CN108693355A - Antihuman globulin detection reagent card and preparation method thereof - Google Patents
Antihuman globulin detection reagent card and preparation method thereof Download PDFInfo
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- CN108693355A CN108693355A CN201710872096.4A CN201710872096A CN108693355A CN 108693355 A CN108693355 A CN 108693355A CN 201710872096 A CN201710872096 A CN 201710872096A CN 108693355 A CN108693355 A CN 108693355A
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- G—PHYSICS
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- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
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Abstract
The present invention provides a kind of zirconium oxide (ZrO2) microballon antihuman globulin detection reagent card and preparation method thereof, the detection reagent card includes:Microtrabeculae pipe, pipe is interior to be equipped with the zirconium oxide microballon containing anti-igg+C3d antibody.Broad spectrum antihumanglobulin detection reagent card high sensitivity prepared by the present invention, specificity be good and stable quality.
Description
Technical field
The present invention relates to medical detection fields, more particularly to a kind of group of broad spectrum antihumanglobulin detection reagent card
At, preparation method and applications.
Background technology
The inventive gel antiglobulin methods first of France Yves Lapierre in 1984, with traditional test tube method antiglobulin
Experiment is compared, and the cumbersome washing step of test tube liquid medium is omitted, and also eliminating must be added not in negative findings test tube
The red blood cell of complete antibody sensitization, to confirm the whether correct step of negative findings, therefore, operation letter few with sample dosage
Just, time saving, human factor is small, be easy to standardize and automate, the easy interpretation of result and can long-term preservation the advantages that, be suitable in high volume
The detection of sample.
Routine, which is had become, in external gel antiglobulin detection reagent card is widely used in bracket for blood grouping, irregular antibody inspection
The various blood types serological test such as survey and cross matching.In recent years, it is domestic oneself start to use gel antiglobulin detection reagent card
Carry out screening and identification, the detection of sensitized erythrocyte etc. of irregular antibody.But mostly use sephadex.But glucan
Gel can by temperature, salinity, osmotic pressure, pH variation and cause the aperture of particle that can change, the space outside bed can also change, and lead
Cause centrichromatography result impacted.Due to being a kind of gelatinous particle, physical strength is poor, with the extension of storage life, meeting
Cause to deform, non-specific adsorption is high, can influence test result.In addition, price is also more expensive.
Therefore, there is an urgent need in the art to research and develop high sensitivity, specificity well and the new antiglobulin detection of stable quality
Reagent card.
Invention content
It is an object of the invention to overcome the shortcomings of traditional test tube method, provide that a kind of high sensitivity, specificity be good and quality
The preparation method of stable broad spectrum antihumanglobulin detection reagent card.
The present invention provides a kind of antihuman globulin detection reagent card, and the detection reagent card includes:
Microtrabeculae pipe, pipe is interior to be equipped with the zirconium oxide microballon containing anti-igg+C3d antibody.
Above-mentioned microtrabeculae pipe is managed for 4-12.Above-mentioned microtrabeculae pipe is 8 pipes.
Above-mentioned zirconium oxide bead particles are 63-125 microns a diameter of.
The volume ratio of above-mentioned zirconium oxide microballon and antibody is 2:1.
There is aluminium foil mounting at above-mentioned microtrabeculae tube opening.
Label paper on mentioned reagent card.
Second aspect of the present invention provides the preparation method of above-mentioned zirconium oxide microballon antihuman globulin detection reagent card, described
Method includes step:
(a) the zirconium oxide microballon that particle diameter is 63-125 microns is filtered out, is suspended to be situated between with microballon and be washed 3-5 times, removed
Microballon breakage fragment and the bead particles of aggregation collect microballon that obtain even particle size and completely spherical in shape;It is described
The component of microballon suspension media is:
(b) microballon and antibody prepared step (a) is respectively according to volume ratio 2:1 ratio mixing, is configured to containing anti-
The microballon of IgG+C3d antibody;
(c) according to the amount of 22-28 microlitres of every pipe, the microballon that step (b) is prepared is dispensed into the microtrabeculae pipe of detection card respectively
In.
The above method is further comprising the steps of:
(d) with aluminium-foil paper by press mold mode by the sealing suitable for reading of microtrabeculae pipe.
