CN1755367B - Reagent for detecting sperm membrane surface antigen by immuno-magnetic sand method, its preparation and detection method - Google Patents
Reagent for detecting sperm membrane surface antigen by immuno-magnetic sand method, its preparation and detection method Download PDFInfo
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- CN1755367B CN1755367B CN 200410040814 CN200410040814A CN1755367B CN 1755367 B CN1755367 B CN 1755367B CN 200410040814 CN200410040814 CN 200410040814 CN 200410040814 A CN200410040814 A CN 200410040814A CN 1755367 B CN1755367 B CN 1755367B
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Abstract
The invention discloses a semen film surface antibody immunity method testing agent and preparing and testing method. The semen film surface antibody immunity method testing agent comprises: 1.8um-2.2um immunity bead which can possesses human antibody IgG or IgA or IgM micromole rubber latex particle, the washing liquid which contents 0.1%-0.5% bonnie serum albumin and the scale dissolver which contents 4%-6% bonnie serum albumin. The agent can also comprise semen extracting liquid with high activity. The invention provides the method for preparing the semen film surface antibody immunity method testing agent.
Description
[technical field]
The present invention relates to a kind of in-vitro diagnosis usefulness reagent and preparation thereof, detection method, be specifically related to a kind of sperm membrane surface antibody immunobead method detectable and preparation thereof, detection method.
[background technology]
Immune factor is one of cause of disease of male sterility, so its Clinical detection is a crucial problem.The World Health Organization's regulation mixed agglutination reaction (MAR) in 2000 and immunobead test (IBT) are the code test of diagnosis immune infertility.
Because immunobead method detectable obtains than difficult, and domestic mainly is to adopt enzyme linked immunological experiment (ELISA) to detect serum or the interior antisperm antibody of refining, this detection has little significance for the male, and false positive rate is very high at present.And in the practical operation of immunobead test, it is not of uniform size also to exist immunobead, and testing result is difficult to interpretation, as shown in Figure 1.Simultaneously, there are shortcomings such as sample for azoospermia, oligospermatism can't directly detect.
[summary of the invention]
The technical problem to be solved in the present invention provides a kind ofly is convenient to preparation, the easy interpretation of testing result, and further can directly detect azoospermia, oligospermatism sample sperm membrane surface antibody immunobead method detectable and preparation, detection method.
For achieving the above object, the technical solution used in the present invention is a kind of sperm membrane surface antibody immunobead method detectable, comprises
1) immunobead: diameter is the crosslinked polymer emulsion granule that anti-human IgG or IgA or IgM antibody are arranged of 1.8～2.2 μ m;
2) cleaning mixture: the tyrode's solution that contains 0.1～0.5% bovine serum albumin;
3) multiple suspension: the tyrode's solution that contains 4～6% bovine serum albumin;
Above-mentioned detectable also can further comprise
4) high motile sperm extracting solution: improvement Earl
1The s culture fluid.
The concrete composition of described high motile sperm extracting solution can be: sodium chloride (NaCl) 0.6～0.8%, potassium chloride (KCl) 0.04～0.05%, calcium chloride dihydrate (CaCl
22H
2O) 0.02～0.03%, Magnesium sulfate heptahydrate (MgSO
47H
2O) 0.01～0.02%, sodium bicarbonate (NaHCO
3) 0.2～0.3%, sodium dihydrogen phosphate dihydrate (NaH
2PO
42H
2O) 0.01～0.02%, Sodium Pyruvate 0.002～0.003%, 60% is syrupy sodium lactate 0.3～0.4%, sodium azide (NaN
3) 0.01～0.02%, the aqueous solution of glucose 0.1～0.15%, Citric Acid Monohydrate 0.03～0.04%, calf serum 0.8～1.2%.
Described immunobead can be stored in the diluent, and the main component of this diluent is to contain Na
+Phosphate buffer.
Described diluent also can contain the bovine serum albumin of 1～2g/L.
Described polymer emulsion is Carboxylated Polystyrene latex preferably.
