Principle:

The ImmunoSpheres^® method can be used to detect the presence or absence  of all classes of  immunoglobulins on the surface of sperm using  latex  beads coated with  antibodies that bind to human IgA, IgG and IgM antibodies.

In the Direct ImmunoSpheres^®,  live  motile sperm are mixed  with a  suspension of bead reagent.  As the sperm swim through the bead suspension, the beads will bind to the sperm if antibodies are present on the sperm.

In the Indirect ImmunoSpheres^®,  live  motile sperm are incubated with diluted  serum.  Any antibodies to sperm present in the serum will bind to the sperm.  Then  unbound antibodies and serum proteins are washed away.

In the next step,  these sperm are mixed with a  suspension of bead reagent. The protocol proceeds as in the Direct ImmunoSpheres^®.

Reagents:

Anti-Ig(H&L) Beads:  0.8 ml white  latex beads coated with (goat) anti-human immunoglobulin heavy and light chains in protein buffer with 0.1% sodium azide.

Materials Required But Not Provided:

1.  Sperm washing medium containing 1 - 5% bovine serum albumin.
2.  Positive and negative serum controls.
3.  Centrifuge capable of 1000g.
4.  37^oC incubator.
5.  Conical centrifuge tubes and rack.
6.  Pipettors and tips.
7.  Glass slides and coverslips.
8.  Sperm counting chamber.
9.  56^oC incubator.
10.  Bright-field microscope with 100X - 400X magnification.
11.  Collecting cups.

Storage and Stability:

Store the reagents at 4^oC. They can be used until the expiration date on each label.

Anti-Ig(H&L) Beads should be stored in an upright position.

Once Anti-Ig(H&L) Beads have been washed, they can be stored up to 3 days at 4^oC.  Or, return washed Anti-Ig(H&L) Beads that were suspended in sperm washing medium but  not used in the experiment to the original bottles rather than discarding them.

Warning and Precaution:

All semen and serum specimens should be considered potentially infectious.  Handle all specimens as if capable of transmitting HIV or hepatitis.  Specimens should be disposed of in accordance with OSHA guidelines.

Specimen Collection:

Semen should be collected in a clean cup.  The semen sample should be stored at room temperature until use.  Semen should be used within three (3) hours of collecting.

Blood should be collected and stored as serum for up to 7 days at 4^oC.  If storage time exceeds 7 days, frozen storage in a non-defrosting freezer is recommended.   Multiple freeze-thaws should be avoided.  Allow previously frozen serum samples to thaw and mix completely before use.

Limitations:

Direct  ImmunoSpheres^®:  Sperm  with a motility of less than 5 million/ml cannot be used in this test.   Indirect  ImmunoSpheres^®.   At least 50 million motile sperm/ml are needed.

Preparation for Direct ImmunoSpheres^®:

1. Bring all reagents to room temperature.
2. Warm sperm washing medium to 37^oC. Caution:  Do not use medium containing human serum albumin.
3. Preparation of Anti-Ig(H&L) Beads:
         3.1.Gently swirl  the vial containing the Anti-Ig(H&L) Beads,   avoiding foaming, to resuspend the beads.
         3.2. Remove an aliquot (use 10 ul for each sample you will be   testing) to  a conical  centrifuge tube.
         3.3.  Add 2 -3  ml  sperm washing medium.
         3.4.  Centrifuge at 1000g   for 5 -10  minutes, remove supernatant.
         3.5.  Add 2 -3  ml  sperm washing medium.
         3.6.  Centrifuge at 1000g   for 5 -10  minutes, remove supernatant   and  resuspend bead pellet to its original  aliquot volume.
         3.7. Store unused washed  Anti-Ig(H&L) Beads at 4^oC for up to   3 days.
4. Semen preparation:
       4.1.  Allow semen sample to liquify.
       4.2.  Add  sufficient sperm washing medium to equal  twice the volume of the semen sample and mix.  For example, for 2 ml semen, add 4 ml sperm washing medium.
       4.3.  Centrifuge at 600g  for 5 - 10  minutes, remove supernatant, and resuspend sperm pellet in about 3 ml sperm  washing medium.
       4.4.  Centrifuge at 600g  for 5 - 10 minutes, remove supernatant, and  resuspend sperm pellet in a small volume of sperm washing   medium.
       4.5. Count sperm and determine motility of washed sperm.
       4.6.  Dilute sperm to give a final concentration of 10  million motile sperm/ml.

