CN108693359B - Zirconia microbead Rh blood type typing reagent card and preparation method thereof - Google Patents
Zirconia microbead Rh blood type typing reagent card and preparation method thereof Download PDFInfo
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Abstract
The invention provides zirconium oxide (ZrO)2) Microbead Rh blood type typing reagent card and itsA method of making, the reagent card comprising: the device comprises a zirconia microsphere micro-column tube containing a monoclonal antibody C with IgM property, a zirconia microsphere micro-column tube containing a monoclonal antibody C with IgM property, a zirconia microsphere micro-column tube containing a monoclonal antibody E with IgM property, a zirconia microsphere micro-column tube containing a monoclonal antibody E with IgM property, a zirconia microsphere micro-column tube containing a monoclonal antibody D with IgM property and a zirconia microsphere micro-column tube containing a zirconia microsphere suspension medium. The zirconia microbead Rh blood type typing reagent card has high sensitivity, good specificity and stable quality.
Description
Technical Field
The present invention relates to the field of medical testing. More particularly, the invention relates to a zirconia microbead Rh blood type typing reagent card and a preparation method thereof.
Background
The Rh blood group was the first blood group system discovered in 1940. The discovery of Rh blood type plays an important role in more scientifically guiding blood transfusion work, further improving experimental diagnosis of neonatal hemolytic disease and maintaining maternal and infant health.
The Rh blood group system contains 6 antigens, namely C, C, D, D, E, E. All erythrocytes are positive for Rh containing D antigen, and negative otherwise. Rh blood group has no natural antibody, and the antibody is mostly generated by blood transfusion or pregnancy immunity, which has important clinical significance. Once antibodies are formed, such as by reinfusion of Rh positive blood, severe transfusion reactions can occur.
The reagent for Rh blood type typing is not standardized at home at present, and Rh blood type typing examination is not carried out in a plurality of medical institutions.
In foreign countries, there are typing reagent cards for Rh blood group, which mostly use dextran gel. However, the size of the particle pore will change due to the change of temperature, salt concentration, osmotic pressure and pH, and the space outside the bed will also change, so that the centrifugal chromatography result will be affected. The gel-like particles have poor physical strength, cause deformation with prolonged shelf life, have high nonspecific adsorption, and affect the test results. In addition, the price is relatively expensive.
Therefore, there is a need in the art to develop a Rh blood typing reagent card with low cost and good stability.
Disclosure of Invention
The invention aims to overcome the defects and provide the zirconia microbead Rh blood type typing reagent card which is high in sensitivity, good in specificity and stable in quality.
In a first aspect, the present invention provides a reagent card for typing blood group comprising zirconia beads Rh, the reagent card comprising:
a microcolumn tube filled with zirconia beads containing monoclonal anti-C antibodies with IgM properties;
a microcolumn tube filled with zirconia beads containing monoclonal anti-c antibodies with IgM properties;
a microcolumn tube filled with zirconia beads containing monoclonal antibody E with IgM properties;
a microcolumn tube filled with zirconia beads containing monoclonal antibody E with IgM properties;
a microcolumn tube filled with zirconia beads containing monoclonal anti-D antibodies with IgM properties.
A micro-column tube filled with zirconia microspheres containing zirconia microsphere suspension medium.
The diameter of the zirconia microsphere particles is 63-125 m.
The titer of the monoclonal antibody C with the IgM property is more than or equal to 16; the titer of the monoclonal antibody c with the IgM property is more than or equal to 16; the titer of the monoclonal antibody E with the IgM property is more than or equal to 16; the titer of the monoclonal e antibody with the IgM property is more than or equal to 16; the titer of the monoclonal antibody D with the IgM property is more than or equal to 64 and is equal to DVIThe phenotypic response was negative.
The volume ratio of the zirconia beads to each antibody was 2: 1.
An aluminum foil sealing sheet is arranged at the opening of the micro-column tube.
The number of the zirconia micro-bead micro-column tubes containing the antibody is 1; the number of the zirconia micro-bead micro-column tubes containing the zirconia micro-bead suspension medium is 1-3.