Described tyrode's solution is to contain CaCl
20.1～0.2g/L, KCl0.1～0.2g/L, NaH
2PO
40.04～0.06g/L, MgCl
26H
2O0.1～0.2g/L, NaCl6～8g/L, NaHCO
31～1.5g/L, the deionized water solution of glucose 1～1.5g/L.
The present invention also provides a kind of method for preparing sperm membrane surface antibody immunobead method detectable, may further comprise the steps,
1) preparation of immunobead and dilution
A) preparation immunobead diluent: in concentration is in the phosphate buffer of 0.01～0.02mol/L, presses Na
+Concentration is that the ratio of 100～150mmol/L adds Na salt, filters the back and preserves;
B) preparation immunobead: in concentration is in the carbonate buffer solution of 0.1～0.2mol/L, adds 0.2～0.4% acrylic resin S-100, is that 1.8～2.2 μ m polymer emulsions mix with diameter, is placed to abundant reaction;
The gained mixed liquor is centrifugal, abandon supernatant, add 0.2～0.3mol/L phosphate buffer, add the anti-human IgG of response magnitude or the antibody of anti-people IgA or anti-people IgM again, be placed to abundant reaction behind the mixing;
The volume ratio of pressing 1:100 adds 1～1.5mol/L ammonia chloride, and fully reaction back recentrifuge is abandoned supernatant, adds the phosphate buffer of 0.02～0.03mol/L;
C) dilution immunobead emulsion: use the immunobead diluted b that a) makes) immunobead that makes, with immunobead after the dilution and the reaction of positive sperm, observed result, getting positive sperm surface, to adhere to the diluted concentration of immunobead number when being 5～15 be optimum concentration, with the immunobead emulsion with the immunobead diluted to optimum concentration;
2) preparing washing liquid: in tyrode's solution,, preserve after the Entkeimung in the ratio adding bovine serum albumin of 1～5g/L;
3) the multiple suspension of preparation: in tyrode's solution,, preserve after the Entkeimung in the ratio adding bovine serum albumin of 40～60g/L.
The bovine serum albumin that before filtration, also can add 1～2g/L in a) process of preparation method step 1) of the present invention.
The polymer emulsion that uses in the preparation method of the present invention is Carboxylated Polystyrene latex preferably.
The present invention also provides a kind of aforementioned detection method that contains the sperm membrane surface antibody immunobead method detectable of high motile sperm extracting solution of using, comprise the detection sample preparation steps, carry out immunobead experimental procedure and interpretation step as a result detecting sample, described detection sample preparation steps comprises the washing step to semen sample, it is characterized in that: in detecting sample preparation steps, the preparation of oligospermatism or azoospermia sample is also comprised the steps
1) the semen sample upper strata after washing adds high motile sperm extracting solution, and thermophilic leaves standstill, and the higher sperm of vigor is moved about to the upper strata from bottom;
2) get solution that the upper strata contains motile sperm detection sample as subsequent step.
Adopt technical scheme provided by the invention, following advantage is arranged: 1) adopting diameter is that the polymer emulsion granule of 1.8～2.2 μ m is as antibody carrier, make that immunobead size for sperm is more suitable, avoid the too small non-specific adsorption that causes of immunobead or excessive cause influence motility of sperm, make testing result be easy to interpretation; 2) further be equipped with high motile sperm extracting solution, can have detected the sample of azoospermia, oligospermatism, overcome the shortcoming that this type of sample can not directly detect because of reasons such as power of washing or concentration; 3) the reagent preparation material all is easy to obtain, and method is simple.
[description of drawings]
Fig. 1 is to use the testing result of available reagent at microscopically.
Fig. 2 is to use the testing result of reagent of the present invention at microscopically.
[specific embodiment]
Below the present invention is described in further detail.