Procedure for Direct ImmunoSpheres^® of Sperm:

1. Pipette 5 ul of the sperm suspension onto a prewarmed glass slide.
2. Pipette 5 ul of the washed Anti-Ig(H&L) Beads onto the sperm suspension.  Use the pipette tip to mix the suspension and  Anti-Ig(H&L) Beads thoroughly.
3.  Place a coverslip on top of the mixture.
4.   After  2 - 5 minutes examine the slide using a microscope.
5.  Count 100 free-swimming sperm and determine if and where any beads are bound to the surface of the sperm.

Preparation for Indirect ImmunoSpheres^®of Serum:

1.  Bring all reagents to room temperature.
2.  Warm sperm washing medium to 37^oC. Caution: Do not use medium containing human serum albumin.
3.  Preparation of Anti-Ig(H&L) Beads:
        3.1. Gently swirl  the vial  containing the Anti-Ig(H&L) Beads,   avoiding foaming, to resuspend the beads.
        3.2. Remove an aliquot (use 10 ul for each sample you will be   testing) to a conical centrifuge tube.
        3.3.  Add about 10  ml  sperm washing medium.
        3.4.  Centrifuge at 1000g   for 5 -10  minutes, remove supernatant.
        3.5.  Add 2 -3  ml  sperm washing medium.
        3.6.  Centrifuge at 1000g   for 5 -10  minutes, remove supernatant   and  resuspend bead pellet to its original  aliquot volume.
        3.7. Store unused washed  Anti-Ig(H&L) Beads at 4^oC for up to   3 days.
4.   Serum preparation:  heat inactivate serum by incubating at 56^oC for 30 minutes.
5.  Semen preparation:
       5.1.  Allow semen sample to liquify.
       5.2.  Add sufficient sperm washing medium to equal twice the volume of the semen sample and mix.  For example, for 2 ml semen, add 4 ml sperm washing medium.
       5.3.  Centrifuge at 600g for 5 - 10  minutes, remove supernatant, and resuspend sperm pellet in about 3 ml sperm  washing
 medium.
       5.4.  Centrifuge at 600g  for  5 - 10 minutes, remove supernatant, and  resuspend sperm pellet in a small volume of sperm washing   medium.
       5.5.  Count sperm and determine motility of washed sperm.
       5.6.  Dilute sperm to give  a final concentration of 50 million motile   sperm/ml.

Procedure for Indirect ImmunoSpheres^® of Serum:

1.  Pipette 50 ul of  the following into separate  test tubes:
            a known  positive control serum,
            a  known  negative control serum, and
            each  unknown serum.
2.  Pipette 400 ul of the sperm washing medium into each test tube.
3.  Pipette  50 ul of the donor sperm suspension into each test tube.  Mix gently. Cover each  test tube and incubate 60 minutes at 37^oC.
4.  Pipette 2 ml sperm washing medium  into each test tube and mix.
5.  Centrifuge at 600g for 5 - 10 minutes.
6.  Discard supernatant.  Resuspend sperm pellet in 2 ml sperm washing medium.
7.  Centrifuge at 600g for 5 - 10 minutes.
8.    Discard supernatant.  Resuspend  sperm pellet  in about 250 ul  sperm washing medium.
9.  Pipette 5 ul of this sperm suspension onto a prewarmed glass slide.
10.Pipette 5 ul of the washed Anti-Ig(H&L) Beads onto the sperm suspension.  Use the pipette tip to mix the suspension and  Anti-Ig(H&L) Beads thoroughly.
11.  Place a coverslip on top of the mixture.
12.   After  2 - 5 minutes examine the slide using a microscope.
13.  Count 100 free-swimming sperm and determine if and where any beads are bound to the surface of the sperm.

Calculation of Percent Total Binding:

Count moving sperm and score as follows:
             free = no beads attached
             head = bead(s) attached to sperm head
             midpiece = bead(s) attached to sperm midpiece
             tail = bead(s) attached along tail length
             entire = beads attached to more than one location on sperm

Calculate  the percent total binding of beads as the sum of all percent bound beads from the various locations on the sperm surface per 100 sperm.

% total binding    =  No. sperm with bound beads     x    100%
                               Total no. sperm counted

Example:  At 400X  the following data  were obtained for an unknown sperm sample mixed with Anti-Ig(H&L) Beads:
             free = 50
             head = 12
             midpiece = 10
             tail = 18
             entire = 10

Applying the formula:

         12 + 10 + 18 + 10  X 100% = 50% total binding of Anti-Ig(H&L)
                 100

Selected Reference:

World Health Organization. 1999.  WHO laboratory manual for the examination of human semen and sperm-cervical mucus interaction. Cambridge University Press. Fourth Edition.