" water " is distilled water, purified water or deionized water, preferably distilled water in microballon suspension media of the present invention.
The beneficial effects of the invention are as follows:
The first aspect of the invention, it is proposed that a kind of standardized zirconium oxide microballon suspension media system, for aoxidizing
The washing and suspension of zirconium microballon, and the stabilization of antibody and zirconium oxide microballon can be maintained for a long time, feature is:
1, the buffer system with very strong buffer capacity is devised, using potassium phosphate, sodium salt and amino acid composition
Buffer system makes the pH value of system maintain 6.6-6.8.With the single citric acid buffer system phase of blood transfusion field generally use
Than having the characteristics that buffer capacity is stronger, being conducive to that whole system is kept to be in desired buffering range, while ensure that entire
The ionic strength of zirconium oxide microballon suspension media.
2, there is significantly more efficient low salt concn system, from its composition as can be seen that the zirconium oxide microballon of the present invention suspends
Medium is used as additive using amino acid and sodium chloride, and auxiliary maintains low ionic strength environment with micro phosphate, holding
Zirconium oxide bead particles are in ball particle, and maintain the diameters of the zirconium oxide bead particles (63- in required range
125 microns).
3, the present invention has unique lubricating system, using certain density bovine serum albumin(BSA) so that red blood cell passes through
Suitable ravine is obtained when zirconium oxide microballon gap and slides ability, and the red blood cell of no agglutination is made to have completely by zirconium oxide bead particles
The ability in gap, and the red blood cell being aggregated cannot then pass through.
4, the present invention has superior protective system, selects sodium benzoate as preservative.Using polyvinylpyrrolidone
Synergistic effect, thus it is different in the ability of penetration cell film, and its antibacterial action site is also just different, so various lists
Ester is just different for the rejection ability of different types of microorganisms.Several esters it is compound, have better anti-corrosion ability, effectively prevent
The procreation of bacterium obtains the storage life of long period, while not only avoiding using Sodium azide chemical preservative to zirconium oxide microballon
The ionic strength of suspension media system is promoted to the sensitivity and specificity to zirconium oxide microballon rare blood type detection reagent card
The harmful effect of generation also avoids the intermediate for being metabolized generation during preservation using antibiotics dilute to zirconium oxide microballon
There is the harmful effect that the specificity of Blood grouping reagent card generates.
On the other hand, it in order to ensure the quality of zirconium oxide microballon antihuman globulin detection reagent card of the present invention, was preparing
It also needs to screen zirconium oxide microballon in journey, that is, first have to select zirconium oxide microballon appropriate, the item that should generally meet
Part is:It is 63-125 microns to select particle diameter, is washed, is suspended to the zirconium oxide microballon raw material that screening obtains, mesh
Be that the zirconium oxide bead particles, the zirconium oxide bead particles of aggregation, internal diameter for making the washing removal of zirconium oxide bead particles damaged exist
Other impurity components other than super large or extra small zirconium oxide bead particles and zirconium oxide microballon other than 63-125 microns.Washing
After the completion with zirconium oxide microballon suspension media come suspension oxidation zirconium microballon.
In another aspect, in order to ensure the quality of zirconium oxide microballon antihuman globulin detection reagent card of the present invention, preparing
Also pair antibody starting material mixed with zirconium oxide microballon screens in journey.
In conclusion why the zirconium oxide microballon antihuman globulin detection reagent card of the present invention can have excellent spy
Anisotropic, sensitivity and the storage life up to 1 year, are the synergistic effects for being whole system various composition.Containing 8 anti-igg+
The zirconium oxide microballon microtrabeculae pipe of C3d, which is arranged on a card, can ensure that a zirconium oxide microballon antihuman globulin, which is used only, to be detected
Reagent card can complete the antihuman globulin detection of most 8 samples.Buffer system can maintain detection reagent card reaction system
The pH needed.Low salt concn system can ensure zirconium oxide bead particles diameter in required range.Lubricating system can be with
Lubricating ability appropriate between guarantee zirconium oxide bead particles.Sodium benzoate preservative can prevent zirconium oxide microballon or antibody because
It fails for bacterial reproduction.Standardized zirconium oxide microballon can ensure the suitable gap between zirconium oxide bead particles.Mark
The antibody of standardization can ensure effective detection of antigen.
