JPH03111760A - Testing tool and testing method for fertility of sperm - Google Patents

Abstract

PURPOSE:To make it possible to evaluate fertility by coupling a specific antibody to each solid-state granule at an antigen part which appears at a human sperm that has gained fertility, obtaining the granule for testing, coupling the sperm to the granule, and counting the coupled number of the sperm. CONSTITUTION:An antibody (e.g. protein A) having specificity with respect to an antigen part on the acrosome of a human sperm (including film) is coupled to the surface of each solid-state granule (e.g. glass bead), and the granule for testing fertility is manufactured. When a test is conducted, it is convenient to prepare one kit for containing the testing granule, a magnet, sperm incubation media and others. In the test, the sperm is picked up from a person under test, and a sperm suspension is obtained. The test granules are added into the suspension and incubated. Thereafter, the granules are separated and washed. Since the antigen part on the acrosome of the sperm having the fertility is exposed, the part is coupled with the antibody on the granule. The number of the coupled sperm is measured with a means such as a microscope. When the number of couplings is less than 5 immediately after the sperm has been picked up and more than 25 after 24 hours, it is judged that the fertility is excellent.

Description

DETAILED DESCRIPTION OF THE INVENTION [Field of Industrial Application] The present invention relates to test granules (beads) used in a human sperm fertility test, a test kit containing the same, and a fertility test method.

[Conventional technology]

Mammalian sperm do not have fertilizing ability in the ejaculated state,
It is known that the female reproductive tract undergoes a photobody reaction, undergoes physiological functional changes, and acquires fertilization potential. Fusion occurs between the acrosome and the acrosome enzyme is released, and both membranes separate from the sperm, exposing the endosome membrane.

Fertility has also been found to be acquired by incubating ejaculated sperm.

Conventional methods for measuring human sperm fertility, for example for the purpose of diagnosing or treating infertility, include methods that measure semen volume, sperm concentration, sperm motility, etc. However, this is not a method of observing fertility itself, so it is completely inaccurate. Another recent method is the hamster test, which examines whether incubated sperm fuses with hamster eggs (which have the ability to fuse with human sperm), but this is a complicated procedure and requires a The drawback is that reproducibility is poor, with different values appearing each time. In addition, there is a method (triple stain) that combines various staining methods, but this method also requires complicated operations and the results are not reliable.

[Problem to be solved by the invention]

Therefore, it is simple, reproducible, and has a theoretical basis.
There was a desire to develop a method for measuring the fertility of human sperm.

[Means to solve the problem]

The inventor focused on the use of antigen-antibody reactions in order to improve the method for measuring human sperm fertility. As a result of repeated research, we succeeded in producing polyclonal and monoclonal antibodies specific to the antigenic sites that appear in human sperm that have acquired fertilization potential, and we also developed these antibodies using labeled antibody methods (fluorescence or enzyme antibody methods). succeeded in specifically staining spermatozoa with fertilizing ability using
No. 300) further developed granules bound to the above-mentioned antibodies, thereby making it possible to easily and quickly diagnose male infertility.

[Structure of the invention]

That is, the present invention provides: (1) Granules for testing sperm fertility, which are formed by binding to the surface of solid granules an antibody that has specificity for the antigenic site on the human sperm acrosome (including the membrane). , (2) (a) A kit for testing sperm fertility, which includes the test granules (b) magnet (c) sperm incubation medium described in item 1 above, (3) for the test in (a) in item 2 above. A kit for testing sperm fertility, comprising instead of granules, an antibody having specificity for an antigenic site on the human sperm acrosome (including the membrane) and solid granules capable of binding said antibody to the surface; (4) A method for testing sperm fertility, which comprises bringing test sperm into contact with the test granules described in item 1 above, and observing sperm with exposed antigenic sites on the acrosome bound to the granules. This is what we provide.

The above antibodies are produced by culturing hybridomas produced by fusion of antibody-producing cells of mammals (excluding humans) and permanently proliferating cells, which are immunized with antigenic sites on the human sperm acrosome (including the membrane). It is produced by separating and collecting antibodies that have specificity for the antigenic site on the human sperm acrosome (including the membrane) (Japanese Patent Application No. 1-63300).

Hereinafter, the above invention will be explained in detail.

