The author(s) shown below used Federal funding provided by the U.S. 

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Document Title: Development of an Immuno-Magnetic 

Procedure for the Separation of 

Spermatozoa from Vaginal Epithelial Cells

Author(s): Mechthild Prinz, Ph.D.

Document Number: 251645

Date Received: April 2018

Award Number: 2011-NE-BX-K547

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Justice. This resource is being made publically available through the 

Office of Justice Programs’ National Criminal Justice Reference 

Service.

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do not necessarily reflect the official position or policies of the U.S. 

Department of Justice.
2011-NE-BX-K547 Final Report Draft 

1

US Department of Justice - National Institute of Justice

2011-NE-BX-K547 

Development of an Immuno-Magnetic Procedure for the Separation of Spermatozoa from 

Vaginal Epithelial Cells

FINAL REPORT

Submitted by

Mechthild Prinz, PhD

Associate Professor John Jay College of Criminal Justice 

New York, NY

May 2015

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2011-NE-BX-K547 Final Report Draft 

2

Abstract

Forensic biology laboratories have long been searching for a more efficient, automatable method 

for processing sexual assault evidence. With some minor modifications a manual differential 

lysis extraction first described over 20 years ago is still the most commonly used method for 

separating sperm cells for vaginal epithelial cells. In this project, the goal was to utilize 

immuno-magnetic bead “capture and release” chemistry to specifically bind spermatozoa and 

sperm heads while all other cell types are removed. Such an assay could easily be moved to a 

robotic platform and allow for a more efficient workflow.

Several promising sperm membrane specific antigens were identified based on published 

research and the human gene compendium. Target proteins for the antibodies tested included 

ADAM2, AKAP3, MFGE8, MOSPD3, Ropn1l, SPAM1, and UBAP2L. One type of capture 

chemistry tested in this study was magnetic beads covered with Protein G, which exhibits a high 

affinity for immunoglobulin (IgG) and will bind this antibody domain. The other bead type was 

covered with streptavidin thus reacting with any biotinylated antibodies. Two different biotin 

conjugates (DSB-X Biotin and EZ-link sulfo-NHS-LC-Biotin) were tested for this labeling step. 

Varying numbers of single source sperm cells and female vaginal epithelial cells were processed 

with different antibodies and both types of beads. In order to separately evaluate the captured 

cells bound to the beads and the remaining cells still in solution, both the “capture” fraction and 

the “supernatant” fraction were subjected to DNA extraction and quantitation with a real time 

PCR assay specific for a human DNA target and a male Y-chromosome sequence. Overall this 

project failed to demonstrate sperm cell specific enrichment for either magnetic bead capture 

assay. Protein G beads were shown to retain DNA, but in a non-specific fashion including 

vaginal epithelial cells and control samples with no antibody addition. For the streptavidin coated

beads almost the entire recovered DNA resided in the supernatant fraction that was taken off the 

immobilized beads. Based on the results for single source samples the project never matured to 

the stage, where real cell separation for sperm/vaginal cell mixtures was attempted. 

 

The DNA recovered by the Protein G beads is probably due to unplanned lysis of cells in 

suspension and unspecific retention of DNA and DNA-protein complexes by the bead surface. 

Microscopic examination failed to detect any intact cells adhering to the Protein G beads. Both 

biotinylation strategies were unsuccessful in capturing sperm cells on the streptavidin coated

beads. Again, except for a single isolated observation, microscopic examination failed to detect 

spermatozoa-bead connections. Antibody quality and lack of antigen specificity could have 

played a role, but at least two of the antibodies had shown a positive sperm cell agglutination 

reaction. Streptavidin covered beads are routinely used for cell sorting and this assay had been 

successfully used for sperm separation with the MOSPD3 antibody. The failure to duplicate 

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2011-NE-BX-K547 Final Report Draft 

3

these results is most likely due to technical problems, especially with the biotinylation step and

insufficient antibody labeling. 

This resource was prepared by the author(s) using Federal funds provided by the U.S. 

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2011-NE-BX-K547 Final Report Draft 

4

Table of Contents 

Abstract .............................................................................................................................. 2 

Executive Summary............................................................................................................ 5 

Main Body 

 Introduction .................................................................................................................. 10 

 Statement of the Problem ....................................................................................... 10 

 Literature Review .................................................................................................... 10 

Rationale for the Research ...................................................................................... 14 

 Material and Methods .................................................................................................. 15 

 Results .......................................................................................................................... 20 

 Agglutination ......................................................................................................... 20 

Magnetic Bead Capture ......................................................................................... 20 

Discussion..................................................................................................................... 30 

 Conclusions and implications for further research ....................................................... 32

 References ..................................................................................................................... 33 

Dissemination of Research Findings ............................................................................. 37 

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2011-NE-BX-K547 Final Report Draft 

5

Executive Summary

Introduction

Forensic biology laboratories have long been searching for a more efficient, automatable method 

for processing sexual assault evidence. The most commonly used method is still the manual 

differential lysis extraction first described over 20 years ago (1). In this project the goal was to 

utilize magnetic bead capture and release chemistry to specifically bind spermatozoa and sperm 

heads while all other cell types are removed. This approach is routinely used in many clinical 

and basic research assays aiming at specific cell types in whole blood or cell culture specimen 

(for example 2-4). Several commercially available products offer universally applicable beads 

that enable research teams to use their own antibodies. New England Biolabs has a magnetic 

bead that is covalently coupled to a truncated form of recombinant Protein G, which exhibits a 

high affinity for immunoglobulin (IgG) from many species including human, rabbit, mouse and 

sheep (5, 6). Other types of customizable beads are being offered by Invitrogen 

Lifetechnologies (7). These beads are either coated with streptavidin, thus reacting with any 

biotinylated antibodies, coupled with secondary antibodies against immunoglobulin domains, or 

epoxy coated. The epoxy coated surface immobilizes ligands containing amino, thiol and 

hydroxyl functional groups; a property that can be used to cover the beads with any custom made 

antibody (8). 

Early work on the forensic application of this type of assay includes the use of epoxy beads and 

photoactivation by Eisenberg (9, 10), and IgG antibody coupled beads by Anslinger et al (11). 

While there were some encouraging results, neither project resulted in either a peer reviewed 

publication or an optimized forensic laboratory assay for sperm capture. The latest published 

sperm-magnetic bead results were generated using the biotin-streptavidin bead approach and an 

antibody against MOSPD3 (motile sperm domain containing protein 3) (12). This antibody is 

thought to be involved in spermatogenesis (13) and encodes a multi-pass membrane protein with 

a major sperm protein (MSP) domain (14). This assay was shown to effectively separate fresh 

sperm cells from female buccal cells but displayed decreased recovery for sperm cells on vaginal 

swabs that had been stored for up to 10 days (12). 

Research Design

Experiments in the present project tested a panel of five different sperm membrane specific 

antibodies identified via the human gene compendium and a review of the relevant literature for 

their ability to capture human sperm via the New England Biolabs Protein G assay. This bead 

type was chosen based on promising preliminary results for a similar bead type also from New 

England Biolabs (15). In a later stage of the project, a two more antibodies were added to the 

panel and a second type of bead, a streptavidin coated Lifetechnology bead, was utilized. 

Planned experiments included: antibody reactivity testing, sperm capture from single source 

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2011-NE-BX-K547 Final Report Draft 

6

samples, sperm capture from mixtures, optimization of the magnetic bead assay and comparison 

to current manual differential lysis methods. In order to separately evaluate the captured cells 

bound to the beads and the remaining cells still in solution, both the “capture” fraction and the 

“supernatant” fraction were subjected to DNA extraction and quantitation with a real time PCR 

assay specific for a human DNA target and a male Y-chromosome sequence. To prove the 

suitability of the recovered DNA for subsequent STR analysis, DNA fractions for several 

experiments were typed using the AmpFlSTR Identifiler Plus Kit from Applied Biosystems – 

Lifetechnologies. 

The following antibodies were included in the study: Ferrtilin β ADAM 2) a single-pass type I 

sperm membrane disintegrin and metalloproteinase domain-containing protein involved in spermoocyte binding (14, 16, 17); sperm adhesion molecule (SPAM1), a GPI-anchor hyaluronidase 

localized on the sperm surface and inner acrosomal membrane involved in sperm-zona pellucida

adhesion (14, 16); MFGE8 or SED1 (secreted protein containing EGF repeats and

Discoidin/F5/8 complement domains) which coats sperm as they traverse though the epididymis

and is required for sperm binding to the egg coat (14, 18); ubiquitin-associated protein 2 like 

protein (UBAP2L) interacting with the ZP3 receptor (14, 19), and AKAP3 or A kinase anchor 

protein 3, which is expressed in spermatozoa and localized to both acrosomal region of the sperm 

head as well as the principal region of the flagella (14, 20). MFGE8 and ADAM2 were tested 

both in monoclonal and polyclonal form, resulting in a panel of seven antibodies total. The 

additional antibodies later added to this panel were specific to AKAP associated Sperm Protein 

or Ropn1l (21, 22) acting on sperm motility and MOSPD3 (motile sperm domain containing 

protein 3), which is thought to be involved in spermatogenesis (12, 13) and encodes a multi-pass 

membrane protein with a major sperm protein (MSP) domain (14). 

The project can be divided in four phases: phase 1 – single source experiments with the full 

initial panel of seven antibodies and Protein G beads; phase 2 - single source experiments with a 

reduced panel of the four most promising antibodies and Protein G beads; phase 3 - duplication 

of the phase 2 single source experiments with fresh biological specimen and two of the 

antibodies, and phase 4 – single source experiments using streptavidin coated beads and three of 

the original panel antibodies, a new candidate Ropn1l, and the antibody to MOSPD3 that was 

selected based on the 2014 publication on sperm capture (12). 

Results

Protein G bead results were originally interpreted as displaying specificity differences between 

the seven antibodies. The tested samples came from two different semen donors with normalized 

cell counts from 20 to 20,000 sperm. Four of the seven antibodies samples had more DNA in the 

sperm cell capture fraction than in the supernatant. These four antibodies were further 

investigated for both semen donors and different amounts of antibody, including a “no antibody” 

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2011-NE-BX-K547 Final Report Draft 

7

control where 1xPBS was substituted for the antibody addition. Several of the samples, including 

the “no antibody” controls displayed a higher DNA yield in the capture than the supernatant 

fraction. This unspecific binding and DNA recovery was confirmed through two experiments 

with female vaginal epithelial cells. Most of the DNA was found associated with the Protein G 

beads and present in the capture fraction. Phase 3 duplicated the phase 2 results for MFGE8 and 

UBAP2L and fresh biological samples. Again, most if not all of the recovered DNA was in the 

bead capture fraction for both the male and female cell types and including the “no antibody” 

control. 

 

The streptavidin-coated beads successfully utilized in the publication by Li et al (12) are 

available as the Dynabead FlowComp Flexi kit from Invitrogen-Life Technologies Carlsbad, 

CA, USA). The biotin labeling chemistry included in this kit is based on DSB-X biotin, which 

was used to biotinylate the antibody to MOSPD3 introduced by Li et al (12) and three of the 

antibodies in the original panel. Single source experiments using the Dynabead FlowComp Flexi 

beads were performed for two fresh semen samples and two new female vaginal swab donors. 

For this type of beads for all of the antibodies and the “no antibody” controls almost none of the 

DNA resided in the bead capture fraction. The amount of DNA recovered in the supernatant 

correlated to the positive control samples, but there was no enrichment through bead capture. 