Rare blood type is detected with this zirconium oxide microballon antihuman globulin detection reagent card, the positive reaction of generation is not less than
1+.Generally the term of validity is preserved under the conditions of 18-25 DEG C to be not less than 12 months.
In short, the implementation of the present invention provides standardized zirconium oxide microballon antihuman globulin detection reagent card product, respectively
Hospital and Blood Transfusion Services can directly from production supplier from obtain the consistent zirconium oxide microballon antihuman globulin detection of standard
Reagent card is precise Identification rare blood type parting, ensure safe transfusion, diagnosing and treating neonatal hemolytic disease creates item as early as possible
Part.
Description of the drawings
Fig. 1 is zirconium oxide microballon microscope figure;
Fig. 2 is the structural schematic diagram of antihuman globulin detection reagent card of the present invention.
1-8. zirconium oxide microballon microtrabeculae pipes;9. detection reagent card;11. zirconium oxide microballon;12. anti-igg+C3d antibody;13.
Microtrabeculae pipe;14. blank card.
Specific implementation mode
Below by way of the specific embodiment real rotation that the present invention will be described in detail and possessed advantageous effect, it is intended to which help is read
Understanding spirit and substance of the present invention, any restriction can not be constituted to the practical range of the present invention.
The preparation and preservation of 1 zirconium oxide microballon antihuman globulin detection reagent card of the present invention of embodiment
Step 1: the preparation of zirconium oxide microballon suspension media
The distillation water dissolution of the above reagent, tune pH value are 6.6-6.8.
Step 2: the preparation of zirconium oxide microballon
It is 63-125 microns to select zirconium oxide microballon, particle diameter, the zirconium oxide microballon suspension media prepared with step 1
Washing 3-5 times, removes zirconium oxide breakage fragment and aggregation zirconia particles, and collection obtains even particle size and complete
Zirconium oxide microballon spherical in shape.
Step 3: the selection of antibody
Select broad spectrum antihumanglobulin reagent (anti-igg+C3d).
Wherein anti-igg ingredient sensitivity:It is required that according to volume ratio 1:2 and volume ratio 1:4 diluted IgG antihuman globulins blood
Volume ratio 1 must be compareed clearly:The red blood cell of 128 diluted anti-D (IgG) sensitization has naked eyes visible agglutination;
The sensitivity of its moderate resistance C3d ingredients:It is required that according to volume ratio 1:4 and volume ratio 1:8 diluted antihuman globulin examinations
Agent (anti-igg+C3d) must have naked eyes visible agglutination to the red blood cell of C3d sensitization.With the broad spectrum antihumanglobulin reagent stoste
Response intensity >=1+;
Broad spectrum antihumanglobulin reagent (anti-igg+C3d) generates positive reaction with the anti-D sensitized erythrocytes of IgG properties, with C3d
Sensitized erythrocyte generates positive reaction, is reacted with the red blood cell not only without IgG antibody but also without C3d antibody sensitizeds for feminine gender.
Step 4: the preparation of zirconium oxide microballon
The antibody that zirconium oxide microballon prepared by step 2 is selected with step 3 is according to volume ratio 2:1 ratio mixing, matches
The zirconium oxide microballon of reagent containing broad spectrum antihumanglobulin (anti-igg+C3d) is made.
Step 5: packing
According to 22~28 microlitres of amount of every pipe, the zirconium oxide microballon that step 4 is prepared is added separately to one piece of blank card
Nine microtrabeculae pipes in, formed with 9 zirconium oxide microballon microtrabeculae pipes zirconium oxide microballon antihuman globulin detection reagent card.For
Convenient for identification label, below reagent card label paper.
