Archives of Andrology

Journal of Reproductive Systems

ISSN: 0148-5016 (Print) (Online) Journal homepage: https://www.tandfonline.com/loi/iaan19

Antisperm Antibody Tests: Traditional Methods

Compared to Elisa

M.-L. Windt, P.-J. D. Bouic, C. J. Lombard, R. Menkveld & T. F. Kruger

To cite this article: M.-L. Windt, P.-J. D. Bouic, C. J. Lombard, R. Menkveld & T. F. Kruger (1989)
Antisperm Antibody Tests: Traditional Methods Compared to Elisa, Archives of Andrology, 23:2,
139-145, DOI: 10.3109/01485018908986836

To link to this article: https://doi.org/10.3109/01485018908986836

Published online: 09 Jul 2009.

Submit your article to this journal

Article views: 610

View related articles

Citing articles: 2 View citing articles

Full Terms & Conditions of access and use can be found at

https://www.tandfonline.com/action/journalInformation?journalCode=iaan20
 ANTISPERM ANTIBODY TESTS: TRADITIONAL
METHODS COMPARED TO ELISA

M.-L. WINDT, P.-J. D. BOUIC, C. J. LOMBARD,

R. MENKVELD, and T. F. KRUGER

Enzyme-linked immunosorbent assays (ELISAs) to detect sperm antibodies can be a useful addition to
other tests. ELISAs allow more quantitative and detailed information than some other tests, but results
should be compared to those of other tests. This study was conducted to correlate results of an ELISA
that used a sperm membrane extract as antigen with those of other tests. Semen, seminal plasma, and
serum of 34 sperm-antibody-positive and 36 sperm-antibody-negative men were tested. There was
poor correlation between the ELISA results and those of the other tests. Only the results of a direct
immunobead test done on semen correlated fairly well with results from ELISAs done on serum.
Further studies are needed on fertility-specific antigens that can be identified and isolated and then
studied by means of ELISAs.

Key Words: Antibody; Antigen; Sperm; ELISA; Seminal plasma.

INTRODUCTION
Several techniques have been used to detect sperm antibodies in serum and seminal plasma
and on the sperm membrane. These include the tray agglutination test (TAT) [5] and the sperm
immobilizing test (SIT) [9]. Antibodies detected with these tests do not necessarily play a role
in infertility, however [3]. Newer antibody tests that detect sperm antibodies bound to the
sperm membrane are more likely to influence fertility. These include the mixed antiglobulin
reaction (MAR) test [lo], the sperm cervical mucus contact (SCMC) test [ll], and the direct
immunobead test (d-IBT) [4]. An indirect immunobead test (i-IBT) has also been developed to
detect the immunoglobulin class of sperm antibody in serum and seminal plasma [4].
Enzyme-linked immunosorbent assays (ELISAs) for sperm antibody detection that make
use of either whole sperm as antigen [6-8, 12, 14, 19, 201 or a sperm membrane extract as
antigen [l] have been proposed. The ELISA is quantitative and qualitative and can detect
various immunoglobulin classes. Detection of secretory immunoglobulin A (IgA), which is not
possible with any of the other methods, is possible with the ELISA [7]. Some investigators
have found the ELISA to be superior to the other methods [ l , 7, 12, 201, whereas others have
reported poor correlation of ELISA results with those of other accepted tests [2, 81.

Accepted March 27, 1989.

From the Reproductive Biology Unit, Department of Obstetrics and Gynaecology, Tygerberg Hospital, Tyger-
berg, 7505, Republic of South Africa (M.-L. W., R.M., T.F.K.); the Institute for Biostatistics, Medical Research
Council, Tygerberg, 7505, Republic of South Africa (C.J.L.), and the Immunology Unit, Department of Microbiol-
ogy, Tygerberg Hospital, Tygerberg, 7505, Republic of South Africa (P.J.D.B.).

ARCHIVES OF ANDROLOGY 23: 139-145 (1989)

Copyright 0 1989 by Hemisphere Publishing Corporation 139
140 M.-L. Windt et al.