This lack of capture result was duplicated for MOSPD3 and Ropn1l using an alternative 

biotinylation assay, the EZ-Link Sulfo-NHS-LC-Biotin kit from Thermoscientific utilized by Li 

et al (12). 

Overall this project failed to demonstrate sperm cell specific enrichment for either magnetic bead 

capture assay. Protein G beads were shown to retain DNA, but in a non-specific fashion 

including vaginal epithelial cells and control samples with no antibody addition. For the 

Dynabead FlowComp Flexi kit almost the entire recovered DNA resided in the supernatant 

fraction that was taken off the immobilized beads. 

Discussion

For the New England Biolabs Protein G beads the problem was not a lack of DNA recovery, but 

rather the unspecific retention of DNA from sperm and non-sperm cells, with and without the 

addition of sperm membrane receptor specific antibodies. It is unlikely that the beads were 

binding whole cells. No cell to bead adhesion was seen during microscopic examination of a 

Protein G sperm extraction bead fraction after bead immobilization and washing. While there has 

been some discussion on cell free DNA (23), it is more likely that cells were lysed during the 

magnetic bead incubation step. Cells should have been intact after suspension in phosphate 

buffered saline, but the Protein G sodium phosphate binding buffer combined with the 

mechanical mixing in the presence of metal beads may have disrupted not only epithelial cell but 

also sperm head membranes. These magnetic beads may have an affinity to DNA or DNAThis resource was prepared by the author(s) using Federal funds provided by the U.S. 

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2011-NE-BX-K547 Final Report Draft 

8

histone complexes either via the Protein G coating or possibly gaps in this coat. No experiments 

with cell lysates or previously extracted DNA were performed to explore this. Anslinger at el 

working with anti-IgG antibodies also reported on this unspecific extraction of female cell DNA 

(11). 

For the assay with streptavidin coated beads, one can think of three possible areas where this 

assay could fail: The first factor that would prevent any specific binding is the choice and the 

quality of the purchased antibodies. Most antibody manufacturers only test their antibody 

performance for Western Blot applications and researchers have encountered vast differences in 

quality and binding capability for the same type of antibody from different companies. The 

agglutination tests showed a strong reaction at least to two antibodies in the panel (AKAP3 and 

MOSPD3), but this did not translate into successful magnetic bead capture. 

Under the assumption that antibodies were sufficiently labeled, sufficiently specific to an 

accessible sperm membrane antigen, and buffer and temperature conditions were suitable for 

efficient binding, the last factor prohibiting cell enrichment is the strength of the non-covalent 

biotin-streptavidin bond and its reaction to shearing effects during the required wash steps. As 

described by Eisenberg (10) agitation and stringent washing led to loss of previously bound 

material for the epoxy-antibody bead conjugates, which was the reason for adding the stabilizing 

photolysis step. It remains a fact that many other research teams have successfully recovered 

their target cell population (2-4). Overall performance will also be affected by the density of 

antigens available on the target for binding, number of biotin molecules incorporated per 

antibody and density of streptavidin receptors on the beads. For many of these applications there 

is abundant starting material and a certain loss of cellular material is expected and tolerated, but 

is unlikely that lack of thermodynamic stability and physical disruption of already formed bonds 

is the explanation for the total failure to capture sperm cell DNA on the streptavidin coated 

beads. 

The third source of failure is biotin labeling reaction which could have been unsuccessful leading 

to unlabeled and thus ineffective antibodies. The initial labeling chemistry used in this study was 

DSB-X biotin succinimidyl ester, which was part of the Dynabead FlowComp Flexi kit and was 

run for 1.5 hours as instructed by the manufacturer. Li et al (12) deviated in this step and labeled 

the MOSPD3 antibody with sulfo-NHS-LC-biotin obtained from Pierce Protein Biology 

Products, Thermo Scientific, Rockford, IL, USA; the labeling reaction was allowed to incubate 

for 72 hours. While there were no obvious issues with the DSB-X biotin succinimidyl ester

reaction, it must be stated that the actual binding of the biotin label was not confirmed via 

Western blot and labeled streptavidin probes. To address this possible source of failure, 

MOSPD3 and Ropn1l were labeled following Li et al (12) as closely as possible and the labeling 

efficiency was monitored using a biotin quantitation kit. The quantitation revealed low 

incorporation and the subsequent capturing experiments failed to show any enrichment. 

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9

Overall the failure to duplicate the results presented by Li et al (12) may be based on a 

combination of factors with antibody quality and some of the buffers and incubation conditions 

failing to facilitate optimal binding. But at least for the EZ-Link sulfo-NHS-LC-biotin kit 

reaction, the most likely step to have failed in our hands was the antibody biotinylation, which 

means the work presented here was compromised by technical issues and cannot be used to 

decide on the overall scientific merit of the magnetic bead sperm capture method. Unfortunately 

it was not possible to spend more time on optimizing this critical step. 

Conclusions and implications for further research

This study clearly demonstrated that Protein G beads are not suitable for magnetic bead based 

sperm separation. For the streptavidin coated beads, the failure to duplicate the results described

by Li et al (12) may be based on a combination of factors with antibody quality and some of the 

buffers and incubation conditions being possible factors. But at least for the EZ-Link sulfo-NHSLC-biotin kit reaction the most likely step to have failed in our hands was the antibody 

biotinylation, which means the work presented here was compromised by technical issues and 

cannot be used to decide on the overall scientific merit of the magnetic bead sperm capture 

method. 

The paper by Li et al is the only peer reviewed publication on the successful recovery of sperm 

cells and male DNA from mixtures with female cells using streptavidin covered beads (12). But 

they also found that the assay was better suited for fresh sperm and describe their success rate 

declining rapidly for dried vaginal swabs after short storage periods. It is unclear, if further 

research would be able to improve on these results. Surface antigens are easily compromised and 

capture release assays are always accompanied by a certain amount of loss. Forensic evidence by 

nature does not consist of pristine and abundant cells and even with very effective antibodies and 

under optimized conditions magnetic bead assays will not be able to recover the highest possible 

amount of male DNA. This makes the idea of an automated immuno-magnetic sperm capture 

assay problematic for compromised samples and evidence with low sperm content. If a working 

assay could be developed its application is likely to be limited to fairly fresh and abundant 

samples. 

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Introduction

Statement of the problem

In the United States, sexual assault occurs every two minutes and more than 200,000 women are

sexually assaulted each year (24, 25). The case backlog of unanalyzed sexual assault cases has 

been deemed a human rights issue and there have been demands to make the timely processing 

of this evidence a priority for criminal justice policy for many years (26). The most probative 

evidence in such cases is the identification of the semen donor from spermatozoa recovered from

vaginal swabs. The mixed nature of the sample (semen from the suspect and vaginal epithelial

cells from the victim) complicates DNA analysis. In these mixed samples, vaginal epithelial cells

vastly outnumber the sperm and separation of the male and female components in these samples

is critical for subsequent genetic analysis by PCR-STR. Overall there has been a lot of progress 

in forensic DNA evidence processing and almost all techniques have been getting faster, more 

sensitive and more discriminatory (27) with one notable exception. The DNA extraction from

mixed samples from sexual assault evidence using the differential extraction procedure (1) has 

remained virtually unchanged for the past 20 years. This separation procedure is time-consuming 

and labor intensive; the separation of the male and female fractions is oftentimes incomplete

which complicates the interpretation of the DNA typing results. A simple, robust, cost and time 

efficient method for sperm cell separation from sexual assault swabs would be of practical 

forensic value.

The differential extraction procedure (1) is a physical-chemical method that exploits the 

differences in membrane stability between the sperm and vaginal epithelial cells to separate the 

cellular components. This multi-step process involves lysis of epithelial cell membranes using 

mild conditions and collection of sperm cells (predominantly heads) by centrifugation. The

pelleted sperm cells are washed several times using phosphate buffered saline (PBS), 

resuspended and lysed in a buffer containing dithiothreitol (DTT). The DNA in the resultant

sperm and epithelial cell fractions is extracted using a method based on either an organic resin

(Chelex, BioRad, Hercules, CA), silica (QIAamp, Qiagen, Valencia, CA), organic phase 

separation (phenol:chloroform) or paramagnetic beads (DNA IQ, Promega, Madison, WI). The

separation procedure is time-consuming (approximately two hours and labor intensive. 

Separation of the male and female fractions is oftentimes incomplete and thus complicates the 

interpretation of the DNA typing results. The repeated washes can result in sperm cell loss and 

the multiple steps increase the risk of contamination. Several alternative methods of cell

separation have been proposed. 

Literature review

Physical, Chemical and Enzymatic Separation Techniques

Schoell et al (28) used a fluorescence-activated cell sorting method to separate sperm and vaginal 

cells. The method has limited applicability to forensic casework since it would require a change

in the method of collection of evidentiary samples from vaginal swabs to vaginal lavages. Laser

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11

capture micro-dissection (LCM) has also been used to separate sperm cells from vaginal epithelial

cells (29-31). Although LCM provides a very precise, clean separation, the technique is laborintensive, time-consuming and costly and thus not amenable to high- throughput analysis of 

casework samples. Filtration-based methods of separation have also been developed (32, 33). The

separation was incomplete and DNA from spontaneously lysed vaginal cells was present in the 

sperm fraction. Clogging of the membrane by debris (cotton fibers from the vaginal swab) was

also problematic. Horsman et al (34) used a micro-fabricated device to separate sperm and

vaginal epithelial cells. Although the method shows promise, the collection (30 min/sample) is

time-consuming. Bienvenue, et al (35) took the microfluidic device a step further by developing a 

microchip- based cell lysis and extraction model. Full DNA profiles were obtained. While this

appears to be a successful approach, both extraction and separation techniques are labor intensive. 

Selective degradation of female DNA in mixed samples via nuclease (DNase I) digestion shows 

promise (36). However, the method is limited due to the long digestion incubation time (5 hrs.) 

prior to DNA purification and thus not amenable to high-throughput analysis of casework 

samples. The DifferexTM System developed by Promega (Madison, WI) uses phase separation and 

differential centrifugation to separate sperm and epithelial cells. Although the method produces 

cleaner separations than the conventional differential extraction method, the technique is still 

labor intensive (37, 38), as is another optimized differential lysis protocol utilizing a modified 

elution buffer (39). Casework samples need a more rapid and possibly automated technique. 

Haak, Porsche, Vollack, Zimmermann, & Pflug (40) developed a semi-automatic, magnetic-bead

based DNA extraction method using the ChargeSwitch® Forensic DNA Purification Kit

(Invitrogen, Carlsbad, CA) and two Te-MagS magnetic bead separation modules (Tecan, Durham,

NC). The system was interfaced with a Freedom EVO 150 liquid handling workstation (Tecan,

Durham, NC) equipped with an eight-channel liquid handling arm and a PosID barcode sample 

identification device (Tecan, Durham, NC). This completely automated system was compared to 

the manually based GEN-IAL First Magnetic Forensic kit (GEN-IAL GmbH, Troisdorf, 

Germany) method. While both methods produced similar DNA yields, neither produced a 

definitive separation of cell types, leaving an “enormous excess” of female DNA (38). This is a 

significant disadvantage when working with casework samples.

More recently the DNA team working for the California Department of Justice published a highthroughput semen evidence workflow that seems promising (41). The method relies on the 

observed propensity of sperm cells to adhere to a substrate such as a vaginal swab, which makes 

it possible to lyse cells and remove non-sperm DNA using an initial alkaline lysis step. 