Embodiment one, a kind of sperm membrane surface antibody immunobead method detectable comprise
1) be stored in immunobead in the diluent: the main component of described diluent is that pH is 7.4, contains Nacl125mmol/L, bovine serum albumin 1.5g/L, NaN
30.15%, the phosphate buffer of phosphate solute 0.02mol/L, described immunobead are that diameter is about 2.0 μ m, the crosslinked Carboxylated Polystyrene latex particle that anti-human IgG or IgA or IgM antibody are arranged;
2) cleaning mixture: the tyrode's solution that contains 0.3% bovine serum albumin;
3) multiple suspension: the tyrode's solution that contains 5% bovine serum albumin;
4) high motile sperm extracting solution: contain NaCl0.7%, KCl0.045%, CaCl
22H
2O0.025%, MgSO
47H
2O0.015%, NaHCO
30.25%, NaH
2PO
42H
2O0.015%, Sodium Pyruvate 0.0025%, 60% are syrupy sodium lactate 0.35%, NaN
30.015%, the aqueous solution of glucose 0.13%, Citric Acid Monohydrate 0.035%, calf serum 0.09%;
Wherein, tyrode's solution is that pH is 7.3, contains CaCl
20.15g/L, KCl0.15g/L, NaH
2PO
40.05g/L, MgCl
26H
2O0.15g/L, NaCl7g/L, NaHCO
31.25g/L, the deionized water solution of glucose 1.25g/L.
In reagent of the present invention,
(1) neccessary composition in the diluent is phosphate buffer and certain density (100～150mmol/L) Na
+Ion, they play appropriate pH (7.3～7.5) that keeps the immunobead storage and the effect of keeping antibody activity respectively, and each is worth the too high or too low activity that all can cause adhering to antibody and reduces.In all the other compositions, bovine serum albumin plays the better protection effect, the desirable 1～2g/L of its content, available ovalbumin, replacements such as Polyethylene Glycol or gelatin as protein stabiliser; NaN
3As antiseptic, play the effect that prolongs the reagent storage time, its content is desirable 0.1～0.2%, replacements such as available Sodium Mercurothiolate, lysozyme or antibiotics.Reagent of the present invention can be preserved more than 1 year under the state that adds antiseptic, did not add also and can preserve about one month.
(2) effect of Carboxylated Polystyrene latex provides carboxyl and antibody linked, and its granular size all is relatively more suitable between 1.8～2.2 μ m.Also can replace with the satisfactory polymer emulsion of other granular sizes, for example, polyacrylamide.
(3) effect of high motile sperm extracting solution is that the sperm that will have higher activity power in the seminal fluid extracts it by its upstream, can adopt existing improvement Earl
1S culture fluid, neccessary composition wherein are certain density Na
+(0.23～0.32%), K
+(0.02～0.026%), Ca
2+(0.005～0.008%), MgSO
4(0.005～0.01%), HCO
3 -1(0.14～0.22%), acetone acid radical anion (0.0016～0.0024%), lactic acid anion (0.095～0.127%) and glucose (0.1～0.15%), ion, acid-base value and energy etc. that they provide sperm to need in active procedure.Other compositions are as an amount of NaH
2PO
42H
2O (0.01～0.02%), NaN
3(0.01～0.02%), Citric Acid Monohydrate (0.03～0.04%), calf serum (0.08～0.1%) etc. can make solution play the effect of better extraction motile sperm and the holding time of prolongation solution etc.
(4) tyrode's solution also can adopt other those component fit systems in the known technology as a kind of physiological solution commonly used (pH desirable 7.2～7.4), for example, uses MgSO
47H
2O replaces MgCl
26H
2O etc.
(5) the best storage temperature of reagent of the present invention is 2～8 ℃, is lower than 0 ℃ reagent was lost efficacy, at room temperature then reagent effect duration shortening.
(6) based on application need, reagent of the present invention can be made into the form of test kit, and the concrete amount that is equipped with of each several part is in the test kit: can store immunobead 4～6 μ l, cleaning mixture 8～10ml, multiple suspension 0.15～0.25ml, high motile sperm extracting solution 1～3ml.
Embodiment two, a kind of method for preparing sperm membrane surface antibody immunobead method detectable among the embodiment one may further comprise the steps,
1) preparation of immunobead and dilution:
A) preparation immunobead diluent: in concentration is in the phosphate buffer of 0.02mol/L, adds the Nacl of 125mmol/L, the bovine serum albumin of 1.5g/L and 0.15% NaN
3, transferring pH is 7.4, filters the back with 0.22 μ m filter and preserves.