(Production of Antibody) 1. Immunization To produce the antibody used in this invention, a mammal is first immunized with the antigenic site on the human sperm acrosome (including the membrane). Human spermatozoa with exposed photophores can be prepared by pre-incubating the ejaculated sperm in a medium containing e.g. human serum albumin, or with an anionic surfactant such as sodium deoxycholate, or with a cationic, non-ionic, It can be obtained by treatment with an amphoteric surfactant or with an ionophore such as A23187. Immunization is carried out, for example, as follows. Collect sperm and immunize mammals such as mice and rats. It is preferable that the mammal be of the same lineage as the permanently proliferating cell of the partner for cell fusion. The age of the animal is preferably 5 to 10 weeks for mice, for example. The sex can be either male or female. The number of human sperm used for immunization is, for example, 5X10' to 2XI per mouse in the case of mice.
O' number is preferable. Sperm can be suspended in e.g. PBS or mixed with Freund's complete adjuvant at l=1
It is preferable to mix the emulsions at a ratio of 1 to 1 and administer the emulsion to animals intraperitoneally, intravenously, subcutaneously, etc. This immune manipulation 1~
Repeat 1 to 5 times at 3-week intervals. The final immunization is carried out, for example, by suspending sperm in PBS and administering it intravenously or intraperitoneally to the animal. Polyclonal antibodies can be obtained from the body fluids or antibody-producing cells of animals immunized in this way.

The antibody titer of the animal is measured, and when it has risen sufficiently, the antibody or producing cells are collected.

2. Cell fusion Antibody-producing cells are taken out from the animal immunized with human sperm as described above. Antibody-producing cells can be obtained from the pancreas, lymph nodes, peripheral blood, etc., but pancreas is preferred. For example, the pancreas is aseptically removed 2 to 5 days after the final immunization, cut into small pieces with scissors in Dulbecco's MEM medium to suspend the pancreatic cells, and then centrifuged to collect the pancreatic cells for use.

As the permanently proliferating cell to be fused, any permanently proliferating cell can be used, but myeloma cells are frequently used. Permanently proliferating cells are preferably derived from the same species of animal as the antibody-producing cells. For example, in the case of a mouse, P3UIP3X63Ag8. UL (P3Ul), P3
/NSI/I-Ag4-1 (NS-1), SP210
-Agl 4 (SF3), P3X63Ag8 (X63)
, P3X63-Ag8°653 (653), etc. are used. Moreover, as permanently proliferating cells, those having characteristics that can be used as markers during selection, such as 8-azaguanine resistant cell lines and hypoxanthine guanine phosphoribosyltransferase-deficient cell lines, are preferable. These cell lines are available from, for example, American Type Culture Collection (ATCC), Fujisawa Pharmaceutical Co., Ltd., or Dainippon Pharmaceutical Co., Ltd. For fusion, one of these permanently proliferating cells is cultured in a growth medium;
Before fusion, the cells are washed with, for example, Dulbecco's MEM medium and collected by centrifugation.

The fusion is performed, for example, as follows. Mix antibody-producing cells (e.g., pancreatic cells) and permanently proliferating cells (e.g., myeloma cells) at a cell number ratio of 2 to +0:1 and incubate at 37°C.
A fusion accelerator such as polyethylene glycol (e.g., average molecular weight 1300-7500.20-40%) is gradually added while maintaining the temperature, or an electric pulse (e.g.
High voltage direct current (such as V/cm) is applied for a short period of time to cause cell fusion. Fusion is stopped by adding culture medium and diluting the fusion promoter, and cells are separated by centrifugation.

Next, cells are treated with hypoxanthine, aminopterin, etc.
suspended in HAT medium with thymidine added to the growth medium;
Dispense 200 μl/well into a 96-well microtest plate and store at 37°C, COt 5%, and humidity 95% CO2.
, in an incubator (hereinafter referred to as CO; the culture conditions in the incubator are all the same as above). Replace half of the culture medium with fresh HAT medium every two days. After culturing for about one week, the medium is changed to HT medium, which is a growth medium supplemented with hypoxanthine and thymidine.

3. Screening and cloning of hybridomas Next, culture in HT medium for several days, and when the hybridoma colonies have spread to about half of the wells of the microtest plate, determine which wells' hybridomas have produced monoclonal antibodies against human sperm. Screening to see if

Screening is performed, for example, as follows. A portion of the culture supernatant from a well in which hybridomas are growing is taken, and whether or not it reacts with human sperm is examined using a known labeled antibody method such as an enzyme antibody method or a fluorescent antibody method.

Next, hybridomas producing monoclonal antibodies that react with human sperm are cloned using known techniques such as the limiting dilution method and the soft agar method to create a population of hybridomas that produce a single monoclonal antibody. Select. It is desirable to repeat cloning and screening two or more times.