Subsequent DNAase digestion is used to eliminate remaining non-sperm DNA on the soaked 

substrate prior to the sperm lysis step. The authors describe increased yields over the standard 

DTT based differential lysis but caution about possible loss of sperm DNA for aged swabs and 

other stain substrates (41). 

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Sperm Surface Receptors

An alternative to the methods discussed above is a separation procedure based on the antigenic 

properties of human spermatozoa. Anti-sperm antibodies have been studied extensively in the 

field of human development and reproduction. Sperm cells possess a high degree of cell surface

differentiation. Each region is designated for a specific function that is mediated by antigens 

located on the head, acrosome, equatorial, midpiece, and tail sections of spermatozoa (42).The

complex process of sperm binding to the zona pellucida of oocytes has been a highly researched

topic in the field of reproduction and development. Human zona pellucida (ZP) glycoproteins

have been found to play the most important role in sperm binding. The human zona pellucida is

a protein matrix surrounding the oocyte which contains four glycoproteins – ZP1, ZP2, ZP3 and

ZP4, respectively. ZP3 has been identified as the primary sperm binding protein and plays a 

complex role in the initial binding of the sperm head ZP3 receptor (ZP3R) to the zona pellucida 

and subsequent induction of the acrosomal reaction necessary for fertilization (43).

While the ZP3 receptor has been shown to play a critical role in sperm-zona pellucida binding, 

it is only one component of a complex reaction that mediates sperm fusion to the oocyte. 

Ferrtilin β (ADAM 2) is a single-pass type I sperm membrane disintegrin and metalloproteinase

domain-containing protein involved in sperm-oocyte binding (14, 16, 17). Sperm adhesion 

molecule (SPAM1/PH-20) is a GPI-anchor hyaluronidase localized on the sperm surface and

inner acrosomal membrane involved in sperm-zona pellucida adhesion (14, 16). MFGE8 or 

SED1 (secreted protein containing EGF repeats and Discoidin/F5/8 complement domains) is an

EGF repeat and discoidin domain protein that coats sperm as they traverse though the 

epididymis and is required for sperm binding to the egg coat (14, 18). Ubiquitin-associated 

protein 2 like (UBAP2L) protein interacts with the ZP3 receptor (14, 19). AKAP3 is also 

known as a kinase anchor protein 3, is expressed in spermatozoa, and localized to both 

acrosomal region of the sperm head as well as the principal region of the flagella (14, 20). A 

similar target is the human AKAP associated sperm protein Ropn1l (21, 22). Motile sperm 

domain containing protein 3 (MOSPD3) is thought to be involved in spermatogenesis (12, 13) 

and encodes a multi-pass membrane protein with a major sperm protein (MSP) domain (14).

Theoretically the antigenic properties of spermatozoa can be used to develop methods 

applicable to the field of forensic science. It should be possible to use antibodies directed

against sperm surface antigens to capture sperm cells and facilitate the separation of cellular 

material in mixed samples.

Immunological Separation Techniques

Several authors have explored magnetic bead separation procedure based on the antigenic

properties of human spermatozoa as an alternative to the physical, chemical and enzymatic

methods discussed above (9, 10, 12, 44-47). Eisenberg (9, 10) developed an antibody-based

separation method using epoxy coated magnetic beads with covalently bound sperm-specific

antibodies to capture spermatozoa. Although promising for casework, the technique has several

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2011-NE-BX-K547 Final Report Draft 

13

limitations. First, the three monoclonal anti-sperm antibodies used were directed against antigens 

located on the tail, acrosome and acrosomal cap. In most instances only sperm heads are isolated

from vaginal swabs- the tails are frequently degraded. Acrosomal antigens are displayed on the 

surface of capacitated and acrosome-reacted sperm cells and not in ejaculates (48). While the 

antibody coated beads were specific to sperm cells, the strength of the bond was not sufficient to 

retain the sperm cells during the required wash steps. This was addressed by photoaffinity 

labeling of the antibody- magnetic bead complex and photoactivation to form covalent spermantibody bonds. Although covalent bond formation increased the avidity of the antibodies, the 

effect was mitigated by a reduction in the affinity of the antibodies. Fewer sperm were captured

but those captured were tightly bound (9, 10). A different choice of antibody may maximize the 

binding and retention of sperm. 

Anslinger, Bayer, Danilove, & Metzger (11) devised an immuno-magnetic bead procedure using 

monoclonal antibodies (mAbs) against the testicular isoform of the angiotensin-converting 

enzyme (tACE). This antigen is located on the neck, mid-piece and flagella of the sperm. These

antibodies were coupled to Mouse IgG Dynabead (Invitrogen - Lifetechnologies, Carlsbad, CA) 

magnetic bead particles. The ability of each of three mAbs to separate sperm cells from vaginal 

epithelial cells from dried swab samples was tested. Results demonstrated a lack of antibody 

binding due to loss of sperm midpiece and flagellum (11). However, positive results were

obtained from vaginal swabs stored in PBS buffer. This is an unrealistic situation because 

casework samples are stored dry and are typically days to months old. Also, depending on the 

ratio of cells in the mixed samples, most male DNA profiles showed varying amounts of female

alleles. Overall, a major male component was found in samples that contained at least 105 sperm

cells. This is a large number of cells and highly unlikely to be found in casework samples.

Again, there is the potential that this assay can be optimized using different antibodies. 

A different type of magnetic bead coated with Protein A (New England Biolabs, Ipswich, MA)

was used in unpublished thesis research at John Jay College of Criminal Justice (15). Protein A 

displays a high affinity for immunoglobulins (IgG) from several species including rabbit and 

mouse and will thus bind to antibodies produced in these animals. Spermatozoa were incubated 

with polyclonal rabbit anti-human sperm antibody, anti-ZP3R (Aviva Systems Biology, San 

Diego, CA), directed against sperm zona pellucida glycoprotein 3 receptor (43) and the antibody 

titer was determined to be 1:256. All samples tested were single source and the endpoint determination

in these studies was the amount of DNA recovered from Chelex extracts of captured sperm and

buccal cell samples. The initial results confirmed the specificity of the antibody. The Protein A 

beads could be shown to capture more male than female DNA but overall most of the DNA was 

recovered in the wash fraction and many samples yielded only partial DNA profiles (15).

 

The latest research describing successful sperm capture using a magnetic bead procedure was 

published in 2014. Li et al (12) employed biotinylated monoclonal antibodies towards motile 

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2011-NE-BX-K547 Final Report Draft 

14

sperm domain protein 3 (MOSPD3) and streptavidin coupled beads available in the Dynabead 

FlowComp Flexi kit (Invitrogen - Lifetechnologies, Carlsbad, CA). They tested fresh sperm and 

female buccal epithelial cells mixed at various ratios as well as dried semen containing vaginal 

swabs. The team was able to microscopically demonstrate magnetic bead adhesion to both the 

sperm head and the tail region. They obtained full male single source STR profiles from all 

mixtures containing 105 sperm cells per mL and good quality partial profiles for 103 and 104

sperm cells per mL. The dried vaginal swabs seem to have been real casework samples and were 

microscopically examined for the presence of spermatozoa. The authors did not define if 

“successful detection” again meant single source male DNA profiles, but it is stated that 

successful detection was correlated to storage time. While dried swabs stored for only 1 day had 

100% successful detection, this rate decreased to16.67% for swabs preserved for 10 days (12). 

Rationale for the Research

Immunological cell separation techniques are widely used for basic and clinical research 

applications and show the same promise for forensic evidence, especially for the separation of 

sperm from vaginal epithelial cells. In order to allow for stringent wash steps and successful 

removal of epithelial cells a cell capture method must combine two features. Antibodies must

display a high level of specificity towards antigens not only reliably present on the sperm head 

membrane, but also reactive and preserved in dried specimen. The antibody-sperm complex must 

form a strong bond to the chosen magnetic bead. It was anticipated that it should be possible to 

optimize one or more of these immuno-magnetic methods and thus provide a more efficient,

rapid, and cost-effective alternative to the current differential lysis methods. 

Based on published data the following antibodies were selected for further testing: antibodies 

directed against ZP3R, ADAM2 -fertilin β, SPAM1, MFGE8-SEDI, AKAP3, UBAP2L, and a 

later stage human AKAP associated sperm protein or Ropn1l and MOSPD3. Unfortunately 

the ZP3R antibody was no longer commercially available and had to be eliminated from the 

study. This project employed Protein G magnetic beads (New England Biolabs, Ipswich, MA, 

USA) instead of the previously tested Protein A beads to take advantage of the higher 

reported IgG affinity for goat based antibodies (9). For phase 4 the bead assay to be evaluated 

was the Dynabead FlowComp Flexi kit (Invitrogen - Lifetechnologies, Carlsbad, CA) 

successfully used by Li et al (12). This antibody bead bond is based on the high affinity 

biotin-streptavidin protein-ligand interaction (12). Planned experiments included: antibody 

titer determination, optimum amount of anti-sperm antibodies, sperm capture from single 

source samples, DNA extraction and quantitation, sperm capture from mixtures, DNA 

amplification and genotyping, optimization of the magnetic bead assay and comparison to 

manual differential lysis employing a Chelex or Differex (37, 38, 47) approach. 

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2011-NE-BX-K547 Final Report Draft 

15

Material and Methods 

Samples 

Sperm samples, vaginal swabs and buccal reference swabs were obtained from three male and 

four female volunteers. Volunteer recruitment and sample collection procedures had been 

approved by the City University of New York Institutional Review Board. In addition to the 

volunteer samples, two single source fresh semen samples were purchased from Lee 

Biosolutions, St. Louis, MO, USA. Semen samples were divided into 100μL aliquots and 

stored refrigerated for up to two weeks and at -20°C for long-term storage. The two fresh 

semen samples were also used to produce 10μL semen stains on cotton material and cotton 

swabs. These stains and swabs were individually packaged in coin envelopes and stored at 

room temperature. Vaginal swabs were air dried and stored under refrigeration; epithelial cells

were obtained from the vaginal swabs by incubation in phosphate buffered saline (PBS) at room 

temperature with agitation (Eppendorf Thermomixer, Daigger, Vernon Hills, IL) for 2 hrs. to

dislodge the cells from the substrate. The cell suspensions and swabs were stored in separate

sterile tubes at 4 oC. 

Cell counting was performed by staining aliquots of spermatozoa and vaginal epithelial cell

suspensions with 1X phosphate buffered saline-Methylene Blue (PBS-MB) solution (4:1) to

visualize the cells and by using a Neubauer

® hemocytometer.

Antibodies 

Antibodies were obtained from the following sources: Abnova, Taipei, Taiwan: SPAM1 (mouse 

polyclonal), MFGE8 (mouse polyclonal and monoclonal), ADAM2 (mouse polyclonal and 

monoclonal), UBAP2L (goat polyclonal). Antibodies-Online Inc, Atlanta, GA, USA: AKAP3 

(rabbit) and Abcam Inc, Cambridge, MA, USA: MOSPD3 (rabbit polyclonal), AKAP associated 

Sperm Protein or Ropn1l (rabbit monoclonal and polyclonal).. Antibody storage buffers were 

checked for sodium azide and bovine serum albumin (BSA) concentrations. No pre-purification 

of antibodies was performed since all levels were found to be below the critical thresholds for the 

two biotinylation assays employed.