Step 6: semi-finished product measure
It is required that the anti-D potency of zirconium oxide microballon antihuman globulin detection reagent card (anti-igg+C3d) detection is not less than
The indirect antihuman globulin method measurement result of test tube;Zirconium oxide microballon antihuman globulin detection reagent card (anti-igg+C3d) detection
Anti- FyaAntibody titer is not less than the indirect antihuman globulin method measurement result of test tube;Zirconium oxide microballon antihuman globulin detection reagent
Block red blood cell response intensity >=1+ of (anti-igg+C3d) and complement sensitization.It is required that being generated with O-shaped RhD positive red blood cells positive solidifying
Collection reaction, and be feminine gender with the reaction of O-shaped RhD feminine genders red blood cell;With O-shaped FyaPositive red blood cell reaction for the positive, and with O-shaped Fya
Negative red blood cell reaction is feminine gender;It is reacted with the red blood cell of C3d sensitization for the positive, and not only without IgG antibody but also without the red of C3d sensitization
Cell effect is feminine gender.
Step 7: sealing
With aluminium-foil paper by press mold mode by the sealing suitable for reading of microtrabeculae pipe.In 18-25 DEG C of preservation after labelling label.
Step 8: food preservation test
Above-mentioned broad spectrum antihumanglobulin cards save 1 year or more, during preserving herein, broad spectrum antihumanglobulin cards tool
There is following testing result:
(1) appearance:
Zirconium oxide microballon on zirconium oxide microballon antihuman globulin detection reagent card in micro-pipe should be in flat-white, zirconium oxide
There is 1~2 millimeter of as clear as crystal liquid in microballon upper end, should not have bubble and foreign matter between zirconium oxide bead particles.
(2) sensitivity:
The sensitivity of anti-igg zirconium oxide microballon:It is required that zirconium oxide microballon antihuman globulin detection reagent card (anti-igg+C3d)
The anti-D potency of the Rh systems of detection and the anti-Fy of Duffy systemsaAntibody titer is not less than the indirect antihuman globulin of test tube
Method measurement result.
Anti- C3d sensitivity:It is required that zirconium oxide microballon antihuman globulin detection reagent card (anti-igg+C3d) and complement sensitization
Red blood cell response intensity >=1+.
(3) specific:
The specificity of anti-igg ingredient:Detect IgG property anti-Ds, it is desirable that generated with O-shaped RhD positive red blood cells positive solidifying
Collection reaction, and be feminine gender with the reaction of O-shaped RhD feminine genders red blood cell;Detect the anti-Fy of IgG propertiesaAntibody, it is desirable that with O-shaped FyaIt is positive red
Cell effect be the positive, and with O-shaped FyaNegative red blood cell reaction is feminine gender.
The specificity of anti-C3d ingredients:It is required that being reacted with the red blood cell of C3d sensitization for the positive.With not only without IgG antibody but also nothing
The red blood cell reaction of C3d sensitization is feminine gender.
2 detection reagent card of the present invention of embodiment using and detecting
1, zirconium oxide microballon broad spectrum antihumanglobulin reagent (anti-igg+C3D) detection card has 8 oxidations in the present embodiment
Zirconium microballon microtrabeculae pipe, 8 zirconium oxide microballon microtrabeculae pipes are all the zirconium oxide microballon microtrabeculae pipe containing anti-igg+C3d antibody.
2, the human red cell and serum (or blood plasma) in whole blood are detached, it will be to be checked red thin with physiological saline or LISS solution
Born of the same parents are configured to 1%-2% (percent by volume) solution of red blood cells, for use.
3, Direct antiglobulin test (DAT):
3.1 take 20 microlitres of solution of red blood cells to be checked to be added in 1 to No. 7 pipe, and 1%-2% (volume basis is added in No. 8 pipes
Than) 20 microlitres of Red Blood Cells of Normal Persons solution, as negative control
3.2 are centrifuged with special centrifugal machine, and 1200rpm is centrifuged 4 minutes, take out naked eyes judgement result.
3.3 result judgement:No. 8 pipe is negative, and experimental result is set up, otherwise experimental result is invalid.1 to No. 7
Be negative result in pipe, shows red blood cell sample in vivo not by sensitization;Be positive result in 1 to No. 7 pipe, shows red thin
Born of the same parents' sample is in vivo by sensitization;
4, indirect antihuman globulin test (IAT)
4.1 take the O-shaped solution of red blood cells of 1%-2% standards to be separately added into 1 to No. 8 pipe, often pipe 20uL.
4.2 take 20-25 microlitres of serum to be checked to be added in 1 to 7 pipe, and AB type people's negative control seras 20-25 is added in No. 8 pipes
Microlitre.