4. Production of monoclonal antibodies Monoclonal antibodies are produced by culturing the hybridomas obtained as described above in vitro (in a culture device or in a nutrient medium) and in vivo (in a living body or in an animal tissue). Cultivation is performed, for example, as follows. For in vitro cultivation, hybridomas are cultured in a suitable medium such as a growth medium, eg, in a CO incubator. When the hybridomas have grown to their growth limit, the culture solution is collected, and the hybridomas and the culture supernatant are separated using a solid-liquid separation means such as centrifugation. The monoclonal antibody in the culture supernatant can be used without purification depending on the purpose, but if it is to be separated, it can be salted out with ammonium sulfate, and then added to a 0.02M phosphate buffer (pH 7,
After dialysis in step 2), it is purified by passing through a diethylamidiethyl cellulose column or the like.

Hybridomas isolated from the culture supernatant can be treated with, for example, dimethyl sulfoxide (5-10% V/V) and fetal bovine serum (
Cells were suspended at a density of 1-10XIO8 cells/mQ in a suitable medium such as Dulbecco's MFM supplemented with 10-20% v/v), placed in a suitable ampoule, and slowly incubated at -80°C.
It is possible to preserve the animal alive for a long period of time by freezing it. In particular, hybridomas can be preserved semi-permanently under ultra-low temperatures such as liquid nitrogen.

When culturing a hybridoma in vivo, the hybridoma is transplanted into any animal, but it is preferable to use the same species as the animal from which the pancreatic cells used for cell fusion were collected. For example, in the case of BALB/c mice, 2.6,1
0.14-Tetramethylpentadecane (pristan) 0
．． 5m(2) is injected intraperitoneally, and 2 to 10XIO" hybridomas per mouse are injected intraperitoneally. After 1 to 2 weeks, ascites containing a high concentration of monoclonal antibodies will be present in the mouse's peritoneal cavity. As the abdominal fluid accumulates and becomes enlarged, the ascites is collected and purified in the same manner as the culture supernatant.

5. Characteristics of monoclonal antibodies The characteristics of the monoclonal antibodies obtained as described above are investigated, for example, as follows.

First, in order to investigate which site of human sperm the monoclonal antibody reacts with, a known labeled antibody method, such as a fluorescent antibody method or an enzyme antibody method, is performed.

Next, the specificity of the monoclonal antibody is examined by a known label immunoassay method (eg, enzyme immunoassay method) that examines reactivity with human sperm, mouse sperm, and the like.

(Production of test granules) To produce the test granules used in the present invention, the antibodies used in the present invention are physically or chemically bound to appropriate granules, such as chromatographic gels. Antibodies having specificity for the antibodies used in this invention (hereinafter referred to as secondary antibodies) and protein A1 are added to the granules in advance.
In this method, protein G or the like is chemically bonded, and the antibody used in the present invention is bonded to the second antibody through specific binding to the antibody used in the present invention.

As granules, beads made of glass, agarose, Sepharose, agarose-filled porous diatomaceous earth, hydrophilic copolymerized acrylic gel, polystyrene, etc. are used.
Preferably, it is made superparamagnetic by including a magnetizable substance (eg Fe, 03) in the core. The shape of the granules can be arbitrary, such as spherical or irregularly crushed.
Spherical shapes are preferred. The particle size is not particularly limited, and may be, for example, from several to several tens to several hundred micrometers.

In order to chemically bind a secondary antibody, protein A or protein G to the above-mentioned granules, it is preferable to activate the granules before binding. Activation of the granules can be carried out by any activation method for binding proteins to granules of this type. Such activation methods include the tosyl chloride method, the bromucyan method, the bromoacetylation method, the geltaraldehyde method, and the like. Some tosyl activated granules are commercially available. Such activation and binding of the activated granules to proteins such as secondary antibodies, protein A, protein G, etc. can be performed by conventional methods.

Furthermore, granules bound with secondary antibodies, protein A1 protein G, etc. are already commercially available. Examples of such granules include Dynabeads M-450 and M-280, imported by Nippon Dynal Co., Ltd. and sold by Veritas Co., Ltd., which are sheep anti-mouse IgG coated type, goat anti-mouse IgG coated type, and sheep anti-rat IgG, respectively. Coated type, sheep anti-rabbit IgG coated type, Polyscience Inc. goat anti-mouse IgG (H&L) carboxylate beads, goat anti-rabbit 1gG (H&L) carboxylate beads, protein A carboxylate beads,
They include goat anti-rabbit IgG (H&L') micromagnetic particles, protein A micromagnetic particles, and sheep anti-mouse IgG (H&L) micromagnetic particles.