We performed most of the available sperm-antibody tests on spermatozoa, serum, and

seminal plasma in infertile patients with sperm-antibody-positive (ASA') and sperm-
antibody-negative (ASA-) serum. These results were compared to results of an ELISA that
used a sperm membrane extract as antigen [l] that was performed with serum and seminal
plasma of two groups of patients. The aim of the study was to evaluate the correlation of
results from the ELISA and other methods of sperm-antibody detection.

MATERIALS AND METHODS

Thirty-four ASA' men (MAR test > 50%) and 36 ASA- men (MAR test = 0%)were selected for
study. Semen samples were produced by masturbation into sterile containers. Semen samples were
allowed to liquefy at 37°C and then were mixed well for 10 min. A portion sufficient for the perfor-
mance of a MAR test and a d-IBT was taken from each sample. The rest of the semen sample was
subjected to centrifugation at 1200 g for 25 min to obtain seminal plasma. Seminal plasma samples, after
inactivation at 56°C for 30 min, were frozen ( - 70°C) for later use in the TAT, SIT, i-IBT, and ELISA.
Sixteen ASA' and 20 ASA- samples were tested. Blood samples were collected in vacuum tubes and
subjected to centrifugation at 800 g for 10 min, inactivated at 56 "C for 30 min, and frozen ( - 70 "C) for
later use in the TAT, SIT, i-IBT, and ELISA. Thirty-four ASA' and 36 ASA- samples were tested.

Tests on Semen. MAR Test [lo, 17, 181. One drop of semen was placed on a microscope slide and
mixed with one drop of monospecific antihuman IgG and one drop of red blood cells (type 0, Rh-
positive, R,R,, sensitized with anti-D blood serum). Results were read after 10 min at 25°C. The
percentage of motile spermatozoa involved in mixed agglutinates with red blood cells was determined
(MAR %) ( > l o % positive, >80% strongly positive) [lo, 151.
d-IBT[3, 4 , 17, 181. One drop of washed semen (3 times in Tyrode medium plus 0.3% bovine serum
albumin [T-BSA] and resuspended in Tyrode medium plus 5% BSA [T-BSAd]) was mixed with one
drop of the immunobead reagent coated with either anti-IgA or anti-IgG and read after 10 minutes at
25 "C. The percentage of motile sperm with one or more immunobeads attached was evaluated micro-
scopically (d-IBT %) (>20% positive).

Tests on Serum and Seminal Plasma. TAT [S, 161. Motile donor spermatozoa (30 x 106/ml)were
obtained by a swim-up procedure into a layer of Baker medium plus 1% BSA. Serially diluted serum and
seminal plasma samples (5 pl each) were transferred to a disposable microchamber (Design AB, Moss,
Norway) containing 3 ml of liquid parafin. Donor spermatozoa (1 pl) were mixed with the samples and
incubated at 37°C for 2 h. Known positive and negative control samples were included in the test
procedure. After a 1-h incubation period, samples were examined at a magnification of lOOX for
agglutination. The test was considered positive when an obvious agglutination of donor sperm was noted.
If all spermatozoa were agglutinated, the result was scored as 3 + ; large agglutinates together with free
+
spermatozoa were scored as 2 , and scattered small but still apparent agglutinates were scored as 1 . +
+
Titer values represented the highest dilution at which a 1 agglutination of donor sperm was still
observed.
SZT(9, 161. The SIT is usually performed together with the TAT so that the same semen sample and
sample dilution are used. Donor semen preparations and test sample dilutions were therefore exactly as
for the TAT. Only a 1 : 4 dilution of each test sample was screened for immobilization. Guinea pig
complement (diluted 1 : 8 in Baker medium + 1% BSA) was divided into two portions; one was used as
positive complement source and the other as negative source after inactivation at 56°C for 1 h. A test
sample (5 pl of 1 : 4 dilution) was transferred in duplicate to a disposable microchamber containing 3 ml
of liquid parafin (as for TAT). Motile donor sperm suspension (2 pl, 30 x 106/ml) was added to each 5-
 Antisperm Antibody Tests 141