Reagent card after 4.3 sample-addings, sets in 37 DEG C of couveuses 15 minutes.
4.4 are centrifuged with special centrifugal machine, and 1200rpm is centrifuged 4 minutes, take out naked eyes judgement result.
4.5 result judgement:No. 8 pipes are negative, and experimental result is set up, otherwise experimental result is invalid.1 to No. 7 pipe
In be negative result, show the incomplete antibody that the O-shaped red cell antigens specificity of the standard is not contained in tested serum;1 to 7
Be positive result in number pipe, shows the incomplete antibody containing the O-shaped red cell antigens specificity of the standard in examinee's serum;
As a result interpretation standard:For red blood cell suspension in being positive findings in bead surface or microtrabeculae, red blood cell is all settled down to micro-pipe bottom
For negative findings.
Each sample can do 2-3 pipes simultaneously, so that experimental result is accurate and reliable.
Usual sephadex price is more than 30 times of zirconium oxide.In addition, zirconium oxide is white odorless, tasteless crystal, it is insoluble
Yu Shui, hydrochloric acid and dilute sulfuric acid.Chemical property torpescence, and high-melting-point, high resistivity, height analyse and penetrate rate and the property of low thermal coefficient of expansion
Matter is important heat-resisting material, ceramic insulating material.
The characteristics of zirconium oxide microballon is chemical physical stability height, and non-specific adsorption is low, and the microorganisms such as bacterium will not give birth to
Long, filler back-pressure spy is low.
Keep this field special by the foregoing description of the disclosed embodiments for the description to the embodiment of the present invention above
Industry technical staff can realize or use the present invention.Various modifications to these embodiments carry out those skilled in the art
Say be it will be apparent that the general principles defined herein can without departing from the spirit or scope of the present invention,
It realizes in other embodiments.Therefore, the present invention is not intended to be limited to the embodiments shown herein.
Claims (10)
1. a kind of antihuman globulin detection reagent card, which is characterized in that the detection reagent card includes:
Microtrabeculae pipe, pipe is interior to be equipped with the zirconium oxide microballon containing anti-igg+C3d antibody.
2. antihuman globulin detection reagent card as described in claim 1, which is characterized in that the microtrabeculae pipe is managed for 4-12.
3. antihuman globulin detection reagent card as described in claim 1, which is characterized in that the microtrabeculae pipe is 8 pipes.
4. the antihuman globulin detection reagent card as described in claim 1,2 or 3, which is characterized in that the zirconium oxide bead particles
It is 63-125 microns a diameter of.
5. the antihuman globulin detection reagent card as described in claim 1,2 or 3, which is characterized in that the zirconium oxide microballon with it is anti-
The volume ratio of body is 2:1.
6. antihuman globulin detection reagent card as claimed in claim 4, which is characterized in that the body of the zirconium oxide microballon and antibody
Product is than being 2:1.
7. antihuman globulin detection reagent card as claimed in claim 5, which is characterized in that set aluminum foil sealing at the microtrabeculae tube opening
Piece.
8. antihuman globulin detection reagent card as claimed in claim 7, which is characterized in that label paper on the reagent card.
9. the preparation method of antihuman globulin detection reagent card as claimed in claim 4, which is characterized in that the method includes steps
Suddenly:
(a) the zirconium oxide microballon that particle diameter is 63-125 microns is filtered out, is washed 3-5 times with microballon suspension media, is removed micro-
Pearl breakage fragment and the bead particles of aggregation collect microballon that obtain even particle size and completely spherical in shape;It is described micro-
The component of pearl suspension media is:
(b) microballon and antibody prepared step (a) is respectively according to volume ratio 2:1 ratio mixing, be configured to containing anti-igg+
The microballon of C3d antibody;
(c) according to the amount of 22-28 microlitres of every pipe, the microballon that step (b) is prepared is dispensed into respectively in the microtrabeculae pipe of detection card.
10. the preparation method of zirconium oxide microballon antihuman globulin detection reagent card as claimed in claim 7, which is characterized in that institute
It is further comprising the steps of to state method:
(d) with aluminium-foil paper by press mold mode by the sealing suitable for reading of microtrabeculae pipe.
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