Table 1 lists sources and catalog numbers for all antibodies 

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2011-NE-BX-K547 Final Report Draft 

16

Table 1 Antibody information 

Antibody Origin Company Catalog Number

SPAM1 mouse polyclonal Abnova, Taipei, Taiwan H00006677-A01

MFGE8 mouse polyclonal Abnova, Taipei, Taiwan H00004240-A01

MFGE8 mouse monoclonal Abnova, Taipei, Taiwan MAB3634

ADAM2 mouse polyclonal Abnova, Taipei, Taiwan H00002515-M02

ADAM2 mouse monoclonal Abnova, Taipei, Taiwan H00002515-A01

UBAP2L goat polyclonal Abnova, Taipei, Taiwan PAB22848

AKAP3 rabbit Antibodies-Online Inc, 

Atlanta, GA

ABIN652454 

MOSPD3 rabbit polyclonal Abcam Inc, Cambridge, MA GR184886

UBAP2L rabbit polyclonal Abcam Inc, Cambridge, MA GR169595

Ropn1l rabbit polyclonal Abcam Inc, Cambridge, MA GR189438

Ropn1l rabbit monoclonal Abcam Inc, Cambridge, MA GR130918

Antibody titer was determined using the modified slide agglutination test (50, 51) at a constant 

sperm concentration and Terasaki optical microtiter plates. A series of two-fold dilutions (1:1

to 1:256) of the anti-sperm antibody was made using 1X phosphate buffered saline-Methylene

Blue (PBS-MB) solution in a total volume of 10 μl. Five μl of antibody or 1X PBS-MB 

(negative control) and 1μl of neat or diluted sperm suspension were added to each well, 

covered and incubated for two hours at 37 oC. The slides were then observed microscopically

at 200 or 400 X magnification. Agglutination results were scored as follows: +++, complete

agglutination, ++, large agglutinates, +, scattered agglutinates, -, no agglutination. 

Dynabead FlowComp Flexi magnetic bead process 

Antibody labeling was performed using the DSB-X Biotin Protein Labeling kit included in the 

Dynabead FlowComp Flexi magnetic bead kit from Invitrogen/Life Technologies, Carlsbad, CA, 

USA. 100μL of a 0.5 - 1μg/μL antibody solution and 50μL of freshly prepared 1M sodium 

bicarbonate solution were added to a 2mL reaction tube containing a stir bar provided in the kit. 

200μL of dimethyl sulfoxide (DMSO, component B of the kit) were added to the kit’s vial of 

DSB-X biotin succinimidyl ester (component A) and 6μL of this DSB-X biotin succinimidyl 

mixture was immediately added to the antibody solution. The reaction was allowed to stir at 

room temperature for 1.5 hours. The labeled antibodies were purified from unincorporated biotin 

using the spin columns and purification resin provided in the kit following the manufacturer’s 

instructions. Based on the manufacturer’s efficiency estimates antibody concentrations were 

calculated as follows: mg/mL DSB-X biotin-labeled protein = initial mg of protein x 0.85/mL 

collection volume. 

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Alternatively, antibody labeling was performed with the EZ-Link Sulfo-NHS-LC-Biotin kit from 

Pierce/Thermoscientific Rockford, IL. MOSPD3 was incubated with a 30 fold molar excess of 

biotin (11.5μL of 10mM freshly prepared biotin reagent to 100μL or 100μg of antibody), a 50 

fold excess was used for monoclonal Ropn1l (5.2μL of 10mM biotin reagent to 100μL or 27.4μg

of antibody) and a 20 fold molar excess of biotin was used for the polyclonal Ropn1l preparation 

(27.5μL of 10mM biotin reagent to 100μL or 180μg of antibody). Following Li et al (12) the 

reaction was set for 72 hours at 4oC. Excess biotin reagent was removed via dialysis in potassium 

and amine free BupH PBS (phosphate buffered saline: 0.1M sodium phosphate, 0.15M NaCl, pH 

7.2) Pierce/Thermoscientific Rockford, IL and using Slide-A-Lyzer Dialysis cassettes with a 10k 

molecular weight cut off also from Pierce. Again, following Li et al (12) dialysis was performed 

for 24 hours at 4o

C with two buffer changes. 

Biotin incorporation or mole to mole ratio was measured using the Pierce Biotin Quantitation kit, 

now sold by Pierce/Thermoscientific Rockford, IL. The kit measures the change of absorbance at 

500nm that occurs when the HABA (4’-hydroxyazobenzene-2-carboxyl acid) – avidin complex 

provided in the kit is disrupted in the presence of biotin. A biotinylated horseradish peroxidase 

(1mg/mL) included in the kit serves as the positive control. To preserve material, all antibodies 

were tested in a 1:10 dilution. Following the kit instructions HABA/avidin premix tubes were 

equilibrated to room temperature and resuspended in 100μL of ultrapure water. For each sample 

and the positive control a 1mL cuvette was filled with 800μL of BupH PBS, 100μL of 

HABA/avidin premix was added and mixed well. The A500 HBA/avidin absorption was 

measured at 500nm. Afterwards 100μL of the neat positive control and 1:10 dilutions for the 

positive control and labeled antibodies were added to each cuvette and measured at 500nm. The 

Pierce online calculator incorporates molecular weights and sample concentrations to translate 

the two absorption measurements into a ratio of biotin molecules per protein molecule. 

For the magnetic bead capture 1μL of neat and 1:10 dilutions of sperm (representing 4,000 - 

40,000 cells) were incubated with 10μL (5μg) DSB-X biotinylated antibody at 37°C on an 

Eppendorf Thermomixer at 800rpm for 1 hour. For the EZ-Link Sulfo-NHS-LC-Biotin labeled 

antibody the initial experiment with MOSPD3 employed three different amounts (10, 1.5 and 

0.5μg) while the remaining experiments used 5μg of antibody as before. After incubation, 

unattached antibody was removed by adding 100μL of cold isolation buffer (phosphate buffered 

saline with 0.1% bovine serum albumin and 2mM Ethylenediaminetetraacetic acid -EDTA), 

brief mixing, pelleting the sperm at 6,000g for 10 min and removing the supernatant. The sperm 

pellet was resuspended in 100μL of cold Isolation Buffer and 10μL of FlowComp Dynabeads 

and incubated for 30 min at 4°C under constant motion on the Life Technologies HulaMixer. 

After incubation 100μL of isolation buffer were added and the samples were placed on a six tube 

DYNAL MPC-S magnet to immobilize the beads. The supernatant containing the non-target 

cells was removed and saved for extraction (Supernatant Fraction). The magnetic beads were 

resuspended in isolation buffer and immobilized twice more for a total of three washes. For the 

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2011-NE-BX-K547 Final Report Draft 

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final step, the magnetic beads were suspended in 200μL release buffer (provided in FlowComp 

Flexi kit), incubated for 30 min at 4°C on the HulaMixer and placed on the magnet. The release 

buffer now containing the captured cells was transferred to a fresh tube and stored for DNA 

extraction (Capture Fraction). 

Protein G Magnetic Bead captures 

1μl of sperm dilution (20-20,000 cells) was mixed with 5μl of unlabeled antibody 

(concentrations ranging from 0.1 to 1μg/μL) and incubated in an Eppendorf thermomixer for 

2hrs at 37°C. After incubation the volume was adjusted to 50μl by adding 1X phosphate buffered 

saline (PBS). 25μl of resuspended Protein G beads (New England Biolabs, Ipswich, MA, USA) 

were mixed with 80μl of binding buffer ((0.1M sodium phosphate buffer pH 8.0) and the 50μl of 

sperm-antibody complex and incubated the sample at 4°C with 550rpm agitation for 30min. For 

phase III of the project this step was performed using the Life Technologies Hulamixer allowing 

for multidirectional mixing rather than horizontal agitation. After incubation the samples were 

placed on a DYNAL MPC-S magnet to immobilize the beads. The supernatant containing the 

non-target cells was removed and saved for extraction (Supernatant Fraction). The magnetic 

beads were resuspended in 500μl of binding buffer and immobilized twice more for a total of 

three washes. The beads were the stored in 20μL of 1X PBS prior to extraction (Capture 

Fraction). 

Chelex extraction

Addition of Chelex beads and 1M DTT was modified based on the cell type and starting volume 

of the cell fractions as follows. 

- Sperm cell supernatant and Dynal FlowComp Flexi capture fractions (starting volume 

180-210μL): 20μL of 20% Chelex (Bio-Rad Laboratories, Hercules, CA), 50μL of sterile 

bidestilled water, 1μL of Proteinase K (20mg/mL) and 7μL of DTT (1M) were added to 

each sample. 

- Sperm cell Protein G bead capture fractions (starting volume 20μL): 200μL of 5% 

Chelex, 1μL of Proteinase K (20mg/mL) and 7μL of DTT (1M) were added to each 

sample.

- Female cell supernatant and Dynal FlowComp Flexi capture fractions (starting volume 

180-210μL): 20μL of 20% Chelex, 50μL of sterile bidestilled water, and 1μL of 

Proteinase K (20mg/mL) were added to each sample. 

- Female cell Protein G bead capture fractions (starting volume 20μL): 200μL of 5% 

Chelex and1μL of Proteinase K (20mg/mL) were added to each sample.

All samples were incubated for 45min in a 56°C thermomixer. After incubation samples were 

vortexed for 10sec, then centrifuged for 15sec at 14,000g, incubated at 100°C for 8min, vortexed 

again, centrifuged for 3min at 14,000g, and stored at 4°C. 

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2011-NE-BX-K547 Final Report Draft 

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Amicon Filter Concentration 

Amicon 50k Ultra-0.5 devices (EMD Millipore, Billerica, MA, USA) were pre-rinsed with

100μL TE-4 buffer. Samples were added and centrifuged at 14,000g for 8min. The reversed spin 

was 2min at 1,000g to transfer the concentrated sample from the device to a clean sample

collection tube. Collection volumes ranged from 20-25μL.

QIAamp DNA Mini Kit DNA Extraction 

20μL of Proteinase K (2omg/mL) and 200μL of sample were mixed with 200μL Qiagen Buffer 

AL (part of QIAamp DNA Mini kit; QIAGEN, Germantown, MD, USA) and 18.3μL of 1M 

DTT and incubated at 56 ̊C for 20min. Samples were cooled to room temperature and 200μL 

ethanol (96-100%) was added. Samples were added to the QIAamp Mini spin column (in a 2mL 

collection tube), and centrifuged at 6,000g for 1 min. The QIAamp Mini spin column was placed 

in a clean 2mL collection and after adding 500μL Qiagen Buffer AW1 centrifuged at 6,000g for 

1 min. This step was repeated with 500μL Qiagen Buffer AW2. DNA was recovered by placing 

the QIAamp Mini spin column in a clean 1.5mL microcentrifuge tube and adding 200μL Qiagen 

Buffer AE, incubating at room temperature (15-25 ̊C) for 1 min, and then centrifuging at 6,000g 

for 1 min. All samples were concentrated using Amicon 50k Ultra devices as described above. 

Process controls

The following controls were added to the experimental set-up. 

Agglutination test:

- Negative control: semen dilutions were prepared and incubated as described above 

without adding any antibody. 

Magnetic bead capture:

- Negative control: all reagents including antibodies were subjected to the full procedure 

with all wash steps, extraction and quantitation without adding any human cells. 

- No antibody control: biological test samples were subjected to the full procedure with 

all wash steps, extraction and quantitation with 1x PBS replacing the antibody volume in 

the initial antibody binding reaction. 

- Positive full process control: corresponding cell counts for each donor were incubated 

in the same buffer, with the same antibody and identical conditions parallel to the bead 

binding step, but without beads. 