p1 sample and incubated at 37°C for 30 min to allow antibody-antigen binding. After 30 min, 5 p1 of
active, fresh complement was added to one sample drop. To the duplicate drop, 5 pl of the inactivated
complement was added. Microchambers were incubated for another hour. Results were read under an
inverted light microscope (as for TAT) at a magnification of 100 x . The percentage of motile spermato-
zoa in duplicate sample drops was estimated. The sperm-immobilization value (SIV) was calculated. A
SIV of more than 2 was regarded as positive.
i-IBT(3, 4, 61. Serum or seminal plasma samples (0.2 ml) were incubated with a portion (20 to 50 pl)
of donor semen (negative on d-IBT and containing lo6 to 2 x lo6 motile spermatozoa). Motile sper-
matozoa were obtained by a layering and swim-up procedure into T-BSA-0.3 for 1 h at 37°C. After
incubation with test samples, the spermatozoa were washed 3 times and tested as for the d-IBT (i-IBT %)
( > 2 0 % = positive).
ELISA. An antigen extract [l, 161 was prepared. Microtiter plates were coated with LIS antigen
extract (15 pglml; 100 p1 per well) in 0.05Mcarbonate buffer (pH 9.6) by incubating trays for 1 h at
37 "C and then overnight at 4 "C. Blocking was conducted with phosphate-buffered saline (PBS, 100 pl
per well) plus 1% BSA for 1 h at 37 "C. Wells were then washed 3 times with PBS (200 pl per well) plus
0.05 % Tween-20 before being loaded with 100 pl of serum samples (1 : 20 dilution) and seminal plasma
samples ( 1 : 3 dilution). These dilutions were optimal [7, 141. The dilution buffer used was PBS plus
0.5% BSA. Triplicate wells were loaded, and the samples were incubated on plates for 1 h at 37°C.
Plates were again washed 3 times before the conjugates or monoclonal antibodies were added.
For the detection of IgG antibody, goat antibody to human IgG conjugated to alkaline phosphatase
(Cappel-Cooper Biomedical [l : 10001) was added to the wells (100 p1 per well), and the samples were
incubated for 1 h at 37 "C. For total IgA (IgA-t) and secretory IgA (IgA-s) detection, 100 p1 of a 1 : 400
dilution of mouse monoclonal antibody to human IgA-t and IgA-s (Bio-Yeda; clone GA-112 and GA-1,
respectively) was added, and the samples were incubated for another hour at 37°C. These plates were
washed 3 times and incubated again with goat antibody to mouse immunoglobulin (Zymed Laboratories
[I : 5001) conjugated to alkaline phosphatase (100 pI per well).
After further washes, the plates were developed with substrate @-nitrophenylphosphate disodium in
diethanolamine buffer, pH 9.8, 1 mg/ml) for approximately 1 h in the dark at 37°C until a known
positive sample reached a sufficient optical density (OD) when read at 405 nm with an EIA reader (Bio-
Tek Instruments Inc.). The EIA reader was calibrated at zero for the wells containing antigen and
substrate only. Background values (OD in wells containing antigen, conjugate, and substrate) were
subtracted from all sample values in each assay. To achieve an internal standard, the same positive
sample was included each time that an assay was set up. ELISA results had an intra-assay coefficient of
variation (CV) of 4.9% and an interassay CV of 11.4%.

Statisticul Analysis. The distributions of the two relevant samples (ASA' and ASA-) were com-
pared by means of Wilcoxon's two-sample test. The logistic regression discriminant procedure was used
to evaluate how the ELISA method could discriminate between positive and negative sperm-antibody
serum and seminal plasma samples according to the different traditional sperm antibody tests.