The second aspect of the invention provides a preparation method of the zirconia microbead Rh blood type typing reagent card, which comprises the following steps:
(a) screening out zirconia microspheres with the particle diameter of 63-125 microns, washing for 3-5 times by using a microsphere suspension medium, removing microsphere damaged fragments and aggregated microsphere particles, and collecting to obtain microspheres with uniform particle size and complete spherical shape;
(b) mixing the microbeads prepared in the step (a) with each antibody according to a volume ratio of 2:1, and preparing the mixture into microbeads containing the antibodies;
(c) respectively mixing the microbeads prepared in the step (a) with a microbead suspension medium according to a volume ratio of 2:1, and preparing microspheres containing a suspension medium of zirconia microspheres;
(d) and (c) respectively subpackaging the microbeads prepared in the step (b) and the step (c) into the micro-column tubes of the detection card according to the amount of 22-28 microliters per tube.
The above method further comprises the steps of:
(e) and sealing the upper opening of the micro-column tube by using aluminum foil paper in a film pressing mode.
The micro-bead suspension medium comprises the following components:
the pH value is 6.6-6.8.
The 'water' in the microbead suspension medium is distilled water, purified water or deionized water, and preferably distilled water.
The invention has the beneficial effects that:
in a first aspect of the present invention, a standardized suspension medium system of zirconia beads is provided, which is used for washing and suspending zirconia beads and can maintain the stability of antibodies and zirconia beads for a long time, and is characterized in that:
1. a buffer system with strong buffer capacity is designed, and the pH value of the buffer system is maintained at 6.6-6.8 by adopting the buffer system consisting of potassium phosphate, sodium salt and amino acid. Compared with a single citric acid buffer system commonly adopted in the field of blood transfusion, the buffer system has the characteristic of stronger buffer capacity, is favorable for keeping the whole system in a required buffer range, and simultaneously ensures the ionic strength of the whole zirconia bead suspension medium;
2. the suspension medium of the zirconia microspheres adopts amino acid and sodium chloride as additives, and helps to maintain a low ionic strength environment by using a trace amount of phosphate, so that the zirconia microspheres are kept to be spherical particles, and the diameter of the zirconia microspheres is maintained within a required range (63-125 micrometers);
3. the invention has a unique lubricating system, adopts bovine serum albumin with a certain concentration, so that the red blood cells obtain proper lubricating capability when passing through the gaps of the zirconia microspheres, and the non-agglutinated red blood cells have the capability of completely passing through the gaps of the zirconia microspheres, but the agglutinated red blood cells cannot pass through;
4. the invention has an excellent antiseptic system, selects sodium benzoate as an antiseptic, adopts polyvinylpyrrolidone as a synergistic effect, and has better antiseptic capability. Effectively prevent the multiplication of bacterium, obtain the retention period of longer time, thereby not only avoid using sodium azide chemical preservative to promote the ionic strength of zirconia microballon suspension medium system and consequently to the sensitivity and the specificity production adverse effect of Rh blood group typing reagent card, also avoid using the antibiotic to produce the midbody to the specificity production of zirconia microballon Rh blood group typing reagent card in the save process.
On the other hand, in order to ensure the quality of the Rh blood type typing reagent card of the zirconia beads of the present invention, the zirconia beads need to be screened in the preparation process, i.e., firstly, a proper zirconia bead needs to be selected, and generally, the conditions that: selecting particles with the diameter of 63-125 microns, and washing and suspending the screened zirconia microsphere raw materials, so that the zirconia microsphere particles are washed to remove damaged zirconia microsphere particles, aggregated zirconia microsphere particles, oversized or ultra-small zirconia microsphere particles with the inner diameter of 63-125 microns and other impurity components except zirconia microspheres.
On the other hand, in order to ensure the quality of the zirconia microbead Rh blood type typing reagent card, the antibody raw materials mixed with the zirconia microbeads are also screened in the preparation process.