B) preparation immunobead: in 2.5ml concentration is in the carbonate buffer solution of 0.5mol/L, adds 0.3% acrylic resin S-100, and transferring pH is 9.5, is that Carboxylated Polystyrene latex about 2.0 μ m mixes with particle diameter, puts 5 ℃ and makes sufficient reacting in 1 hour.
Described particle diameter is that to take from concentration that U.S. Sigma company produces be 2.5% Carboxylated Polystyrene latex to the Carboxylated Polystyrene latex about 2.0 μ m.Getting this product 500 μ l, is 9.5 with pH, and concentration is that the carbonate buffer solution of 0.15mol/L washes twice, removes its protection composition in storage process and can use.
The gained mixed liquor is centrifugal, abandon supernatant, add the phosphate buffer of 2.5ml0.25mol/L, contain the antibody of the anti-human IgG of 0.15ml or anti-people IgA or anti-people IgM in this phosphate buffer, transferring pH is 4.4, mixing made sufficient reacting in 3 hours for rearmounted 5 ℃;
Add 1.25mol/L ammonia chloride 55 μ l, 5 ℃ of reaction 10min, the carboxyl of binding antibody not on the sealing latex.Recentrifuge is abandoned supernatant, adds the phosphate buffer of 0.025mol/L, and transferring pH is 7.3;
C) dilution immunobead emulsion: use the immunobead diluent that a) makes with b) the immunobead emulsion dilution that makes is a series of concentration, with immunobead after the dilution and the reaction of positive sperm, observed result, getting positive sperm surface, to adhere to the diluted concentration of immunobead number when being 10 be optimum concentration, with the immunobead emulsion with the immunobead diluted to optimum concentration.
2) preparing washing liquid: taking by weighing the 0.35g bovine serum albumin, to be dissolved in 100ml pH be in 7.3 the tyrode's solution, with preserving after the 0.22 μ m microporous membrane Entkeimung;
3) the multiple suspension of preparation: taking by weighing the 5g bovine serum albumin, to be dissolved in 100ml pH be in 7.3 the tyrode's solution, with preserving after the 0.22 μ m microporous membrane Entkeimung;
(step 2), 3) in tyrode's solution as a kind of physiological solution commonly used, can adopt those common in known technology manner of formulation, for example: press CaCl
20.15g/L, KCl0.15g/L, NaH
2PO
40.05g/L, MgCl
26H
2O0.15g/L, NaCl7g/L, NaHCO
31.25g/L the ratio of glucose 1.25g/L is dissolved in each solute in the deionized water, transferring pH is 7.3.)
4) the high motile sperm extracting solution of preparation: press NaCl7g/L, KCL0.45g/L, CaCl
22H
2O0.25g/L, MgSO
47H
2O0.15g/L, NaHCO
32.5g/L, NaH
2PO
42H
2O0.15g/L, Sodium Pyruvate 0.025g/L, contain 60% syrupy sodium lactate 3.5ml/L, NaN
30.15g/L, the ratio of glucose 1.25g/L, Citric Acid Monohydrate 0.35g/L is dissolved in each solute in the distilled water successively;
Add 56 ℃ of calf serum 9ml/L after the 30min deactivation again, mixing, transferring pH is 7.4, with preserving after the 0.22 μ m microporous membrane Entkeimung.
In preparation method of the present invention,
(1) to adopt microporous membrane to filter be for Entkeimung and guarantee not contain in the solution and impurity that immunobead shape size is similar, in order to avoid the result becomes immunobead with the impurity particle misidentification when observing), the microporous membrane specification all suits between 0.2～0.36 μ m;
(2) addressing " transferring pH to be " and can adopt the method for those common in known technology adjustment pH value of solution, for example, generally is to use KH to phosphate buffer
2PO
4, NaH
2PO
4Deng, generally be to use Na to carbonate buffer solution
2CO
3, NaHCO
3Deng.