In order to bind the antibodies used in the present invention to the granules described above, the granules are suspended in a suitable medium such as a buffer, preferably treated with a protein solution to remove non-specific adsorption, and then the antibodies are added. Mix the containing ascites fluid or purified antibody solution.

(Test method) To carry out the test method of this invention, semen is collected from a subject and used as a sperm suspension. Immediately after collection and after 24 hours of incubation, test granules prepared as described above are added, incubated for 10-30 minutes, the granules are separated and washed, and the number of bound spermatozoa per 100 granules is determined by means such as a microscope. Measure. Since the antigenic site on the acrosome of sperm with fertilizing ability is exposed, the antibody on the granule reacts with the antibody at 1:1, 1:2.2:1, 1:3.3:11.
Combine at a ratio of 2:3.3:2, etc. Note that since the bonds are likely to come off, centrifugation should be avoided for separating and washing the granules. If the binding amount is, for example, 5 or less immediately after collection and 25 or more 24 hours later, the fertility is determined to be good.

(Kit) To conduct the above tests, it is convenient to prepare a kit containing the necessary materials. Such a kit may include test granules, a magnet and sperm incubation medium as described above. The incubation medium may contain inorganic salts, organic salts, sugars, serum albumin, antibiotics, indicators, and the like. In addition, the kit may include (d) test tubes, centrifuge tubes, and other similar glass containers, (e) pipettes or similar suction instruments, and (f) microscopes.

Note that instead of the test granules described above, solid granules that are the raw material for their production can be combined with antibodies.

[Effect] According to this invention, fertilization ability can be evaluated by binding antibodies to solid granules to form test granules, binding sperm to these, and counting the bound sperm. Fertility can be tested easily and reliably in a short time without requiring complicated measurements such as fluorescence. Additionally, since sperm can be counted directly under a microscope, the results are highly reliable. Therefore, this invention greatly contributes to speeding up and objectifying the diagnosis of infertility.

〔Example〕

Hereinafter, this invention will be explained in more detail with reference to Reference Examples and Examples.

Reference Example 1 (Preparation of anti-human sperm monoclonal antibody) Generally, before combining with an egg, sperm undergo a reaction called a photosome reaction to expose the internal endophotomembrane (hereinafter abbreviated as lAM).

An antibody that can detect the antigen specifically present in this IAM will be able to react with the sperm surface as the sperm develops fertilization potential.

(Experimental method) The medium used in the human sperm suspension preparation experiment was 0.
．． A modified Piggers-Whiten-Whiting Gum medium (hereinafter referred to as m-BWW) supplemented with 3% human serum albumin (hereinafter referred to as H9A; Sigma, Fr, V) was used.

Semen collected manually from an adult male was kept at 37℃ and 5%C.
O, containing air for 30-60 minutes.

Take each of these 0 and 5 mQ into a small test tube, and add m-
BWW2m (!) was layered. To increase the contact surface with semen, the small test tube was tilted at approximately 30°, covered with parafilm, and allowed to float in the air containing 5% CO7 at 37°C for 60 minutes. The supernatant was sucked out with a micropipettor (Gilson, Pipetman P-1000), and the sperm were transferred to m-BW.
Washed twice with W. The obtained spermatozoa were treated with m-BWW (H9A
A sperm suspension was prepared by adding 1 mf2 of 3.5% concentration.

The obtained spermatozoa were treated with A23187 to acquire fertilization capacity or to expose the IAM.

Treatment with A23187 It has been observed by electron microscopy that spermatozoa treated with A23187 have a higher rate of photoreaction. Therefore, according to the method of Kamiguchi et al., a final concentration of 10μ was added to the medium.
A23187 (Sigma, free acid) was added to give M, and after reacting for 10 minutes, it was washed twice with m-BWW and used for immunization.

Immunization of mice: 1×10 human spermatozoa subjected to the above treatment each time.
' was prepared and immunized against C57BL/6 mice. Immunization was performed on days 0, 21, and 28, and thereafter every two weeks until the antibody titer increased. 1
After creating an emulsion with complete Freund's adjuvant for the first time and incomplete Freund's adjuvant for the second time,
From the third time onward, the drug was administered while suspended in PBS. From the second time onward, serum was collected by fundus blood collection on the third day after administration, and the antibody titer was measured by indirect fluorescent antibody method (described later).

When the antibody titer had risen sufficiently, the tibia was removed 3 days after the final administration and used for fusion.