In conclusion, the excellent specificity, sensitivity and storage life of up to 1 year of the reagent card for typing blood types of the zirconia microbead Rh of the invention is the synergistic effect of various components of the whole system. The arrangement of the zirconia micro-bead micro-column tubes containing 5 Rh typing antibodies on one card can ensure that the examination of all 5 antigens of the Rh blood group system can be completed only by using one zirconia micro-bead Rh blood group typing reagent card. The buffer system can maintain the required pH of the reagent card reaction system. The low salt concentration system can ensure that the zirconia microsphere particles are fully swelled and the diameter of the zirconia microsphere particles is in a required range. The lubricating system can ensure proper lubricating capability among the zirconia microsphere particles. The preservative can prevent the zirconium oxide micro beads or antibodies from being ineffective due to bacterial reproduction. Standardized zirconia beads can ensure proper spacing between bead particles. Standardized antibodies can ensure efficient detection of antigens.
When the zirconia microbead Rh blood type typing reagent card is used for detecting Rh blood type, the generated positive reaction is not less than 1 +. The shelf life is not less than 12 months under the condition of 18-25 ℃ generally.
In conclusion, the implementation of the invention provides a standardized zirconia microsphere Rh blood type typing reagent card product, and various hospitals and blood collection institutions can directly obtain the zirconia microsphere Rh blood type typing reagent card with consistent standards from production suppliers, thereby creating conditions for accurately identifying Rh typing, ensuring safe blood transfusion, diagnosing and treating neonatal hemolytic disease as early as possible.
Drawings
FIG. 1 is a microscopic view of zirconia beads;
fig. 2 is a schematic structural diagram of the Rh blood group testing reagent card of the present invention.
1-8, zirconia micro-bead micro-column tube; 9. detecting a reagent card; 11. zirconia micro beads; 12. a microbead suspension medium; 13. a microcolumn tube; 14. an empty card.
Detailed Description
The following detailed description is provided for the implementation and beneficial effects of the present invention through specific examples, and is intended to help the reader better understand the spirit and essence of the present invention, and should not be construed as limiting the scope of the implementation of the present invention in any way. All reagents and instruments are commercially available.
Example 1: preparation of zirconia microbead Rh blood type typing reagent card
Step one, preparation of suspension medium of zirconia microspheres
The formula of the zirconia microsphere suspension medium is as follows:
the above reagents are dissolved in distilled water, and the pH value is adjusted to 6.6-6.8.
Step two, preparation of zirconia microspheres
Zirconia micro-beads are selected, and the particle diameter is 63-125 microns. And (3) washing for 3-5 times by using the zirconium oxide microsphere suspension medium prepared in the step one, removing the damaged fragments of the zirconium oxide microspheres and the aggregated zirconium oxide microsphere particles, and collecting to obtain the applicable zirconium oxide microspheres which are uniform in particle size and complete and spherical.
Step three, selecting antibodies
Selecting monoclonal antibody C with IgM property, the titer is more than or equal to 16;
selecting monoclonal antibody c with IgM property, and the titer is more than or equal to 16;
selecting monoclonal antibody E with IgM property, the titer is more than or equal to 16;
selecting monoclonal antibody e with IgM property, the titer is more than or equal to 16;
selecting monoclonal antibody D with IgM property, whose titer is greater than or equal to 64 and is identical with DVIThe phenotypic response was negative.
Step four, preparation of zirconia microspheres
Mixing the zirconia micro-beads prepared in the step two with the antibodies selected in the step three according to the volume ratio of 2:1, respectively preparing zirconia beads containing monoclonal antibody C with IgM property, zirconia beads containing monoclonal antibody C with IgM property, zirconia beads containing monoclonal antibody E with IgM property, zirconia beads containing monoclonal antibody E with IgM property and zirconia beads containing monoclonal antibody D with IgM property; mixing the zirconia microspheres prepared in the step two with the zirconia microsphere suspension medium prepared in the step one according to the volume ratio of 2:1, and preparing the zirconia microspheres containing the zirconia microsphere suspension medium.
Step five, subpackaging
And (3) adding the zirconia micro-beads prepared in the step four into micro-column tubes of a blank card respectively according to the amount of 22-28 microlitres per tube. The number of the zirconia micro-bead micro-column tubes containing the antibody is 1; the number of the zirconia micro-bead micro-column tubes containing the zirconia micro-bead suspension medium is 1-3. The test card can perform Rh blood typing on one sample. In order to facilitate the identification of the mark, label paper is attached below the reagent card.