DNA extraction:

- Positive “extraction only” control: corresponding cell counts for each donor were 

extracted together with the test sample fractions without undergoing the bead incubation 

step. 

STR typing:

- Positive control: control DNA provided by the manufacturer was typed in parallel to the 

samples. 

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- Contamination control: reference samples from project workers were typed to test for 

contamination. 

- Amplification negative control: all amplification reagents were amplified and typed 

without DNA addition. 

DNA Quantitation

Male and total DNA in all DNA extracts was quantitated using the Quantifiler Duo® DNA 

Quantification kit (Applied Biosystems - Lifetechnologies, Foster City, CA) for Real-Time 

(RT) PCR Model 7500 according to the manufacturer’s protocol. 

STR Typing 

For phase 1 and 2 samples, references and other controls with quantitation results within the

recommended range of input DNA for the AmpFlSTR® Identifiler Plus Kit (Applied Biosystems - 

Lifetechnologies, Foster City, CA) were amplified on the Gene Amp PCR 9700 System (Applied 

Biosystems) and typed on the 310 Genetic Analyzer (Applied Biosystems – Lifetechnologies,

Foster City, CA) according to the manufacturer’s instructions. DNA profiles were determined

using the GeneMapper

® ID (Version 3.2) software package from Applied Biosystems with the 

analytical threshold set to 50RFU. According to the Identifiler Plus Manual the recommended 

DNA input is 0.5-1.25 ng, and 10μl of extract can be added to the PCR reaction mix, therefore all 

extracts yielding 0.05- 0.125 ng/μl DNA were candidates for STR amplification. Resulting 

profiles were compared to the buccal reference swabs and contamination control samples. 

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Results

Agglutination assay 

The original batch of antibodies obtained from Abnova, Taipei, Taiwan that was tested included: 

SPAM1 (polyclonal), MFGE8/SEDI (polyclonal and monoclonal), ADAM2 (polyclonal and 

monoclonal), and UBAP2L (polyclonal). Most of these showed only weak scattered 

agglutination results for the neat antibody addition and no agglutination for any dilutions. The 

exceptions were SPAM1 which showed no agglutination at all, and ADAM2 (monoclonal) 

showing some scattered agglutination for the neat and 1:2 and 1:4 antibody dilutions, ADAM2 

polyclonal reacting for neat and 1:2. The same titration for AKAP3 (polyclonal) obtained from 

Antibodies-Online Inc, Atlanta, GA, USA resulted in large agglutinates for the 1:4 dilution and 

scattered aggregates were seen down to the 1:256 antibody dilution. An attempt to repeat this 

agglutination test for the additional semen samples acquired in phase 3 was inconclusive due to 

the presence of undefined clumps of material and aggregates resembling agglutination in the 

negative controls. 

The results for the MOSPD3 antibody ordered from Abcam, Cambridge, MA were positive for 

sperm-antibody binding. Large agglutinates were seen for the neat and1:4 samples. This titration 

series stopped at 1:8 and this final dilution showed scattered agglutination. 

Magnetic bead capture results

Phase 1 – full seven antibody panel - Protein G beads 

Experiments were performed as described above using two semen donors (01s and 02S) and five 

different sperm counts (20,000; 2,000; 200; 20 and 2). Two positive controls (one for each semen 

source) were processed using the same cell counts. Extraction yields for the combined capture 

and supernatant fractions were similar for the positive control and correlated well with the cell 

count. For cell counts below 2,000 most of the DNA concentrations were below the quantitation 

assay threshold which was why the Amicon concentration step was added to the procedure. 

Table 2 shows the ratio between the DNA concentrations in the bead capture fraction versus the 

supernatant or first wash fraction. Each value represents a single sample. Ratios above 2 are 

marked. These experiments did not include a no-antibody sperm capture control. Based on these

results, the four antibodies with an increased amount of DNA in the bead fraction versus the 

supernatant fraction (UBAP2L, MFGE8m, ADAM2m and AKAP3) were selected for further 

testing. 

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2011-NE-BX-K547 Final Report Draft 

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Table 2 Calculated ratios between DNA concentrations in capture versus supernatant fraction for 

the 7-Antibody panel and Protein G beads, 

SPAM UBAP2L MFGE8m MFGE8p ADAM2m ADAM2p AKAP3

01S 20k 0.85 2.47 2.18 0.46 2.65 0.74 2.57

01S 2k 1.07 3.29 1.99 0.24 4.34 0.15 1.47

02S 20k 1.09 2.78 3.83 0.18 2.66 0.20 0.66

02S 2K 1.13 0.17 3.59 0.19 2.64 0.29 1.43

nc 01S 20k 0.99 5.40 7.68 1.40 3.95 0.68 5.42

nc 01S 2k 1.30 0.70 35.47 1.34 und 0.24 7.92

nc 02S 20k 2.05 7.86 6.02 0.24 2.94 0.75 2.95

nc 01S 2K 4.39 und 24.50 0.46 und 1.69 10.53

ratio >2 2 5 7 0 6 0 5

“und” indicates no quantitation reading obtained for one of the fractions. 

“nc” not concentrated 

DNA typing of eligible DNA fractions resulted in the expected partial to full single source 

profiles. The number of loci detected strongly correlated to the initial DNA input (data not 

shown). 

Phase 2 – Reduced four antibody panel - Protein G beads 

Subsequent testing included only the four most promising antibodies and omitted the 200 and 20 

cell samples. This means there were now three cell count titration steps for each semen source

(20,000; 2,000 and 200). In this phase the Amicon Ultra 50k filter concentration step was added 

to all Chelex extractions. Other modifications included adding the “no antibody control” and 

doubling the amount of antibody added to the reaction. Samples were tested in parallel using 5uL 

and 10uL of antibody. Overall yields for this set of experiments were low. The positive controls 

yielded between 33 and 86% of the DNA amount expected based on the cell count. For both test 

samples magnetic bead capture and supernatant fractions combined, only recovered 

approximately 16% of the expected DNA. 

As depicted in table 3, for the ADAM2 antibody the bead and wash fractions did not show the 

expected enrichment in the bead fraction seen in the first round of testing. 

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Table 3: Quantitation values of cells captured in the sperm-ADAM2 (monoclonal)-bead 

complex (in pg/μL) 

Beads - 01S Wash - 01S Beads - 02S Wash - 02S

No Ab 20K 37 15 69 3

No Ab 2K 2 7 6 1

No Ab 200 0 0 0 0

5 uL Ab 20K 27 22 100 117

5 uL Ab 2K 6 8 18 6

5 uL Ab 200 1 1 1 0

10 uL Ab 20K 15 196 92 143

10 uL Ab 2K 2 5 5 8

10 uL Ab 200 0 1 0 1

For the MFGE8 antibody only two of the 20K samples for donor 01S showed any enrichment in 

the bead fraction (see highlights), but overall yields were low, the result was not confirmed for 

donor 02S, and the no antibody control also showed more DNA in the bead versus the wash 

fraction (table 4). 

Table 4: Quantitation values of cells captured in the sperm-MFGE8 (monoclonal)-bead complex 

(in pg/μL) 

Beads - 01S Wash - 01S Beads - 02S Wash - 02S

No Ab 20K 37 15 69 3

No Ab 2K 2 7 6 1

No Ab 200 0 0 0 0

5 uL Ab 20K 31 3 100 96

5 uL Ab 2K 2 23 5 6

5 uL Ab 200 1 0 0 0

10 uL Ab 20K 77 11 61 125

10 uL Ab 2K 5 11 10 7

10 uL Ab 200 1 0 0 0

Table 5 shows results for the UBAP2L antibody. Two of the 20K samples for donor 01S and one 

2k sample for donor 02S (see highlights), but none of the other samples with the exception of the 

same “no antibody” control showed any enrichment in the bead fraction. Again overall yields 

were low. 

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Table 5: Quantitation values of cells captured in the sperm-UBAP2L (polyclonal)-bead complex 

(in pg/μL) 

Beads - 01S Wash - 01S Beads - 02S Wash - 02S

No Ab 20K 37 15 69 3

No Ab 2K 2 7 6 1

No Ab 200 0 0 0 0 

5 uL Ab 20K 22 11 130 194

5 uL Ab 2K 9 21 6 3 

5 uL Ab 200 0 0 0 0 

10 uL Ab 20K 89 17 91 147

10 uL Ab 2K 4 3 11 1

10 uL Ab 200 0 0 0 0 

The 5uL and 10ul amounts of antibody were very similar and did not show a trend; these results 

were therefore combined (n=2) to calculate the average capture versus supernatant yield ratio. 

Table 6: Average ratio between DNA concentrations in capture versus supernatant fraction: 

Protein G beads 

UBAP2L* MFGE8 m ADAM2 m

No Antibody 

control 

01S 20k 3.98 7.65 0.20 2.56

01S 2k 0.52 0.22 0.63 0.27

02S 20k 0.65 0.73 0.74 25.75

02S 2K 5.17 1.13 1.65 5.65

* ratios >2 are highlighted 

Clearly the bead fraction captures DNA independent of the presence of antibodies in the assay. 

The experiment testing the AKAP antibody was inconclusive. This run using semen donors 2 

and 3 had been modified by adding 1 hour of incubation of the magnetic bead fraction at 37°C 

prior to the Chelex extraction. Here all ratios between capture and supernatant fraction, including 

for the “no antibody” control, were below 1 signifying less capture by the Protein G beads. But 

all DNA yields for all fractions were very low indicating a problem with this extraction, possibly 

caused by degraded DTT (data not shown). 

The unspecific binding of the protein G beads was confirmed in the experiments using female 

cells. Two independent runs with defined cell counts of 400 vaginal epithelial cells both resulted in 

more DNA in the bead capture fraction. For the second run testing either 5μL or 10μL of each 

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25

antibody, the positive extraction control yielded much more than the expected 2.4ng total yield, 

which indicates problems with adding defined cell counts (data not shown). As seen in the graph in 

Figure 1, there are also variations in the overall DNA amounts recovered for this sample donor. 

Again, results for 5 and 10μL antibody tests were combined so n=2. Figure 1 also shows that all of 

the bead capture fractions contain epithelial cell DNA, which confirms the unspecific binding seen 

for the sperm cell experiments. 

 

Figure 1: Average DNA concentrations in pg/μL for bead capture and supernatant fractions for 

female samples after incubation with four semen specific antibodies. 

Again, DNA typing of eligible DNA fractions resulted in the expected single source male or 

female DNA profiles (data not shown). Profile quality varied with samples with lower DNA 

yields showing partial types. 

This set of cell capture results raised several issues: for all sperm cell experiments DNA yields, 

even when combining the two fractions, were generally very low with often less than 15% 

recovery of the theoretical DNA contained in the defined cell amounts. Then there was the nonspecific binding of cells or DNA to the Protein G beads that could be shown for both the female 

cells and the “no antibody” controls for the sperm and epithelial cell experiments. While silica 

coated beads are routinely used for DNA capture and extraction, Protein G covered beads are 

very specific to mouse or rabbit immunoglobulin (6) and should not have captured intact vaginal 

cells or, if lysis is assumed, any nuclei or chromosomal DNA. In order to troubleshoot these 

results, these Protein G bead experiments were repeated with fresh biological samples and the 

“extraction only” control was introduced. 