Cell fusion and cloning Fusion and cloning were carried out according to standard methods.

The obtained tile cells were fused with P3U1 mouse myeloma cell line (Fujisawa Pharmaceutical Co., Ltd.) in the presence of polyethylene glycol 4°00 (Hirai special grade) to create a hybridoma. Among these, we screened those that produced antibodies that reacted with human sperm, and the positive strains were cloned by limiting dilution to establish monoclonal antibody-producing strains.

Indirect fluorescent antibody method was used to screen antibodies and measure antibody titers.

50 μg of a 20-fold dilution of culture supernatant or antiserum in PBS was added to 50 μg of a sperm suspension of 1×10” sperm/mρ, and the mixture was allowed to react at room temperature for 2 hours. After washing twice with PBS,
FITC-labeled goat anti-mouse Ig (A+M+G) (Cubbell) diluted 125 times in PBS containing 5% newborn bovine serum (hereinafter abbreviated as NBC8) as a second antibody 10μρ
was added and allowed to react at room temperature for 1 hour. Thereafter, it was washed twice with PBS and observed under a fluorescence microscope.

Proliferation of Antibody-Producing Cells (In Vivo) Hybridomas in the logarithmic growth phase were collected and treated with 0.5 m+2 of Bristan (Sigma, p-1403) in advance (10 to 20 days ago), followed by CBFI (Balb/cX).
C57BL/6) 1-2XIO per male mouse
'Cell administration. Since the cells proliferate as ascites-type cancer cells over about two weeks, ascites was collected when the weight reached 40 g or more, frozen at 80° C., and then stored in liquid nitrogen.

Reference example 2 (recognition of anti-human sperm monoclonal antibody)
(1) Cross-reaction The components of seminal plasma are strongly bound to the ejected human sperm, and cannot be removed by ordinary washing alone. These components are highly antigenic, and if sperm is immunized, there is a strong possibility that antibodies against seminal plasma will be produced. Since the antibody targeted by this invention is capable of detecting changes accompanying the acquisition of sperm's fertilization potential, it is desirable that it not react with seminal plasma.

(Experimental Method) Preparation of Human Seminal Plasma Semen obtained from an adult male is liquefied by standing at 37° C. in air containing 5% CO7 for 30 to 60 minutes.

An equivalent amount of m-BWW solution is added to this semen, and the semen is centrifuged at 1500 xg for 5 minutes to remove sperm. The supernatant was centrifuged once more to completely remove sperm and was used as seminal plasma in the experiment.

Examination of reactivity between seminal plasma and antibodies The reactivity with seminal plasma was measured using ELISA (enzyme-linked immunosorbent assay). As a positive control, human sperm immobilized on a plate with geltaraldehyde (Wako Pure Chemical Industries, Ltd.) was used, and as a negative control, m-BWW solution was used.

50 μa of the solution containing the antigen was placed on an EL I SA plate (Falcon, 3911) and left to dry at 37° C. overnight to adsorb the antigen. 0.05% Tween 20
After washing three times with Tris buffer saline (pH 7, below 4, abbreviated as Tween-TBS) containing (Hirai-grade) 5% milk (
Place PB8200μg containing Morinaga skim milk, 1
Blocking was performed by standing at room temperature for an hour.

After washing three times with Tween-TBS, each antibody diluted 1000 times with ascites 1% BSA-PBS was used as the first antibody.
Add 0 μQ, react for 2 hours, wash and add 1% BSA-PB.
50 μg of peroxidase-labeled goat anti-mouse Ig (A+M+GX Kappel) diluted 1000 times with S was added, and the mixture was allowed to react at room temperature for 2 hours.

After washing, color was developed using a substrate. As a substrate solution,
0-phenylenediamine (Hirai-grade), 0°1%, H
O,IM containing 1.2% of 2O, < citrate buffer (pH 4
, 5) was added to the plate for 30 minutes while shielding from light.
Allowed to react for minutes. Then 12.5% H, So, 50u
The reaction was stopped at Q, and the absorbance at 450 nm was measured.

The MH61 antibody showed no reactivity with human seminal plasma.

(2) Changes in the reactivity of sperm with antibodies due to the acquisition of artificial fertilization ability In order to measure the fertilization ability of sperm using antibodies, it is necessary for the antibody to recognize the antigen that appears along with the expression of fertilization ability. It is. Although it cannot be completely proven that the sperm has acquired fertilization ability unless the sperm enters the human egg, Green et al.
It has been reported that the number of spermatozoa treated with 3187 that undergoes a photoreactive reaction increases. There, fresh sperm and A2
We investigated whether a difference in reactivity could be found between spermatozoa treated with 3187.