The sperm membrane surface antibody immunobead method detectable that makes among embodiment three, a kind of embodiment of use two is carried out the method that the sperm membrane surface antibody detects, may further comprise the steps,
1) reagent is prepared
The washing immunobead: face with before can wash immunobead, avoid the non-specific sperm surface that sticks to of immunobead, concrete grammar is
A) in the Eppendorf pipe, add immunobead and the 1.5ml cleaning mixture that 50 μ l are diluted to optimum concentration, the centrifugal 10min of 2000g;
B) abandon supernatant, add multiple suspension to 50 μ l.
Immunobead after the washing is put 2～8 ℃ of preservations, available 1 month.
2) sample preparation
A) common semen sample preparation
1. the bright seminal fluid with certain volume is transferred to the conical bottom centrifuge tube, adds cleaning mixture to 4ml.Seminal fluid application of sample amount can be according to sperm concentration and activity ratio with reference to following table
2. with centrifugal 8 minutes of gained mixed liquor 500g.
3. careful abandoning supernatant adds cleaning mixture 4ml, and sperm is precipitated group's mixing gently, 500g recentrifuge 8 minutes.
4. careful tipping supernatant adds multiple suspension 0.2ml, and sperm is precipitated group's mixing gently.
B) few sperm and the preparation of azoospermia subsample
1. the seminal fluid with certain volume is transferred to the conical bottom centrifuge tube, adds cleaning mixture to 4ml.Seminal fluid application of sample amount can be according to sperm concentration and activity ratio with reference to following table:
2. with centrifugal 8 minutes of gained mixed liquor 500g.
3. careful abandoning supernatant adds cleaning mixture 4ml, and sperm is precipitated group's mixing gently, 500g recentrifuge 8 minutes.
4. careful tipping part supernatant, the about 0.5ml of pipe end liquid stay, mixing is transferred in the motile sperm extraction tube gently.
5. carefully add high motile sperm extracting solution 2ml on the upper strata, test tube is tilted 45 ° to strengthen the contact area of seminal fluid and extracting solution, 37 ℃ leave standstill 50min the higher sperm of vigor are moved about to the upper strata from bottom.
6. gently that test tube is vertical, carefully draw solution 1ml that the upper strata contains motile sperm to another test tube, add the high motile sperm extracting solution of 2ml then, centrifugal 5 minutes of 500g.
4. careful tipping supernatant adds multiple suspension 0.2ml, and mixing is adjusted sperm concentration to 50 * 10 with multiple suspension gently
6About/ml.
C) serum/refining/cervical mucus sample preparations:
1. cervical mucus is earlier with liquefier dissolving (liquefier can be purchased in addition), fresh serum, 56 ℃ of deactivations in 30 minutes of refining.
2. the dilution proportion of serum or refining or cervical mucus being pressed 1:5 with multiple suspension, as the 10ul specimen as the resuspended liquid of 40 μ l.
3) direct immunization pearl test
A) adding on the clean glass slide: process 5ul step 2) a) or b) sperm suspension of preparation and the immunobead (with preceding abundant mixing) after the 5ul washing.
B) with the immunobead after sample injector head or cover plate edge mixing sample and the washing 5 times, covered.
C) room temperature (25～35 ℃) is hatched and was made antigen-antibody fully react in 10 minutes.
4) immunobead test indirectly
A) can select for use following arbitrary specimen compound mode to carry out antisperm antibody detects:
1. male spouse's motile sperm+women spouse's serum or cervical mucus
2. donor motile sperm (antisperm antibody feminine gender)+refining to be checked
3. donor motile sperm (antisperm antibody feminine gender)+cervical mucus to be checked
4. donor motile sperm (antisperm antibody feminine gender)+serum to be checked
B) operational approach
1. get washing back sperm suspension 50ul in the Eppendorf pipe, increase serum or refining or cervical mucus diluent 50ul in addition, test tube is added a cover, mixing.Hatched 1 hour for 37 ℃.
2. add cleaning mixture to 1ml, centrifugal 8 minutes of 500g.
3. careful abandoning supernatant adds cleaning mixture 1ml, and sperm is precipitated group's mixing gently, 500g recentrifuge 8 minutes.
4. careful tipping supernatant is suspended into original volume (100ul), mixing gently with multiple suspension again with the sperm granule.