Step six, semi-finished product determination
The antibody-containing zirconium oxide micro-bead micro-column tube is required to have a positive reaction of more than or equal to 1+ with the red blood cells of the corresponding antigen of the antibody, namely the red blood cells are concentrated on the upper surface of the zirconium oxide micro-bead and are linear. And the red blood cells do not contain the corresponding antigen of the antibody to generate positive reaction, namely, the red blood cells can completely reach the bottom of the microtube through the zirconia micro-beads and are deposited at the bottom of the microtube. And red blood cells in the zirconia micro-bead micro-column tube which does not contain the antibody and only contains the mixture of the zirconia micro-bead suspension medium and the zirconia micro-beads can all reach the bottom of the micro-tube through the zirconia micro-beads and deposit at the bottom of the micro-tube to present negative reaction.
Step seven, sealing
And sealing the upper door of the microcolumn tube by using aluminum foil paper in a film pressing mode. Labeling, and storing at 18-25 deg.C.
Step eight, preservation test
The zirconia microbead Rh blood type typing reagent card is stored for more than 1 year, and has the following detection results during the storage period:
(1) appearance of the product
Zirconia beads in the zirconia bead Rh blood type typing reagent card are uniform and white, clear and transparent colorless transparent liquid with the diameter of 1-2 mm is arranged at the upper end of a bead layer, and no bubbles or foreign matters exist among zirconia bead particles.
(2) Sensitivity of the probe
In the microcolumn tube containing the antibody and the zirconia beads, the positive reaction which is more than or equal to 1+ with the erythrocytes of the antigen corresponding to the antibody is generated, namely the erythrocytes are concentrated on the upper surface of the zirconia beads and are in a linear shape.
(3) Specificity of
In the microcolumn tube containing the antibody and the zirconia microbeads, the erythrocytes with the antigens corresponding to the antibody generate positive reaction, namely the erythrocytes are concentrated on the upper surface of the zirconia microbeads and present a linear shape. And the negative reaction is generated with the red blood cells without the corresponding antigen of the antibody, namely, the red blood cells can completely pass through the zirconia micro-beads to reach the bottom of the microtube and are deposited at the bottom of the microtube. And red blood cells in the zirconia micro-bead micro-column tube which does not contain the antibody and only contains the mixture of the zirconia micro-bead suspension medium and the zirconia micro-beads can all reach the bottom of the micro-tube through the zirconia micro-beads and deposit at the bottom of the micro-tube to present negative reaction.
Usually, the price of the sephadex is more than 30 times that of the zirconia. In addition, zirconia is a white odorless and tasteless crystal, insoluble in water, hydrochloric acid, and dilute sulfuric acid. The ceramic insulating material is an important high-temperature resistant material and a ceramic insulating material due to the characteristics of inactive chemical properties, high melting point, high resistivity, high precipitation rate and low thermal expansion coefficient.
The zirconia micro-bead is characterized by high chemical and physical stability, low nonspecific adsorption, no growth of microorganisms such as bacteria and the like and extremely low back pressure of the filler.
The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention, including any reference to the above-mentioned embodiments. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Accordingly, the invention is not to be limited to the embodiments shown herein.
Claims (7)
1. The utility model provides a zirconia microballon Rh blood type typing reagent card which characterized in that, the reagent card includes:
a microcolumn tube filled with zirconia beads containing monoclonal anti-C antibodies with IgM properties;
a microcolumn tube filled with zirconia beads containing monoclonal anti-c antibodies with IgM properties;
a microcolumn tube filled with zirconia beads containing monoclonal antibody E with IgM properties;
a microcolumn tube filled with zirconia beads containing monoclonal antibody E with IgM properties;
a microcolumn tube filled with zirconia beads containing monoclonal antibody D having IgM properties;
a microcolumn tube filled with zirconia beads containing a zirconia bead suspension medium; the diameter of the zirconia microsphere particles is 63-125 microns; the zirconia microspheres are suspended by microsphere suspension media, and the microsphere suspension media comprise the following components:
the pH value is 6.6-6.8.