(Experimental method) Preparation of human sperm suspension The method of Reference Example 1 was followed. A23187 was added to this suspension to a final concentration of 10 μM and reacted for 10 minutes. Thereafter, A23187 was removed by centrifugation at 500×g, and the sperm was washed twice with m-BWW to obtain A23187 sperm.

Examination of reaction mode with human sperm Sperm were stained by indirect fluorescent antibody method. The spermatozoa were observed using a fluorescence microscope, and the staining pattern of the spermatozoa and the proportion of the spermatozoa present were measured.

(Experiment results) Washing A231g? Treated antibody ITHA
MATN TTHAEMPTEATNDE 98
2----- 908-----2MH61--31
--961l-4522-21-213 (W is the entire body, T is the tail, head, A is the light body, E is the equatorial region, M is the lieutenant colonel, P is the posterior region, and N is unstained) MH61 The antibody hardly reacted with fresh sperm, but showed a reaction when sperm were treated with A23187. The MH61 antibody hardly reacted with washed sperm, but reacted with sperm treated with A23187 at a high rate. Furthermore, its binding site was localized to the acrosomal region and the head. These results were very similar to the capacitated sperm specific OBF 13 antigen reported in mouse sperm. Hello, M
If the antigen recognized by H61 is a substance involved in fertilization, fertilization should be inhibited by the addition of antibodies. Therefore, the effect of MH61 antibody on sperm function was investigated.

Reference Example 3 (Effects of each monoclonal antibody on sperm function)
Some antibodies have strong sperm aggregation activity, and when many sperm are cross-linked, the apparent sperm concentration decreases and, as a result, fertilization is inhibited.

(experimental method) Preparation of human sperm suspension The method of Reference Example 1 was followed.

The collected sperm were reacted with 10 μM of A23187 for 10 minutes and used in the experiment.

Observation of sperm aggregation activity of antibodies Activity was measured by the microtiter method.

PBS containing 1% BSA (Sigma, Fr, V)
Antibody ascites diluted 500 times with (BSA-PBS) was serially diluted 2 times with BSA-PBS in small holes of a hemagglutination plate. 5 to 50μσ of each antibody dilution
0 μg of sperm suspension (I x 1011 sperm/m) was added, diluted 2 times, and reacted for 1 hour at 37 °C in air containing 5% CO2. Sperm in the pores were examined using a phase contrast microscope (
160), aggregation was observed.

As a negative control, ascites fluid from the P3U1 mouse myeloma cell line was similarly diluted and used.

(Experimental result) Table 2 shows the sperm aggregation activity of the antibodies.

Table 2 Dilution rate Ascites 1000 2000 4000 8000 1
6000320003U1 11+p +++ +++ +++
+++ ++ ++M) 161 Strong sperm aggregation activity was observed in the anti-human sperm antibody YP used as a positive control, but no sperm aggregation activity was observed in P3U1 and MH61 used as negative controls.

Example! (Manufacture of test granules) IgG co-binding method to beads (1) Bead activation method (tosyl activation with p-toluenesulfonyl chloride) ■) Undiluted Guinabeads M-450 uncoated (
Add an appropriate amount of 3019/w (Q) to dry acetone and perform continuous washing (for beads of 210 to 1000 u, increase the volume to 7.0 u).

Step 1: Water/Acetone-77310 Step 2:
Water/acetone = 6/4 10g (2 Step 3: Water/acetone - 2/8 10 villages Step 4-6: Water/acetone = O/10 10xC Step 7: Resuspend in acetone Collect (1 minute) and discard the top layer.

Add the following water/acetone mixture to the test tube and suspend for 5 minutes.

2) 0.75 mmol pyridine and 0.3 mmol p
-) Add luenesulfonyl chloride per undiluted Dynabeads M-450 Uncoated 1+f2. (
When using 1000m9, 2x (1 part of pyridine and 2
9p-Toluenesulfonyl chloride/8m (using acetone) The operation is carried out in a fume hood.

3) Incubate for 20 hours at room temperature with stirring.

4) Collect beads with a magnet and resuspend in acetone.

Collect beads and wash three times with acetone.

5) Reverse steps 1 to 3 of ■) (3→2→1) and return to water.

6) Collect beads, discard supernatant, add 1mM HCQ10ic
Resuspend in

The active beads prepared as described above were 1mMHc
Stable for 12 months when stored at 4°C in f2.

