(42) ±: Only very few erythrocytes were agglutinated.
(43) −: Erythrocytes were hemolyzed without being agglutinated.
(44) The results are shown in Table 2 below.
(45) TABLE-US-00002 TABLE 2 Rate of Surfactant agglutination Example 5 Deoxycholic acid ++ Example 6 Sodium cholate ++ Example 7 Sodium taurodeoxycholate hydrate +++ (TDOC)
(46) The results in Table 2 shows that when the hemagglutination reagents contained various cholic acid-based surfactants in combination with the acid, the rates of agglutination were evaluated as “++” or “+++” and excellent agglutination effects were observed. In particular, the effect of sodium taurodeoxycholate hydrate (TDOC) on hemagglutination was most significant.
Example 8
(47) Hemagglutination Depending on Concentration of Cholic Acid-Based Surfactant
(48) Sodium taurodeoxycholate hydrate (TDOC) and citric acid (100 mM) were added to a physiologic saline to prepare hemagglutination reagents (pH3.0), and their abilities to agglutinate erythrocytes were compared in the concentration range of TDOC as shown in the table below.
(49) After adding 60 μL of a whole blood sample to 400 μL of the hemagglutination reagent, the rate of agglutination of erythrocytes was evaluated. The rate of agglutination was evaluated as +++, ++, +, or ± in order of rapidity based on the criteria below. Hemolysis without agglutination was evaluated as −. The following criteria were the same as those in Examples 1 to 7 and Comparative Examples 1-1 to 4-2.
(50) +++: Erythrocytes were instantaneously agglutinated into a sufficient size and the agglutinated erythrocytes were precipitated.
(51) ++: Erythrocytes started to agglutinate after leaving to stand for a few seconds, and agglutinated erythrocytes were gradually precipitated.
(52) +: Erythrocytes were agglutinated, but the agglutinates were very small and the agglutination was incomplete.
(53) ±: Only very few erythrocytes were agglutinated.
(54) −: Erythrocytes were hemolyzed without being agglutinated.
(55) The results are shown in Table 3 below.
(56) TABLE-US-00003 TABLE 3 Concentration of TDOC Rate of (wt %) agglutination 6.0 +++ 1.0 +++ 0.5 +++ 0.1 +++
(57) The results in Table 3 shows that when the concentration of the cholic acid-based surfactant was from 0.1 to 6.0% by weight, the rate of agglutination was evaluated as “+++” and an excellent agglutination effect was observed.
(58) In Examples 1 to 4 described above, after the agglutinated erythrocytes were precipitated, the supernatant was separated and added dropwise to an immunochromatographic assay kit (Quick Navi®-Ebola). When the movement on a membrane was observed, the membrane was not colored red. These results also supported the fact that the above-mentioned separated supernatant did not substantially contain erythrocytes and is suitable as an analysis sample for biochemical tests.