RESULTS
Table 1 shows the number of positive and negative serum and seminal plasma samples
tested with each traditional test and compared with the ELISA results. Positive and negative
groups of patients were determined as follows for the different traditional antibody tests for
semen, serum, and seminal plasma: MAR > 80%,positive; TAT > 1 : 8 titer, positive; SIT
SIV > 2, positive; i-IBT > 20%,positive; and, d-IBT > 20%,positive.
The results of the ELISAs (IgG, IgA-t, and IgA-s) done on serum showed signifiant differ-
142 M.-L. Windt et al.

TABLE 1 Samples (Serum and Seminal Plasma) Compared with the ELISA for Each
Traditional Test

Test n (Total) n (Positive) n (Negative)

Serum
MAR 71 29 42
d-IBT (IgA) 47 27 20
d-IBT (IgG) 47 27 20
i-IBT (IgA) 71 33 38
i-IBT (IgG) 71 33 38
TAT 70 32 38
SIT 70 8 62

Seminal Plasma
MAR 36 16 20
d-IBT (IgA) 18 14 4
d-IBT (IgG) 18 14 4
i-IBT (IgA) 34 12 22
i-IBT (IgG) 34 12 22
TAT 29 5 24
SIT 29 2 22

ences between positive and negative groups as determined by the results of the MAR test,
TAT, d-IBT, and 1-IBT (p c 0.05) (Table 2). Nevertheless, the percentage sensitivity (% sens),
specificity (1% spec), and percentage correct (% cor) were only good in the case when the
ELISA (IgG) results were compared to those for the d-IBT for IgG (74.1 % sens; 70.0% spec;
72.3% cor), when the ELISA (IgA-t) results were compared to those for the d-IBT for IgA
(77.8% sens; 70.0% spec; 74.5% cor), and when the ELISA (IgA-s) results were compared to
those for the d-IBT for IgA (85.2% sens; 80.0% spec; 83.0% cor). None of the ELISA results
could discriminate between positive and negative SIT groups (p > 0.5).
For the seminal plasma samples, the ELISA results only showed significant differences
between the positive and negative i-IBT groups (p < 0.05) (Table 3). All other tests showed
no significant discrimination between groups (p > 0.1). The values for % sens, % spec, and
% cor were poor in all tests compared with those from the ELISA. The few positive SIT
values (n = 2) for seminal plasma made statistical evaluation impossible.

DISCUSSION
That so many tests for sperm antibodies exist is an indication that no single test provides all
the information necessary to give patients an accurate prognosis for their infertility problem.
The advantages and disadvantages of the different methods in use have been discussed [13].
Some tests are only useful in screening for antibodies (MAR and SCMC) and give limited
information about Ig classes, localization, and titers. Methods that rely on agglutination and
immobilization activity in serum and seminal plasma (TAT and SIT) also do not define isotypes
of Ig present. Antibodies in serum and seminal plasma are not necessarily involved in the
reproduction process and may not measure clinically significant antibodies. The i-IBT can
 Antisperm Antibody Tests 143

detect antibody classes in the serum and seminal plasma but is tedious to perform for titer
determinations. The d-IBT is specific and reproducible, detects Ig classes, and shows the
region of sperm to which the antibody is bound. Detection of antibody on the sperm cell itself
is important in the clinical significance of the antisperm antibody involved [13].
Although the ELISA also only detects antibodies in serum and seminal plasma and not on
the sperm membrane, its sensitivity and possible use in future research in combination with
clinically significant antigens are of importance. ELISAs for sperm antibodies are therefore
compared to traditional methods to assess their place in the battery of tests for prognosis of
immunological infertility.
ELISAs that use whole sperm as antigen can be problematic because sperm cells must be
fixed to microtiter wells. Fixatives can damage or change the antigen and also cause high
background values, resulting in false-negative or false-positive results [2, 13, 201. The use of a
sperm membrane extract as antigen solves this problem, but the extraction of antigens not
related to infertility is a matter of concern for some investigators [2].
Comparison of the ELISA with other antibody tests has been described. In a few cases,
good correlation of results was found. Howe and Lynch [7], by using whole sperm fixed with
paraformaldehyde, showed excellent correlation among ELISA, TAT, and SIT results for
seminal plasma. Lynch and colleagues [ 121 reported similar results.
Alexander and Bearwood [ l ] used a LIS-membrane extract and found that those samples
positive for ELISA were also positive for TAT and SIT, although there was no one-to-one