5. adding on the clean glass slide: the immunobead (with preceding abundant mixing) after sperm suspension after the above-mentioned washing of 5ul and the 5ul washing.
6. with sample injector head or cover plate edge mixing sample and washing immunobead 5 times, covered.
7. room temperature (25～35 ℃) was hatched 10 minutes.
5) interpretation as a result
400 * sentence read result to 600 * high power microscope or the phase contrast microscope.Distinguish in the documentary film attached to the percentage of motile spermatozoa on the immunobead (ignoring the combination of tail point).Reporter antibody kind (IgG, IgA or IgM) and immunobead are with the bonded position of sperm (head, stage casing, tail).
Fig. 2 and Fig. 1 have provided employing reagent of the present invention and available reagent (IMMUNBEADREAGENT RABBIT AND ANTI-HUMAN IMMUNOGLOBULIN, the IRVINE SCIENTIFIC of manufacturer (Irving company)) respectively in the observed figure as a result of DMLS2 (Leica of manufacturer) microscopically (shooting of Leica DFC480 special digital camera).Comparison diagram 2 (reagent of the present invention) and Fig. 1 (available reagent) can it is evident that, owing to immunobead in the reagent of the present invention is of moderate size, unanimity, thereby it is the judgement of binding site with combining of sperm or all is very clear and easy in conjunction with the resolution of number, and the impurity of having avoided other shapes and size makes the result be easy to interpretation to interference and influence that observation causes; And among the testing result figure of available reagent, immunobead not of uniform size is deposited in around the sperm, almost can't tell concrete bonded immunobead number on the sperm significantly, and is difficult to tell impurity and microsphere from background, causes erroneous judgement easily.
6) normal reference value
If 50% motile sperm surface adhesion has immunobead, clinical meaning is arranged then.
If, then do not have clinical meaning in conjunction with only limiting to the tail point.
7) clinical applicability
The method is the immune infertility diagnostic method that WHO recommends.
Claims (8)
1. a sperm membrane surface antibody immunobead method detection kit comprises
1) immunobead: the crosslinked polymer emulsion granule that anti-human IgG or IgA or IgM antibody are arranged, institute
The diameter of stating immunobead is 1.8～2.2 μ m;
2) cleaning mixture: the tyrode's solution that contains 0.1～0.5% bovine serum albumin;
3) multiple suspension: the tyrode's solution that contains 4～6% bovine serum albumin;
4) high motile sperm extracting solution: by weight percentage, described high motile sperm extracting solution is sodium chloride-containing 0.6～0.8%, potassium chloride 0.04～0.05%, calcium chloride dihydrate 0.02～0.03%, Magnesium sulfate heptahydrate 0.01～0.02%, sodium bicarbonate 0.2～0.3%, sodium dihydrogen phosphate dihydrate 0.01～0.02%, Sodium Pyruvate 0.002～0.003%, 60% aqueous solution for syrupy sodium lactate 0.3～0.4%, sodium azide 0.01～0.02%, glucose 0.1～0.15%, Citric Acid Monohydrate 0.03～0.04%, calf serum 0.8～1.2%.
2. sperm membrane surface antibody immunobead method detection kit according to claim 1, it is characterized in that: also comprise diluent, described immunobead is suspended from the diluent, and the main component of this diluent is to contain Na
+Phosphate buffer.
3. sperm membrane surface antibody immunobead method detection kit according to claim 2, it is characterized in that: described diluent also contains the bovine serum albumin of 1～2g/L.
4. sperm membrane surface antibody immunobead method detection kit according to claim 2, it is characterized in that: described polymer emulsion is a Carboxylated Polystyrene latex.
5. sperm membrane surface antibody immunobead method detection kit according to claim 2, it is characterized in that: described tyrode's solution is to contain CaCl
20.1～0.2g/L, KCl 0.1～0.2g/L, NaH
2PO
40.04～0.06g/L, MgCl
26H
2O 0.1～0.2g/L, NaCl 6～8g/L, NaHCO
31～1.5g/L, the deionized water solution of glucose 1～1.5g/L.
