Principle:

AcroScreen^TM  measures the total acrosin activity present in spermatozoa relative to the activity of a known amount of protease.

In the first step, a semen sample is subjected to filtration and washing so as to remove the seminal plasma, which contains protease inhibitors, and leave behind spermatozoa.

In the second step, the spermatozoa are incubated with a detergent-substrate solution. The detergent permiabilizes the sperm cell membranes thereby making the intracellular acrosin available to react with the substrate.  At the same time proacrosin is converted to acrosin.  The acrosin converts the substrate to a soluble colored product:
                                                                             acrosin
N-a-benzoyl-DL-arginine p-nitroanilide HCl (BAPNA)  -------------->  p-nitroaniline

The intensity of color from semen sample wells is measured in a microplate reader at 405 - 410 nm. Using a formula, it is possible to calculate the 'acrosin activity index' for each semen sample by knowing the optical density (OD) of the reacted semen sample, the OD of the positive control, and the number of spermatozoa per volume of semen.  Or, using a different formula, it is possible to calculate the acrosin activity per million sperm for each semen sample by knowing the OD of the reacted semen sample and the number of spermatozoa per volume of semen.

Reagents:

Wash:  50 ml phosphate buffered saline, pH 7.2, with sodium azide. Ready to use.  Warning:  Dispose of with care.
Substrate:  2 ml N-a-benzoyl-DL-arginine p-nitroanilide hydrochloride (BAPNA) in solvent. Ready to use. Caution:  Let Substrate liquify completely at room temperature before using.
Detergent:  9 ml Triton x-100 in buffer, pH 8.0, with sodium azide. Ready to use. Warning:  Dispose of with care.
Positive Control:  0.5 ml protease in a suspension. Ready to use.  Mix thoroughly before using.  Warning:  Contains an eye irritant.
Membrane Plate:  Each plate has 96 useable wells.  Caution:  To ensure the useability of all the wells, care should be taken to cover the wells not being used with the Plate Sealer so that the membrane does not get destroyed by repeated vacuuming.
Plate Sealer: Peel off backing before using.

Materials Required But Not Provided:

1.  Microplate reader, capable of reading at 405 - 410 nm.
2.  Clear-bottom microplate wells.
3.  Test tubes and rack.
4.  Pipettors and tips.

Vacuum Manifold attached with tygon tubing to a liquid trap
which is attached to a vacuum generator (GAST).

5.  Vacuum manifold attached to a vacuum source.
6.  Microscope or CASA system and counting chamber.
7.  Paper towels.
8.  Collecting cups.

Storage and Stability:

Store the reagents at 2^o - 8^oC.  They can be used until the expiration
date shown on each reagent label.

Warning and Precaution:

All semen samples 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 using. Semen should be used within three (3) hours of collecting.

Preparation:

1. Bring all reagents to room temperature.
2.  Determine the number of wells needed for running the assay.  Mark off the wells on the assay map you will be using in the assay. Mark off the wells on the Membrane Plate.
3. Cover the wells you will not be using with the Plate Sealer after first peeling off backing.

Procedure:

1.  Allow semen sample to liquify.
2.  Count spermatozoa.
3.   Dilute semen to between 3 million and 6 million spermatozoa per milliliter with Wash using the following suggested protocol (note: 100ul  = 0.1ml.

4.  Pipette 100  ul of the diluted semen into a well according to your assay map.  Repeat for duplicates.
5.  Pipette 100  ul  of Wash into a well as a Negative Control.
6.  Place Membrane Plate onto vacuum manifold and turn on the vacuum gradually.  Allow all liquid to drain from wells. Turn off vacuum.

The Membrane Plate is sitting on the Vacuum Manifold.
The vacuum is turned on and the liquid in the wells goes through
the membrane and into the liquid trap.

7.  Fill up each well with Wash.  Turn on vacuum.  Allow all liquid to drain from wells.
8.  Turn off vacuum.  Remove Membrane Plate and blot its undersurface thoroughly with toweling.
9.  Pipette 20 ul Positive Control into a well. Repeat for duplicate.
10.  Add exactly 5 drops of Detergent to each well.
11.  Add exactly 1 drop of Substrate to each well.
12.  Briefly agitate Membrane Plate (horizontally) to mix contents, cover wells and set on a nonabsorbent surface at room temperature.  Begin timing.
13.  After 90 minutes transfer the entire contents of each well to a clear-bottom microtiter plate well.  Be sure there are no bubbles.
14.  Read the optical density (OD) at 405 - 410 nm within 5 minutes using a microplate reader.  First adjust the microplate reader to zero while reading the Negative Control and then read the other wells.
15.  Record OD readings.

NOTE:   The OD  reading of  the Positive Control should fall between 0.28 and 0.4.  Repeat   the test if your Positive Control is lower (<0.28) or higher (>0.4).

Calculation of Acrosin Activity Index (AAI):

             AAI =  OD_{diluted semen sample} x 10¹⁰        x     Dilution Factor
                       __________________________
                       OD_{Positive Control} x  Sperm Count

Example:  At 405 nm, the following data were obtained for a semen specimen:
               Sperm Count = 80 X 10⁶ cells/ml
               Dilution Factor = 0.4 for a 1:20 dilution of the semen
               OD of Positive Control = 0.360
               OD of Specimen = 0.146

Applying the formula:

               0.146 x 10¹⁰ x 0.4  =     20 (AAI)
               ________________
               0.360 x 80 x 10⁶

Calculation of Acrosin Activity:

Note that this calculation does not use the Positive Control data.

mIU acrosin/10⁶ sperm    =  OD_{diluted semen sample} x Dilution x 10⁶
                                         -----------------------------------------------------
                                          Sperm Count In Millions x 278

where Dilution is the amount sample is diluted up; for example, a 1:5 dilution is diluted 5 times, a 1:10 dilution is diluted 10 times, etc.  The constant is calculated as follows:

                    278  =  9.9 mM^-1cm^-1x 90 min x 0.5 cm
                               -----------------------------------------------
                                0.18 ml x 10

where 9.9 mM^-1cm^-1is the extinction coefficient, 90 min is the incubation time, 0.5 cm is the distance to the cuvette for a microplate reader, 0.16 ml is the volume and 10 is the volume factor of sperm in each well or 1 ml/0.1 ml.

Example:  Using  the same data from the calculation of Acrosin Activity Index, the following were obtained:
                Sperm Count In Millions = 80
                Dilution  = 20 for a 1:20 dilution of the semen
                OD of Specimen = 0.146

Applying the formula using the above example:

 0.146 x 20 x 10⁶  =     131  mIU acrosin/10⁶ sperm
      80 x 278