2. The reagent card for typing Rh blood group with zirconia micro beads according to claim 1, wherein the titer of monoclonal C antibody with IgM property is not less than 16; the titer of the monoclonal antibody c with the IgM property is more than or equal to 16; the titer of the monoclonal antibody E with the IgM property is more than or equal to 16; the titer of the monoclonal e antibody with the IgM property is more than or equal to 16; the titer of the monoclonal antibody D with the IgM property is more than or equal to 64 and is equal to DVIThe phenotypic response was negative.
3. The zirconia microbead Rh blood grouping reagent card of claim 2, wherein the volume ratio of zirconia microbeads to each antibody is 2: 1.
4. The reagent card of claim 3, wherein an aluminum foil sealing is provided at the opening of the microcolumn.
5. The zirconia microbead Rh blood grouping reagent card as in claim 3, wherein there are 1 each of the zirconia microbead microcolumn tubes containing the antibody; the number of the zirconia micro-bead micro-column tubes containing the zirconia micro-bead suspension medium is 1-3.
6. The method for preparing the reagent card of the zirconia microbead Rh blood grouping type according to claim 3, which comprises the following steps:
(a) screening out zirconia microspheres with the particle diameter of 63-125 microns, washing for 3-5 times by using a microsphere suspension medium, removing microsphere damaged fragments and aggregated microsphere particles, and collecting to obtain microspheres with uniform particle size and complete spherical shape;
(b) mixing the microbeads prepared in the step (a) with each antibody according to a volume ratio of 2:1, and preparing the mixture into microbeads containing the antibodies;
(c) mixing the microbeads prepared in the step (a) with a microbead suspension medium according to a volume ratio of 2:1, and preparing microspheres containing a suspension medium of zirconia microspheres;
(d) and (c) respectively subpackaging the microbeads prepared in the step (b) and the step (c) into the micro-column tubes of the detection card according to the amount of 22-28 microliters per tube.
7. The method for preparing the reagent card of the zirconia microbead Rh blood grouping according to claim 6, which further comprises the following steps:
(e) and sealing the upper opening of the micro-column tube by using aluminum foil paper in a film pressing mode.
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| JPH0690206B2 (en) * | 1986-06-16 | 1994-11-14 | 日本赤十字社 | Antigen-antibody complex and method of using the same |
| NL1008738C2 (en) * | 1998-03-27 | 1999-09-28 | Stichting Bloedvoorziening | Solid phase method for antigen and antibody determinations in blood group serology, and test kit. |
| DE19856703C2 (en) * | 1998-12-09 | 2001-02-01 | Deutsches Rotes Kreuz Blutspen | Methods for the detection of antibodies or antigens |
| EP1452865B1 (en) * | 2001-12-04 | 2009-02-18 | Sekisui Chemical Co., Ltd. | Composition for blood serum or plasma separation and vessel for blood examination containing the same |
| JP2008089415A (en) * | 2006-10-02 | 2008-04-17 | Kamakura Techno-Science Inc | Examination reagent and examination method |
| CN101578518A (en) * | 2007-01-24 | 2009-11-11 | 东丽株式会社 | Analysis chip and analysis method |
| US9192915B2 (en) * | 2008-05-10 | 2015-11-24 | Brigham Young University | Porous composite particulate materials, methods of making and using same, and related apparatuses |
| EP2349566B1 (en) * | 2008-10-03 | 2016-01-06 | Micronics, Inc. | Microfluidic apparatus and methods for performing blood typing and crossmatching |
| CN101701961B (en) * | 2009-11-25 | 2013-03-06 | 江阴力博医药生物技术有限公司 | Method for preparing typing detection reagent card of blood types of A, B and O |
| CN101718793B (en) * | 2009-11-25 | 2013-03-20 | 江阴力博医药生物技术有限公司 | Preparation method of Rh blood type typing card |
| CN102445549A (en) * | 2010-10-08 | 2012-05-09 | 上海博冠生物技术有限公司 | Novel column agglutination glass microsphere clamping type blood type detection reagent |
| CN102161716B (en) * | 2010-12-30 | 2014-03-05 | 北京九强生物技术股份有限公司 | Method and reagent for latex sensitization |
| CN103033632B (en) * | 2012-12-12 | 2014-10-22 | 英科新创(厦门)科技有限公司 | Reverse typing colloidal gold kit for ABO blood groups and preparation method thereof |
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