6. the preparation method of a sperm membrane surface antibody immunobead method detectable may further comprise the steps,
1) preparation of immunobead and dilution:
A) preparation immunobead diluent: in concentration is in the phosphate buffer of 0.01～0.02mol/L, presses Na
+Concentration is that the ratio of 100～150mmol/L adds Na salt, filters the back and preserves;
B) preparation immunobead: in concentration is in the carbonate buffer solution of 0.1～0.2mol/L, adds 0.2～0.4% acrylic resin S-100, is that 1.8～2.2 μ m polymer emulsions mix with diameter, is placed to abundant reaction;
The gained mixed liquor is centrifugal, abandon supernatant, add 0.2～0.3mol/L phosphate buffer, add the anti-human IgG of response magnitude or the antibody of anti-people IgA or anti-people IgM again, be placed to abundant reaction behind the mixing;
Add 1～1.5mol/L ammonia chloride by 1: 100 volume ratio, fully reaction back recentrifuge is abandoned supernatant, adds the phosphate buffer of 0.02～0.03mol/L;
C) dilution immunobead emulsion: use the immunobead diluted b that a) makes) immunobead that makes, with immunobead after the dilution and the reaction of positive sperm, observed result, getting positive sperm surface, to adhere to the diluted concentration of immunobead number when being 5～15 be optimum concentration, with the immunobead emulsion with the immunobead diluted to optimum concentration;
2) preparing washing liquid: in tyrode's solution,, preserve after the Entkeimung in the ratio adding bovine serum albumin of 1～5g/L;
3) the multiple suspension of preparation: in tyrode's solution,, preserve after the Entkeimung in the ratio adding bovine serum albumin of 40～60g/L.
7. the method for preparing sperm membrane surface antibody immunobead method detectable according to claim 6 is characterized in that: the bovine serum albumin that also adds 1～2g/L in a) process of step 1) before filtration.
8. the detection method of a sperm membrane surface antibody immunobead method detectable, comprise the detection sample preparation steps, carry out immunobead experimental procedure and interpretation step as a result detecting sample, described detection sample preparation steps comprises the washing step to semen sample, it is characterized in that: in detecting sample preparation steps, the preparation of oligospermatism or azoospermia sample is also comprised the steps
1) the semen sample upper strata after washing adds high motile sperm extracting solution, and thermophilic leaves standstill, and the higher sperm of vigor is moved about to the upper strata from bottom;
2) get solution that the upper strata contains motile sperm detection sample as subsequent step.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0348174A2 (en) * | 1988-06-23 | 1989-12-27 | Bio-Rad Laboratories, Inc. | Sperm antibody test |
| CN1432811A (en) * | 2002-01-06 | 2003-07-30 | 安徽安科生物工程股份有限公司 | Sperm antibody testing reagent |
| CN1755366A (en) * | 2004-09-30 | 2006-04-05 | 深圳华康生物医学工程有限公司 | IgG detection reagent for mixed agglutination reaction of sperm membrane surface antibody, and immuno-microsphere preparation method |
-
2004
- 2004-10-01 CN CN 200410040814 patent/CN1755367B/en not_active Expired - Fee Related
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0348174A2 (en) * | 1988-06-23 | 1989-12-27 | Bio-Rad Laboratories, Inc. | Sperm antibody test |
| CN1432811A (en) * | 2002-01-06 | 2003-07-30 | 安徽安科生物工程股份有限公司 | Sperm antibody testing reagent |
| CN1755366A (en) * | 2004-09-30 | 2006-04-05 | 深圳华康生物医学工程有限公司 | IgG detection reagent for mixed agglutination reaction of sperm membrane surface antibody, and immuno-microsphere preparation method |
Non-Patent Citations (2)
| Title |
|---|
| MacMillan RA, Baker HW..Comparison of latex and polyacrylamide beads for detectingsperm antibodies..Clin Reprod Fertil.5 4.1987,5(4),203-209. |
| MacMillan RA, Baker HW..Comparison of latex and polyacrylamide beads for detectingsperm antibodies..Clin Reprod Fertil.5 4.1987,5(4),203-209. * |
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| CN1755367A (en) | 2006-04-05 |
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