TABLE 2 Comparison between ELISA Results on Serum and Those of other Traditional
Sperm-Antibody Tests

Test Po % Sens % spec % Cor

ELISA (IgG)
MAR 0.0412 24.1 83.3 59.2
d-IBT (IgG) 0.0008 74.1 70.0 72.3
i-IBT (IgG) 0.0069 51.5 76.3 64.8
TAT 0.0063 50.0 76.3 64.3
SIT 0.8175 0.0 100.0 88.6

ELISA (IgA-t)
d-IBT (IgA) 0.0001 77.8 70.0 14.5
i-IBT (IgA) 0.0002 54.5 73.7 64.8
TAT 0.0008 46.9 76.3 62.9
SIT 0.0558 0.0 100.0 88.6

ELISA (IgA-Sj
d-IBT (IgA) O.oo00 85.2 80.0 83.0
i-IBT (IgA) 0.0010 51.5 84.2 69.0
TAT 0.0042 46.9 81.6 65.7
SIT 0.0578 0.0 100.0 88.6

‘Wilcoxon two-sample test.

144 M.-L. Windt et al.

TABLE 3 Comparison between ELISA Results on Seminal Plasma and Those of other
Traditional Sperm-Antibody Tests

Test Pa % Sens % Spec % Cor

ELISA (IgG)
MAR 0.9362 18.8 95.0 61.1
d-IBT (IgG) 0.9568 100.0 0.0 77.8
i-IBT (IgG) 0.0282 33.3 86.4 67.6
TAT 0.621 1 20.0 100.0 86.2

ELISA (IgA-t)
d-IBT (IgA) 0.2880 100.0 0.0 77.8
i-IBT (IgA) 0.0018 50.0 90.9 76.5
TAT 0.3240 0.0 100.0 82.8

ELISA (IgA-S)
d-IBT (IgA) 0.1508 92.9 25 .O 77.8
i-IBT (IgA) 0.0633 25.0 95.5 70.6
TAT 0.6032 0.0 100.0 82.8

“Wilcoxon two-sample test.

correlation. Wolff and Scill [20] used positively charged plates and showed varying concor-
dance (0% to 75%) between their ELISA results and those of an agglutination method and
stated that the concordance was dependent on the Ig class. Bronson and co-workers [2] used
glutaraldehyde-fixed spermatozoa and found that the ELISA results were only comparable
with the i-IBT results when test sera were incubated with spermatozoa before fixation in the
ELISA. Ing and co-workers [8], by using Cytofix spray to fix whole spermatozoa to microtiter
wells, compared their ELISA results with those of TAT and SIT and came to the conclusion
that different tests detect different but an overlapping spectrum of antibodies, making absolute
correlation impossible.
The ELISA described here can indicate differences between negative and positive sperm-
antibody samples but cannot discriminate absolutely among groups. When the ELISA results
were compared to those of the other detection methods by means of the logistic regression
discriminant procedure, high (>80%) values for % sens, % spec, and % cor were evident
only in the case of the d-IBT (all Ig classes). All the other test results correlated poorly with
the ELISA results when thus analyzed.
The much lower antibody concentration in seminal plasma compared to other fluids can be
the reason for the poor discrimination among antibody tests in the seminal plasma (Table 3).
Although the ELISA is a promising t p t , its results seem to correlate poorly with those of other
tests except the d-IBT. Further studies are therefore needed in which solid-phase, antigenic
fractions of solubilized sperm membranes involved in the reproductive process can be used.
Only then can results of the ELISA be compared to those of the established methods (immobi-
lization, agglutination, and immunobead binding) that use intact spermatozoa.
 Antisperm Antibody Tests 145