Phase 3 –Protein G beads result confirmation with fresh biological samples 

The first set of three independent experiments involved 1μL of fresh semen and 5μg of antibody 

No

Antibody

Control

ADAM2m MFG8m UBAP2L AKAP3

Capture 147.04 242.00 23.00 96.65 110.50

Supernatant 22.78 21.95 12.08 10.77 5.98

0.00

50.00

100.00

150.00

200.00

250.00

300.00

DNA concentration pg/uL

Protein G Bead results female cells

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necessarily reflect the official position or policies of the U.S. Department of Justice.
2011-NE-BX-K547 Final Report Draft 

26

MFGE8. Instead of using normalized cell counts, a positive control for each donor was processed 

in parallel (without beads) to serve as a benchmark for the expected yield. Sperm counts for the 

purchased semen samples M1 and M2 had been determined to be 40,000 and 17,000 sperm per μL. 

With the exception of using the multidirectional mixer there were no changes to the previous 

protocol. The ratio between the bead fraction and the cells remaining in the supernatant for both 

donors resulted in an average of 17.4 (n=6). Protein G beads again captured sperm cell DNA. All 

of these experiments unfortunately did not include a “no antibody” control, so this data set in itself 

cannot be used to evaluate unspecific binding. 

Capture fraction DNA recovery was slightly higher than the positive control recovery, but 

overall yields including the “complete process positive control” again were low compared to the 

known sperm count. In order to investigate the low yields, a second set of positive controls was 

added to the Chelex extraction set. These fresh samples were not subjected to the two lengthy 

incubation and multiple bead wash steps necessary for the bead separation. The DNA yield for 

the first experiment using “extraction only” controls, the DNA recovery in the these controls was 

ten times as high as for the other samples (data not shown). 

Figure 2 shows the results for an additional Protein G bead experiment employing both semen 

donors, both antibodies, and again the “extraction only” positive controls. Each bar represents a 

single sample. 

Figure 2: Human DNA quantitation results for positive controls and two semen donors (M1 and 

M2) after Protein G bead procedure; DNA concentrations in ng/μL for bead and supernatant 

fractions after incubation with two semen specific antibodies; “POS Che” indicates the two 

extraction only controls added to the Chelex extraction set after the incubation steps. 

The amount of DNA in the bead/capture fractions is similar to the two positive controls that were 

0

2

4

6

8

10

12

POS M1 POS M2 POS Che M1POS Che M2 M1 A1 M2 A1 M1 A2 M2A2

ng/uL

A1 = Antibody MFGE8, A2 = Antibody UBAP2L

Total DNA or Bead Fraction

Supernatant after bead capture

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2011-NE-BX-K547 Final Report Draft 

27

incubated with the antibodies and processed in parallel with no beads added. There is less DNA in 

the supernatant. The “extraction only” controls vary in yield, but the >10ng/μL concentration seen 

for donor M1 is very close to the yield in the previous experiment mentioned above and it is 

unlikely that this ten-fold difference in DNA recovery is caused by pipetting variation. With the 

regular positive controls, which do not contain any beads, showing the same loss of DNA, the 

yield reduction cannot be caused by the magnetic beads present in the semen samples. A 

quantitation artifact is also excluded as the cause. All samples and controls were concentrated 

using Amicon Ultra filter membranes; a step which normally is also efficient at removing PCR 

inhibitors. The Quantifiler Duo Internal Positive Control (IPC) and the sample curve slopes did not 

indicate any type of inhibition for the regular positive controls and the samples. The only 

remaining option is that one or both of the necessary incubation steps (2h at 37°C with the 

antibody and 30min at 4°C with the beads) cause a certain amount of cellular and DNA 

degradation which leads to a loss in DNA yield. 

The single source cell type Protein G experiment was also repeated for vaginal epithelial cells. 

Cells were eluted from the swab and small aliquots from the cell pellet and the elution fraction 

were used for the capture/extraction procedure. As before, an equivalent positive control was 

utilized to replace the cell counting step. Two experiments were performed for MFGE8. Figure 3 

displays the results for the experiment employing both antibodies, the initial MFGE8 results 

were very similar. Both the pellet (labeled F1A) and the elution buffer fraction (labeled F1B) 

yielded only low amounts of DNA with the pellet being more concentrated. Again, the 

“extraction only” positive control has a much higher yield. For the magnetic bead samples 

almost the entire DNA is in the bead capture fraction which confirms that there is clearly an 

affinity of Protein G beads to human cells (or possibly DNA) that is not related to the presence of 

specific antibodies. 

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2011-NE-BX-K547 Final Report Draft 

28

Figure 3: Human DNA quantitation results for positive controls and two amounts of female cells 

(F1A and F1B) after Protein G bead procedure; DNA concentrations in ng/μL for bead and 

supernatant fractions after incubation with two semen specific antibodies; “POS Che” indicates 

the two controls added to the Chelex extraction set after the incubation steps; each bar 

represents a single sample. 

Protein G Magnetic beads as purchased from New England Biolabs are described as nonporous 

paramagnetic particle covalently coupled to a truncated form of recombinant Protein G. It is 

unclear what exactly causes the unspecific binding and how the surface could be blocked to only 

allow for specific IgG – Protein G reactions. The New England Biolabs protocol for Protein G 

beads, is geared towards the isolation of immunoglobulins and antibody facilitated 

immunoprecipitation of target proteins out of crude cell lysates (6). Similar Pan Mouse IgG 

coated beads are available from Lifetechnologies and marketed as a tool to either positively 

isolate or deplete antibody bound target cells (50). Anslinger et al (11) employed these IgG beads 

in their research and also observed unspecific binding and co-extraction of female cells for their 

mixtures of buccal cells and varying amounts of spermatozoa. 

Theoretically, the assay described here is employing intact cells and, especially for the male 

compact sperm cells with membrane containing strong disulfide bonds, there should have been

no cell lysis until the Chelex extraction. On the other hand, the higher DNA yield for the 

“extraction only” control indicates possible cell and DNA degradation prior to the extraction 

step. It is possible that multidimensional agitation in the presence of metallic beads could have 

led to physical rupture of cell membranes. 

0

0.05

0.1

0.15

0.2

0.25

POS F1A POS F1B POS Che

F1A

POS Che

F1B

F1A A1 F1B A1 F1A A2 F1B A2

ng/uL

A1 = Antibody MFGE8; A2 = UBAP2L

Total DNA or bead fraction

Supernatant after bead capture

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2011-NE-BX-K547 Final Report Draft 

29

Phase 4 – Dynabead FlowComp Flexi magnetic bead capture 

Li et al (12) describe the successful enrichment of sperm cell DNA from fresh single source and 

mixed semen samples, as well as dried vaginal swabs using the Dynabeads FlowComp Flexi kit 

from Invitrogen. In this project the first sets of phase 4 experiments using this kit, labeled 

MFGE8 antibody and both semen and vaginal cell samples, yielded some DNA in the bead 

capture fractions, but all positive controls and the supernatant fractions were negative for human 

or male DNA. This was due to real time PCR inhibition. The Quantifiler Duo Internal Positive 

Control (IPC) designed to indicate PCR inhibition only amplified in sample fractions recovered 

in release buffer (capture fractions). All other samples including the positive controls, which 

prior to the addition of the Chelex beads were still suspended in incubation buffer, showed 

complete amplification failure with no signal for the IPC. 

One component of the isolation buffer must have been co-purified during the Amicon Ultra 0.5 

concentration step and caused this real time PCR inhibition. The isolation buffer as described by 

the manufacturer consists of phosphate buffered saline (PBS) that is supplemented with 0.1% 

bovine serum albumin (BSA) and 2mM ethylenediaminetetraacetic acid (EDTA). EDTA is a 

known PCR inhibitor and may not have been removed by the 50k NMWL (nominal molecular 

weight limit) cut-off membrane. Clearly the Dynabead FlowComp Flexi isolation buffer was 

incompatible with the Chelex single tube extraction even after Amicon filtration; therefore for 

future experiments the downstream DNA purification method was replaced by a silica column 

based DNA extraction method (QIAamp DNA Minikit from Qiagen). 

There are three major differences between the Protein G bead assay as performed for this grant 

and the Dynabead FlowComp Flexi magnetic bead capture as described by the manufacturer and 

Li et al (12). The antibody binds to the beads via a biotin-streptavidin intermediate, the assay 

contains a sperm pelleting step designed to remove unincorporated antibody and instead of 

adding the bead/cell conjugate to DNA extraction, cells are separated from the beads with release 

buffer incubation. All of these steps are designed to make the assay more specific but may also 

result in a certain loss of cellular material. 

Five subsequent magnetic bead capture experiments with four biotin labeled antibodies and 

either single source sperm or vaginal epithelial cells failed to show the expected sperm DNA 

capture. All resulting QIAamp DNA fractions were successfully quantitated but none of the bead 

fractions, including the samples containing sperm and labeled antibodies, indicated any DNA 

enrichment. The resulting DNA yields were comparable to the positive controls but resided 

almost exclusively in the supernatant fraction. While some of the experiments included slight 

variations in incubation conditions and number of sperm cells used, all results were similarly low 

for the bead capture fraction and were pooled (sample numbers range from one to six) to 

calculate the ratios shown in table 7. 

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2011-NE-BX-K547 Final Report Draft 

30

Table 7 Average ratios between DNA concentrations in capture versus supernatant fraction: 

Dynabead FlowComp Flexi 

Cell type Antibody Average Ratio

Sperm

MFGE8 0.22

UBAP2L 0.01*

MOSPD3 0.04

AKAP3 0.17

No-antibody controls 0.24

Vaginal MOSPD3 0.08

No-antibody controls 0.10

 

* This value is not an average but a single measurement. 

For two of the MOSPD3 experiments we microscopically examined the magnetic bead fraction 

after completion of the three wash steps. No adhering sperm cells were seen. It is unclear if this 

failure of sperm capture is based on lack of specificity of the antibody, problems with the biotin 

labeling step or other buffer/temperature incompatibilities. 

As a final protocol modification, new batches of antibody for MOSPD3 and, since AKAP3 was 

not available from Abcam, AKAP associated protein or Rpn1l (polyconal and monoclonal) were 

tested using the biotinylation assay employed by Li et al (12). The biotin quantitation assay gave 

the expected result for the neat positive control, but showed a different result for the 1:10 

dilution of the positive control and less than one biotin molecule per immunoglobulin G for the 

1:10 dilution of the antibodies. Possibly the 1:10 dilutions were below the detection limit for the 

assay. Instead of 100μg as for the neat positive control, only 2.7 to 20μg could be added for each 

antibody, otherwise the samples would have been consumed. Magnetic capture was tested for 

three different input amounts was tested for MOSPD3, but no capture fraction enrichment was 

seen. To possibly remedy the low incorporation rate, all three already labeled antibodies were 

then subjected to another round of labeling followed by biotin quantitation. There was no 

improvement in the quantitation results. Regardless of the biotin quantitation result, all three 

labelled antibodies were then tested in a magnetic bead capture experiment with 5μg of each 

antibody. As can be seen in Figure 4, again almost the entire DNA resided in the supernatant 

fraction. 

For MOSPD3 microscopic examination of the magnetic bead fraction revealed a single isolated 

observation of a sperm head and bead next to each other, but this could have been accidental and 

does not prove an interaction. 

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2011-NE-BX-K547 Final Report Draft 

31

 

Figure 4: Human DNA quantitation results for samples after EZ-Link Biotinylation and controls; 

DNA concentrations in ng/μL for bead and supernatant fractions after incubation with three

semen specific antibodies; “QIAamp” indicates the “extraction only” control added to the 

Qiagen extraction set after the incubation steps; each bar represents a single sample. 