(Note) Products activated using the above method (14003/
14004 Dynabis M-450 Tosil Activated) is commercially available.

(2) Covalent bonding method to beads l) Wash a suspension of active beads in 1 mM HC (2) once with sterile distilled water.

2) Stir briefly if necessary to obtain a homogeneous suspension of active beads.

3) Purified antibodies were added to 0.2M portate buffer (pH 9).

5) to a concentration of 150 μg/ilJ.

4) Add an equal volume of active bead suspension to the above IgG solution (antibody/beads = 75 μ9/l 5 mg).

5) Incubate for 24 hours at 22°C with slow stirring.

6) Collect the beads with a magnet and discard the supernatant with the magnet attached.

7) Clean using the following method.

■ 0.1M PBS 5iC, I 0 minutes.

■ IM ethanol 7mi containing 0.1% Tween 2o: /
-HC (! (pH 9,5) 5x (!, 2 hours (Tween 20 added to buffer immediately before use).

■ 0.1M NaCQ, 0.1% BSA, 0.01%
Merthiolate, 0.05M containing 0.1% Tween 20
Tris (pH 7,5) 5 iL for 12 hours.

■ Buffer solution without Tween 20 for 5112 hours.

8) Collect beads with a magnet. Discard the supernatant and use PBS/US
Approximately 4X108 beads/x12 (30rttq/
rttQ or resuspend at the desired concentration. The obtained IgG-labeled beads are stable for at least 6 months when stored at 4°C. The storage buffer is 0.1M NaCQ, 0.05M Lys containing 0.1% BSA.

Monoclonal antibody purification method: Add saturated ammonium sulfate to the ascites stock solution to obtain a final concentration of 20% ammonium sulfate solution, and let stand for 1 hour (4°C). 100009. Centrifuge for 15 minutes at 4°C. Discard the precipitate, and add saturated ammonium sulfate to the supernatant to make a final concentration of 40% ammonium sulfate solution (4°C). Let stand for 1 hour. 100009. Centrifuge for 15 minutes at 4°C. The supernatant is discarded, and the precipitate is dissolved in an adsorption buffer for protein A column (PIERCE) twice the volume of the ascites stock solution. 1
00009. Centrifuge for 15 minutes at 4°C. Take the supernatant and
Subject to protein A affinity chromatography.

Approximately 3 to 4119 IgG can be obtained per 1 volume of ascites fluid.

The eluate is desalted (dialyzed after passing through a 625 column) and then lyophilized. In this way, purified monoclonal antibodies are obtained.

Bead pretreatment method with skim milk powder Guinabeads M-450 Sheep Antimouse I g
Suspend the 4 x 10''/m (l) suspension of G + (F c) well before use. Centrifuge at 10009 for 5 minutes.

Discard the supernatant, and add the same amount of 5% skim milk powder in PBS (add skim milk powder to PBS to a final concentration of 5%, and incubate at 60°C for 1 hour.
(about 2 mf
2) and warmed with shaking at 37°C for 1 hour. to
Centrifuge at o og for 5 minutes, wash 3 times with PBS, and then
Adjust to approximately 4 x 108/IIIQ with S.

Preparation of test beads Take 50 μa of pretreated beads and add the same amount of MH611 antibody (ascites fluid stock solution).

Add antibody (ascites fluid stock solution). After binding the antibody to the beads while shaking at 37°C for 1 hour, add PBS(-) to the beads.
Add 1 ml of 10009.5 minutes at 4°C and centrifuge. Wash the precipitate two more times with PBS(-).

The MH61-beads thus obtained were added to 4xlO8
/mQ in PBS containing 5% neonatal bovine serum (
-). Before use, axto'/m (2 to m-B
Dilute to WW. Store the beads at 4°C, but use them within one week if possible.

Example 2 (Test method) (Things to be prepared) Sterile tLf (a) (50 mQ, Sumitomo Bakelite) (for containing semen) m-BWW (separate description) Sterile tube (b) (1511172, Sumitomo Bakelite)
For measuring semen volume) Glass test tube (C) (φ12.5yttytt, ]01
(for sperm migration) Glass centrifuge tube (d) (10m (for 2X sperm centrifugal concentration)) Glass test tube (e) (l 0m for CX bead test) Test front pipette... J Toman (o-1000, P- 200,P-2
0) Centrifuge (IOoog output) Microscope (x400, for observation) MH61 beads (3XlO″i (2, (composition of m-BWW) Component 9/ρ in m-BWW) aCf2 Cf2 CaCi2* ・2 HzO KH , P.O.