REFERENCES
1. Alexander NJ, Bearwood D (1984): An immunosorption assay for antibodies to spermatozoa: Comparison with
agglutination and immobilization tests. Fertil Steril 41 :270-276
2. Bronson R, Cooper G, Rosenfeld D, et al(1984): Detection of spontaneously occurring sperm-directed antibodies
in infertile couples by immunobead binding and enzyme-linked immunosorbent assay. Ann NY Acad Sci
438:504-507
3. Clarke GN, Elliot PJ, Smaila C (1985): Detection of sperm antibodies in semen using the immunobead test: A
survey of 813 consecutive patients. Am J Reprod Immunol Microbiol 7:118-123
4. Clarke GN, Stojanof A, Cauchi MN (1982): lmmunoglobulin class of spermbound antibodies in semen. In:
Immunology of Reproduction: Proceedings of the Fifth International Symposium on Immunology of Reproduc-
tion. Branatov K (Ed). Sofia: Bulgarian Academy of Sciences Press, pp 482-485
5. Friberg J (1974): A simple and sensitive micro-method for demonstration of sperm agglutinating activity in serum
from infertile men and women. Acta Obstet Gynaecol Scand 36(suppl):21-29
6. Golomb J, Vardinon N, Homannai ZT, et a1 (1986): Demonstration of antispermatozoal antibodies in varicocele-
related infertility with an enzyme-linked immunosorbent assay (ELISA). Fertil Steril 45:397-402
7. Howe SE, Lynch DM (1986): Quantitation of sperm-bindable IgA and IgG in seminal fluid. Am J Reprod
lmmunol Microbiol 1 1:17-23
8. Ing RMY, Wang S-X, Brennecke AM, et a1 (1985): An improved indirect enzyme-linked immunosorbent assay
(ELISA) for the detection of antisperm antibodies. Am J Reprod Immunol Microbiol 8: 15-19
9. Isojima, Shun T, Ashikata Y (1968): Immunologic analysis of sperm-immobilizingfactor found in sera of women
with unexplained sterility. Am J Obstet Gynecol 101:677-683
10. Jager S, Kremer J, Van Slochteren-DraaismaT (1978): A simple method of screening for antisperm antibodies in
the human male: Detection of spermatozoa1 surface IgG with the direct mixed antiglobulin reaction test on
untreated fresh human semen. Int J Fertil 23: 12-21
11. Kremer J, Jager S (1976): The sperm cervical mucus contact test: A preliminary report. Fertil Steril27:335-340
12. Lynch DM, Leah BA, Howe SE (1986): A comparison of sperm agglutination and immobilization assays with a
quantitative ELISA for anti-sperm antibody in serum. Fertil Steril 46:285-292
13. Mandelbaum SL, Diamand MP, DeCherney AH (1987): The impact of antisperm antibodies on human fertility. J
Urol 138:l-8
14. Paul S, Baukloh V, Mettler L (1983): Enzyme-linked immunosorbent assays for sperm antibody detection and
antigenic analysis. J Immunol Method 56:193-199
15. Van Zyl JA (1986): Manlike infertiliteit. In: Ginekologie. Odendaal HJ (Ed). Cape Town: Juta, pp 111-118
16. Windt M-L, Bouic PJD, Menkveld R, et a1 (1989): Use of monoclonal antibodies to secretory IgA for the
detection of sperm-antibodies in serum and seminal plasma by ELISA method. (submitted for publication)
17. Windt M-L, Menkveld R, Kruger TF, et a1 (1988): The effect of rapid dilution of semen on sperm-bound
autoantibodies. Arch Androl 22:227-23 1
18. Windt M-L, Menkveld R, Kruger TF, et al (1988): The effect of sperm washes and swim-up on antibodies bound
to the sperm membrane: Use of immunobead and sperm cervical mucus contact tests. Arch Androl 22:55-59
19. Witkin SS (1983): Enzyme-linked immunosorbent assay (ELISA) for detection of antibodies to spermatozoa. Res
Reprod 15:l-2
20. Wolff H, Scill WB (1985): A modified enzyme-linked immunosorbent assay (ELISA) for the detection of anti-
sperm antibodies. Andrologia 17:426-439