With the lack of success for single source sperm samples the project never reached the stage of 

testing cell mixtures and dried semen stains. 

Discussion

This project failed to demonstrate sperm cell specific enrichment for either magnetic bead 

capture assay. Protein G beads were shown to retain DNA, but in a non-specific fashion 

including vaginal epithelial cells and control samples with no antibody addition. For the 

Dynabead FlowComp Flexi kit almost the entire recovered DNA resided in the supernatant 

fraction. 

Developing a working magnetic based sperm capture assay has been an elusive goal over many 

years. Early data produced by Eisenberg (9, 10) and Anslinger et al (11) were never published in 

a peer review journal and neither team has pursued this topic with more research. This lack of 

progress cannot have been due to the scarcity of known antibodies. Clinical research concerned 

with human fertility has thoroughly explored sperm surface characteristics and identified many 

possible antigens (53, 54). This number of available target sites has grown exponentially with the 

advent of proteomics research and proteomics databases (55, 56). Magnetic beads have also 

evolved to be more user friendly and easy to use products. The Invitrogen Dynabead FlowComp 

Flexi kit tested in this study and other Dynabead products are now routinely being used for a 

variety of cell enrichment applications (for example 2-4). While it has to be noted that many of 

0

1

2

3

4

5

6

POS

MOSPD3

POS

monc

Rpn1l

POS

polyc

Rpn1l

QIAamp MOSPD3 monoc

Rpn1l

polyc

Rpn1l

No AB

ng/μL

Quantity of antibody used= 5μg

Total DNA or in the Capture Fraction

 Supernatant Fraction

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32

the beads are designed to work with whole blood or cell cultures, and normally require an excess 

of fresh material, it is also true that these assays can be made very specific and that the beads 

successfully retain the target cells (7). Clearly antibody based magnetic bead capture should 

work at least for fresh sperm cells and this was demonstrated for three different types of beads 

with different coating (10-12). The success rate declines for dried specimen, especially with 

increasing age. Older semen evidence mostly contains sperm heads and no intact spermatozoa 

with the tails attached, which means antibodies binding to the tailpiece will lose effectiveness 

(11, 12). Another reason for the loss of recovery could be membrane degradation and loss of 

antigen specific quaternary structure and binding capacity. 

For the Protein G beads investigated here the problem was not a lack of DNA recovery but rather 

the unspecific retention of DNA from sperm and non-sperm cells, with and without the addition 

of sperm membrane receptor specific antibodies. It is unlikely that the beads were binding whole 

cells. The microscopic examination of a Protein G sperm extraction bead fraction after bead 

immobilization and washing did not reveal any cell to bead adhesion. While there has been some 

discussion on cell free DNA (21), it is more likely that cells were lysed during the magnetic bead 

incubation step. For most experiments cells were not frozen prior to use and should have been 

intact after suspension in phosphate buffered saline, but the sodium phosphate Protein G bead 

binding buffer combined with mechanical mixing in the presence of metal beads may have 

disrupted not only epithelial cell but also sperm head membranes. These magnetic beads may 

have an affinity to DNA or DNA-histone complexes either via the Protein G coating or possibly 

gaps in this coat. No experiments with cell lysates or previously extracted DNA were performed 

to explore this. 

Rather than spending more time on the Protein G magnetic bead type, the team decided to 

explore the Dynabead FlowComp Flexi kit as described by Li et al (12). Antibodies tested were 

the four selected for this study and the MOSPD3 antibody employed by Li et al (12). None of the 

antibodies produced any enrichment in the bead capture fraction. There are three possible areas 

where this assay can fail: the biotin labeling reaction could be unsuccessful leading to unlabeled 

and thus ineffective antibodies. The labeling chemistry used in this study was DSB-X biotin 

succinimidyl ester, part of the Dynabead FlowComp Flexi kit and was run for 1.5 hours. Li et al 

(12) deviated in this step and labeled the MOSPD3 antibody with sulfo-NHS-LC-Biotin obtained 

from Pierce Protein Biology Products, Thermo Scientific, Rockford, IL, USA; also the labeling 

reaction was allowed to incubate for 72 hours. Theoretically the labeling should not have been an 

issue. Invitrogen technical support had been consulted on the technical details and, for example, 

Jensen et al (2) used the same chemistry. The company specifically warns against certain 

antibody storage buffer additives that may interfere with antibody labeling and may require prior 

dialysis. In this project these additives were either not present or below the problematic 

threshold. On the other hand the reaction was performed multiple times with different antibodies

without producing any successful DNA capture. Also the actual binding of the biotin label was 

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2011-NE-BX-K547 Final Report Draft 

33

not confirmed via Western blot and labeled streptavidin probes, so a systemic problem in the 

labeling step could not be excluded. To address this possible source of failure, MOSPD3 and 

Ropn1l were labeled following Li et al (12) as closely as possible and the labeling efficiency was 

monitored using a biotin quantitation kit. The quantitation revealed low incorporation and the 

subsequent capturing experiments failed to show any enrichment. Antibody conjugation is not as 

straightforward as indicated in commercial kits and may require antibody specific optimization; 

in addition NHS chemistry has the potential of putting the label in the binding domain of the 

antibody and neutralizing activity (peer review comment). More work could be done to produce 

high density MOSPD3 biotin conjugates. 

The other factor that would prevent any specific binding is the choice and the quality of the 

purchased antibodies. Most antibody manufacturers only test their antibody performance for 

Western Blot applications and researchers have encountered vast differences in quality and 

binding capability for the same type of antibody from different companies (Kappen, DresesWerringloer, personal communication). For the phase 1 antibody panel, only the AKAP3 

antibody obtained from Antibodies Online, Atlanta, GA showed large agglutinates. All of the 

agglutination tests for the antibodies from Abnova, Taipei, Taiwan were either weak (scattered 

agglutination for undiluted antibody) or inconclusive (aggregates in negative control). It was 

unfortunately not possible to purchase the more promising antibodies from different sources and 

compare the specificity for different lot numbers and providers. The MOSPD3 antibody 

employed here was polyclonal, produced in rabbit and from a different company than the 

monoclonal MOSPD3 antibody used by Li et al (12). This antibody was also shown to produce 

large agglutinates. Even with high quality antibodies there is no guarantee that the sperm 

membrane antigen in its mature post ejaculation form will be recognized and many theoretically 

feasible antibodies will not show the expected reaction. For this project, at least the AKAP3 and 

MOSPD3 antibodies had been shown to react with sperm and should have facilitated sperm 

capture. 

Under the assumption that antibodies were sufficiently labeled, sufficiently specific to an 

accessible sperm membrane antigen, and buffer and temperature conditions are suitable for 

efficient binding, the last factor prohibiting cell enrichment is the strength of the non-covalent 

biotin-streptavidin bond and its reaction to shearing effects during the required wash steps. As 

described by Eisenberg (10) agitation and stringent washing led to loss of previously bound 

material for the epoxy-antibody bead conjugates, which is why the stabilizing photolysis step 

was added. It remains a fact that many other research teams have successfully recovered their 

target cell population (2-4). Overall performance will also be affected by the density of antigens 

available on the target for binding, number of biotin molecules incorporated per antibody and 

density of streptavidin receptors on the beads. For many of these applications there is abundant 

starting material and a certain loss of cellular material is expected and tolerated. In this study 

starting amounts of sperm cells were fairly high, which makes it unlikely that that lack of 

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2011-NE-BX-K547 Final Report Draft 

34

thermodynamic stability and physical disruption of already formed bonds is the explanation for 

the total failure to capture sperm cell DNA on the streptavidin coated beads. 

Conclusions and implications for further research

This study clearly demonstrated that Protein G beads are not suitable for magnetic bead based 

sperm separation. For the streptavidin coated beads, the failure to duplicate the results described

by Li et al (12) may be based on a combination of factors with antibody quality and some of the 

buffers and incubation conditions being possible factors. But at least for the EZ-Link sulfo-NHSLC-biotin kit, reaction the most likely step to have failed in our hands was the antibody 

biotinylation, which means the work presented here was compromised by technical issues and 

cannot be used to decide on the overall scientific merit of the magnetic bead sperm capture 

method. 

The paper by Li et al is the only peer reviewed publication on the successful recovery of sperm 

cells and male DNA from mixtures with female cells using streptavidin covered beads (12). But 

they also found that the assay was better suited for fresh sperm and describe their success rate 

declining rapidly for dried vaginal swabs after short storage periods. It is unclear, if further 

research would be able to improve on these results. Surface antigens are easily compromised and 

capture release assays are always accompanied by a certain amount of loss. Forensic evidence by 

nature does not consist of pristine and abundant cells, and even with very effective antibodies 

and under optimized conditions magnetic bead assays will not be able to recover the highest 

possible amount of male DNA. This makes the idea of an automated immuno-magnetic sperm 

capture assay problematic for compromised samples and evidence with low sperm content. If a 

working assay could be developed, its application is likely to be limited to fairly fresh and 

abundant samples. 

References 

1. Gill P, Jeffries A, Werrett D. Forensic applications of DNA fingerprints. Nature 1985; 

318:577-79. 

2. Jensen K, Sletbakk Brusletto K, Dalsbotten Aass HC, Olstad K, Kierulf P, Gautvik KM. 

 Transcriptional profiling of mRNAs and microRNAs in human bone marrow 

 precursor B cells identifies subsetand age-specific variations. PloS one 2013; 8,

 e70721. 

3. Watanabe M, Serizawa, M, Sawada T, Takeda K, Takahashi T et al. A novel flow 

cytometry-based cell capture platform for the detection, capture and molecular 

 characterization of rare tumor cells in blood. J Translational Med 2014; 12:143 

This resource was prepared by the author(s) using Federal funds provided by the U.S. 

Department of Justice. Opinions or points of view expressed are those of the author(s) and do not 

necessarily reflect the official position or policies of the U.S. Department of Justice.
2011-NE-BX-K547 Final Report Draft 

35

4. Zheng Q, Miks-Krajnik M, Yang Y, Xu W, Yuk HG. Real-time PCR method combined 

with immunomagnetic separation for detecting healthy and heat-injured Salmonella

Typhimurium on raw duck wings. Int J Food Microbiology. 2014; 186:6-13. 

5. New England Biolabs, accessed September 24, 2014; https://www.neb.com/tools-andresources/selection-charts/affinity-of-protein-ag-for-igg-types-from-different-species 

6. New England Biolabs. Protein G magnetic beads product Information. Accessed 

September 24, 2014; https://www.neb.com/products/s1430-protein-g-magnetic-beads - 

tabselect1. 

7. Invitrogen- Life Technologies. Dynabeads Cell Separation Guide. 2010. Accessed 

September 26, 2014; 

http://tools.lifetechnologies.com/content/sfs/brochures/Dynabeads_cell_separation_guide

.pdf

8. Invitrogen- Life Technologies. Dynabeads M-450 Epoxy. Product information. 2012. 

Accessed September 26, 2014; 

http://tools.lifetechnologies.com/content/sfs/manuals/dynabeads_m450epoxy_man.pdf 

9. Eisenberg A. Development of a spermatozoa capture system for the differential 

extraction of sexual assault evidence. Fifth Ann Cambridge Healthtech Inst DNA 

Forensics Conf 2002; Washington, DC. 

10. Eisenberg A, Spermatozoa Capture during the differential extraction process for STR 

typing of sexual assault evidence. Final report submitted to US Department of Justice 

2002. Accessed on September 24, 2014; 

https://www.ncjrs.gov/pdffiles1/nij/grants/197532.pdf

11. Anslinger K, Bayer B, Danilov SM, Metzger R. Application of sperm-specific antibodies 

for the separation of sperm from cell mixtures. Forensic Sci Int Genet Suppl Series 2008; 

1(1):394-395. 