Mg5O+・7HtO Na pyruvate Na lactate Glucose phenol red C10m9/11rQ solution) 10 Antibiotics 11 NaHCOs 12 Human serum albumin 4.9LO J56 251 O,162 0,294 0,028 (2,416) 1.0QO 400μQ 10 butts Q 84, QO 78 1,71 1,19 1,19 0,25 21,58 5,56 3,00035,71 3,000 (for extravasation, washing) 35,000 (for incubation) The antibiotic is penicillin G Potassium 50001U/IC and streptomycin sulfate 5y, 9/MQ.

Sodium lactate is in DL, 60% Nrotubu.

Human serum albumin is a fraction (Sigma).

1-1.0 as a stock solution from 40 to
501117! Dispense into portions and store frozen at -20°C. The day before the experiment, bring the required amount to room temperature and add NaHCO3.
, H9A, and equilibrated overnight at 37°C and 5% CO after filter sterilization.

(Preparation of sperm suspension) Semen from healthy adult males is collected manually into a previously sterilized tube. Semen is a mixture of sperm, prostatic fluid, seminal vesicle fluid, etc., and these are not homogeneous immediately after ejaculation. Therefore, it is left to stand at 37°C for 30 to 60 minutes (liquefaction).

30 4 sterilized test tubes (c) (depending on semen volume)
Let stand at ℃ and add m-BWW (0.3% H9A) 2i&. Next, add 0,5 liters of liquefied semen to this lower layer.
Inject mQ slowly. Put on the aluminum cap 3
Let stand for 60 minutes at 7°C and 5% CO2.

As highly motile sperm migrate into the m-BWW, slowly suck out 1.5xlJ of the supernatant into a centrifuge tube (d).
Move to. Centrifuge at 500 g for 5 minutes at room temperature, suck out the supernatant, suspend the precipitated sperm in m-BWW (0.3% H9A), and centrifuge. Repeat this operation once again. Add 1Q of m-BWW (3.5% H5A) to the second precipitate and suspend. Take 10μQ of the precipitate and place it on Toma's blood cell plate to measure the number of sperm.

Centrifuge once again depending on the number of sperm, and m-BWW (3
5% HSA) to prepare a sperm suspension to a final concentration of 4X106/vrQ. A portion of this is used for the 0 hour bead test.

(Acquisition of sperm capacitance) 4 X I O”/ in m-BWW (3,5% HSA)
Human sperm prepared to mQ are precultured at 37°C and 5% COt to acquire fertilization ability.

Fertility acquisition time varies from person to person and depending on the solution.
- Although it cannot be determined generally, most people become fertile after 24 hours, so do a 24-hour bead test after 24 hours.

(Test) Sperm to be incubated with beads is 4 x 10”/z
I2 is desirable (if it is too thin, the probability of reaction with beads will be low). Add 10 uQ of beads of 3 x 106/m (l) to this sperm suspension and let stand at 37°C, 5% CO for 1 hour. Avoid shaking as the beads will stick to the test tube wall and cannot be recovered.

Bring a strong magnet (rare earth magnet) close to the slightly cloudy reaction solution. The beads will gather in 1 to 2 minutes, so remove the supernatant with a pipette while keeping the magnet in place (be careful not to pipette at this time as the sperm will come off the beads). 500μC
Add m-BWW (0.3% HSA) (while keeping the magnet in place) and discard the supernatant again. Repeat once more, place the collected beads on a glass slide (avoid pipetting), and examine under a microscope. Count 100 beads and use the number of attached sperm as the bead test value.

(result) The following results were obtained.

[θ time] Sperm donor A [24 hour time Seiko provider A 9 5 9 8 *Number of sperm bound when counting 100 beads

Claims (4)

[Claims] (1) Granules for testing sperm fertility, which are made by binding to the surface of solid granules an antibody that has specificity for the antigenic site on the human sperm acrosome (including the membrane). (2) (a) Test granules according to claim 1 (b) Magnet (c) Sperm incubation medium A kit for testing sperm fertility, including: (3) In claim 2, instead of the test granules of (a), an antibody having specificity for an antigenic site on the human sperm acrosome (including membrane) and the above-mentioned antibody may be bound to the surface. Kit for testing sperm fertility, containing solid granules. (4) A method for testing sperm fertility, which comprises bringing the test granules according to claim 1 into contact with the test sperm, and observing the sperm with exposed antigenic sites on the acrosome bound to the granules.