12. Li, XB, Wang, QS, Feng Y, Ning, SH, Miao YY, Wang, YQ, Li HW, Magnetic beadbased separation of sperm from buccal epithelial cells using a monoclonal antibody 

against MOSPD3. Int J Legal Med 2014; published online DOI 10.1007/s00414-014-

0983-3 

13. Thaler R, Rumpler M, Spitzer S, Klaushofer K, Varga F. Mospd1, a new player in 

mesenchymal versus epidermal cell differentiation. J Cell Physiol 2011; 226(10):2505 

14. GeneCards. The Human Gene Compendium. Accessed on September 24, 2014; 

http://www.genecards.org.

15. Duda AJ Development of an immune-magnetic procedure for the separation of human 

spermatozoa from vaginal epithelial cells. Unpublished thesis 2010. John Jay College of 

Criminal Justice. City University of New York. 

16. Dickman AB, Herr JC. Sperm antigens and their use in the development of an 

immunocontraceptive. Am J Reprod Immunol 1997; 37(1):111-7. 

17. Chen MS, Tung SK, Coonrod SA, Takahashi Y, Bigler D, Chang A, Yamashita Y, 

Kincade PW, Herr JC, White JM. Role of the integrin-associated protein CD9 in binding 

This resource was prepared by the author(s) using Federal funds provided by the U.S. 

Department of Justice. Opinions or points of view expressed are those of the author(s) and do not 

necessarily reflect the official position or policies of the U.S. Department of Justice.
2011-NE-BX-K547 Final Report Draft 

36

between sperm ADAM2 and the egg integrin α6β1: Implications for murine fertilization. 

PNAS 1999; 96(21):11830-35. 

18. Shur BD, Rodeheffer C, Ensslin MA, Lyng R, Raymond A. Identification of novel 

gamete receptors that mediate sperm adhesion to the egg coat. Mol Cell Endocrinol 

2006; 250 (1-2):138-48 

19. Naz RK, Dhandapani L. Identification of human sperm proteins that interact with human 

zona pellucida3 (ZP3) using yeast two-hybrid system. J Reprod Immunol 2010; 

84(1):24-31 

20. Lea IA, Widgren EE, O’Rand MG. Association of sperm protein 17 with A kinase 

anchoring protein 3 in flagella. Reprod Biol Endrocrinology 2004; 2:57. 

21. Carr DW, Fujita A., Stentz CL, Liberty GA, Olson GE, Narumiya S. Identification of 

sperm-specific proteins that interact with A-kinase anchoring proteins in a manner similar 

to the type II regulatory subunit of PKA. J Biol Chem 2001; 276(20):17332-17338. 

22. PhosphoSite Plus. A protein modification resource. Accessed on February 19, 2015; 

http://www.phosphosite.org/proteinAction.do?id=11995062&showAllSites=true 

23. Vandewostyne M, Van Hoofstat D, Franssen A, Van Nieuwerburgh F, Deforce D. 

Presence and potential of cell free DNA in different types of forensic samples. Forensic 

Sci Int Gen 2013; 7:316-20. 

24. Rape Kit DNA Analysis Backlog Elimination Act (2002). 107

th 

Congress H.R. 3961, U.S. 

Congress, Washington, DC 

25. Statistics. Rape, Abuse and Incest National Network. (2008). 

 Retrieved March 13, 2011 from http://www.rainn.org/statistics 

26. Human Rights Watch Organization. Testing Justice - The rape kit backlog in Los Angeles 

City and County. Human Rights Watch, New York, NY USA. 2009. ISBN 1-56432-461-

3. 

27. Butler J. Advanced Topics in Forensic DNA Typing: Methodology. (2012) Elsevier 

Academic Press, Amsterdam, The Netherlands. 

28. Schoell WM, Klintschar M, Mirhashemi R, Pertl B. Separation of sperm and vaginal 

cells with flow cytometry for DNA typing after sexual assault. Obstet Gynecol 1999;

94(4):623-27. 

29. Elliott K, Hill DS, Lambert C, Burroughes TR, Gill P. Use of laser microdissection 

greatly improves the recovery of DNA from sperm on microscope slides. Forensic Sci 

Int 2003; 137(1):28-36. 

30. Espina V, Wulfkuhle JD, Calvert VC, VanMeter A, Zhou W, Coukos G, Geho GH, 

Petricoin III EF & Liotta L. Laser-capture microdissection. Nat Proto 2006; 1(2):586-

603. 

31. Sanders CT, Sanchez N, Ballantyne J, Peterson DA. Laser microdissection separation 

from epithelial cells for short tandem repeat analysis. J Forensic Sci 2006; 51(4):748-757. 

32. Chen J, Kobilinsky L, Wolosin D, Shaler R, Baum H. A physical method for 

separating spermatozoa from epithelial cells in sexual assault evidence. J Forensic Sci 

This resource was prepared by the author(s) using Federal funds provided by the U.S. 

Department of Justice. Opinions or points of view expressed are those of the author(s) and do not 

necessarily reflect the official position or policies of the U.S. Department of Justice.
2011-NE-BX-K547 Final Report Draft 

37

1998; 43(1):114-18. 

33. Garvin AM. Filtration based DNA preparation for sexual assault cases. J 

 Forensic Sci 2003; 48(5):1084-7. 

34. Horsman KM, Barker SL, Ferrance JP, Forrest KA, Koen KA, Landers JP. Separation of 

sperm and epithelial cells in a microfabricated device: potential application to forensic 

analysis of sexual assault evidence. Anal Chem 2005; 77(3):742-49. 

35. Bienvenue JM, Duncalf N, Marchiarullo D, Ferrance J, Landers J.P. Microchip based cell 

lysis and DNA extraction from sperm cells for application to forensic analysis. J Forensic 

Sci 2006;51(2):266-273. 

36. Garvin AM, Bottinelli, Gola M, Conti A, Soldani G. DNA preparation from sexual 

assault cases by selective degradation of contaminating DNA from the victim. J Forensic 

Sci 2009; 54(6):1297-303. 

37. Tsukada K, Asamura H, Ota M, Kobayashi K & Fukushima H. Sperm DNA extraction 

from mixed stains using the DifferexTM system. Int Cong Series 2006; 1288:700-703. 

38. Valgren C, Edenberger E. Evaluation of the DifferexTM system. Forensic Sci Int: Genet 

 Suppl Series 2008; 1(1):78-79. 

39. Hulme P, Lewis J, Davidson G. Sperm elution: an improved two phase recovery method 

for sexual assault samples. Science and Justice 2013; 53:28-33. 

40. Haak B, Porsche A, Vollack K, Zimmermann P, Pflug WT. Evaluation of a 

semiautomated, magnetic bead-based DNA extraction method for genetic fingerprinting 

of forensic casework samples. Forensic Sci Int: Genet Suppl Series 2008;1(1):35-36. 

41. Hudlow WR, Buoncristiani MP. Development of a rapid 96-well alkaline lysis based 

differential extraction method for sexual assault evidence. Forensic Sci Int: Genet 2012; 

6:1-16 

42. Isahakia M, Alexander NJ. Interspecies cross-reactivity of monoclonal antibodies 

directed against human sperm antigens. Biol Reprod 1984; 30(4):1015-1026. 

43. Rodeheffer C, Shur B. Characterization of a novel ZP3-independent sperm binding ligand 

that facilitates sperm adhesion to the egg coat. Development 2003; 131(3):503-512. 

44. Foresta C, Varotto A, Caretto A. Immunomagnetic method to select human sperm 

without sperm surface-bound autoantibodies in male autoimmune infertility. Arch 

Androl 1990; 24(2):221-25. 

45. Sanjurjo C, Dawidowsky AR, Cameo MS, Echeverria FG, Blaquier JA. Participation of 

human epididymal sperm coating antigens in fertilization. J Androl 1990; 11(5):476-83. 

46. Ohashi K, Saji F, Wakimoto A, Tsutsui T, Nakazawa T, Okabe M, et al. Selection of 

acrosome-reacted sperm with MH61-immunobeads. J Androl 1994; 15(1):78-82. 

47. Hirano M, Kamada M, Maeda N, Yamamoto S, Aono T. Presence of immunoglobulin 

binding factor on human sperm surface as sperm coating antigen. Arch Androl 1996; 

37(3):163-70. 

48. de Lamirande E, Leclerc P, Gagnon C. Capacitation as a regulatory event that primes 

spermatozoa for the acrosome reaction and fertilization. Mol Hum Reprod 1997; 

This resource was prepared by the author(s) using Federal funds provided by the U.S. 

Department of Justice. Opinions or points of view expressed are those of the author(s) and do not 

necessarily reflect the official position or policies of the U.S. Department of Justice.
2011-NE-BX-K547 Final Report Draft 

38

3(3):175-94. 

49. Walsh PS, Metzger DA, Higuchi R. Chelex 100 as a medium for simple extraction of 

DNA for PCR-based typing from forensic material. Biotechniques 1991; 10(4):506-513 

50. Franken DR, Gonin P, Slabber, CF. The Friberg spermagglutination test- a modification. 

Andrologia 1979; 11(1):62-66. 

51. Francavilla F, Catignani P, Romano R, Suntucci R, Francavilla S, Santiemma V. 

Modification of the slide agglutination test for the detection of sperm-agglutination. 

Andrologia 1983; 15(6): 699-704. 

52. Lifetechnologies. Dynabeads PanMouse IgG product information. 2005. Accessed 

September 24, 2014; 

http://tools.lifetechnologies.com/content/sfs/brochures/35_105.F.072.04.pdf

53. Mandal A, Klotz KL, Shetty J, Jayes FL, Wolkowicz MJ, Bolling, LC, et al. SLLP1, a 

Unique, Intra-Acrosomal, Non-Bacteriolytic, C Lysozyme-Like Protein of Human 

Spermatozoa. Biology of Reproduction. 2003; 68:1525-1537. 

54. Shetty J, Wolkowicz MJ, Digilio LC, Klotz KL, Jayes FL, Diekman AB, et al. SAMP 14, 

A novel, acrosomal membrane-associated, glycosylphosphatidylinositol-anchored 

member of the Ly-6/Urokinase-type plasminogen activator receptor superfamily with a 

role in Sperm-Egg Interaction. J Biol Chem 2003; 278:30506-30515. 

55. Johnston DS, Wooters J, Kopf GS, Qiu Y, Roberts KP. Analysis of the Human Sperm 

 Proteome. Annals of the New York Academy of Sciences 2005; 1061:190-202. 

56. Oliva R, Martinez-Heredia J, Estanyol JM. Proteomics in the study of the sperm cell 

composition, differentiation and function. Systems Biology in Reproductive Medicine, 

2008; 54:23-36. 

Dissemination

It is generally not possible to publish negative results, especially if the failure to demonstrate a 

hypothetical mechanism is mostly based on technical difficulties. This would be different, if we 

had been able to identify true scientific reasons why the tested streptavidin coated bead assay did 

not yield the predicted results. Unfortunately that was not the case, and with the exception of this 

final report, we are not planning to submit this material for publication. 

This resource was prepared by the author(s) using Federal funds provided by the U.S. 

Department of Justice. Opinions or points of view expressed are those of the author(s) and do not 

necessarily reflect the official position or policies of the U.S. Department of Justice.