A New Reagent (ZZAP) Having Multiple Applications in
Immunohema tology
DONALD R. BRANCH, MT(ASCP)SBB AND LAWRENCE D. PETZ, M.D.
A reagent (ZZAP) containing a mixture of 0.1 M dithiothreitol
(DTT) plus 0.1% cysteine-activated papain was found to dissociate IgG immunoglobulin from red blood cells (RBC) of patients having a positive direct antiglobulin test (DAT) although
this could not be achieved with either chemical alone. In all 67
patients tested, ZZAP treatment of IgG sensitized RBC reduced the strength of the DAT, and in all 52 instances tested,
this allowed for accurate Rh phenotyping using slide/rapid tube
typing reagents. This included five examples in which spontaneous agglutination that occurred in saline or 6% albumin was
eliminated by ZZAP. Thus, all red blood cell typing for Rh-Hr
antigens was accomplished using slide/rapid tube reagents
making unnecessary the use of saline reactive or chemically
modified antisera. Kidd antigen typing is also possible after
ZZAP treatment of IgG sensitized RBC. In regard to warm
autoabsorption tests, ZZAP treatment of 14 RBC samples having a positive DAT proved preferable to heat elution technics
since equal or greater amounts of IgG were removed by ZZAP
and little or no hemolysis resulted. ZZAP has no effect on
ABH, Rh or Kidd antigens but denatures Duffy, MNSs and all
Kell antigens tested (K:l-K:7, K:ll-K:14, K:18, K:19). This
should prove valuable in certain investigations of multiple alloantibodies and, moreover, may allow for better characterization of Kell antigens. (Key words: ZZAP reagent; Autoimmune
hemolytic anemia; Red blood cell antigens; Autoimmunity; Red
blood cell typing; Warm autoabsorption; Kell antigen denaturation) Am J Clin Pathol 1982; 78: 161-167
TECHNICS FOR dissociating IgG from human red
blood cells have major applications in immunohematology. In 1959, Nisonoff and associates10 described the
use of L-cysteine, a disulfide-bond reducing reagent, in
combination with the proteolytic enzyme pepsin to isolate univalent fragments of rabbit anti-ovalbumin. This
was not possible when using pepsin alone. Nisonoff postulated that the sulfhydryl reagent acted independently
of the enzyme and directly upon the immunoglobulin
molecule and that the combination of the two chemicals
resulted in the split products observed. Based upon this
work, we have developed a reagent consisting of a mix-
Received September 15, 1981; received revised manuscript and accepted for publication October 26, 1981.
Presented in part at the 33rd Meeting of the American Association
of Blood Banks, Washington, D. C, November, 1980.
Address reprint requests to Mr. Branch: Research Associate, Division of Medicine, Department of Clinical and Experimental Immunology, City of Hope National Medical Center, 1500 East Duarte
Road, Duarte, California 91010.
Division of Medicine, Department of Clinical and
Experimental Immunology, City of Hope National Medical
Center, Duarte, California
ture of a sulfhydryl reagent and a proteolytic enzyme
which is effective in dissociating IgG molecules from
human red blood cells. Use of this reagent makes possible certain red blood cell antigen typings in patients
having a positive direct antiglobulin test (DAT) including Rh-Hr and Kidd. Further, red blood cells from
patients with autoimmune hemolytic anemia (AIHA)
may be prepared for use in the warm autoabsorption
technic more quickly and more reliably than by currently available methods. Finally, the reagent denatures
antigens of the Kell blood group system. This characteristic makes it very useful in certain antibody investigations and may lead to better characterization of the
various antigens in the Kell blood group system.
Since this reagent is a mixture of a compound that
reduces disulfide bonds (S-S) and a proteolytic (AP)
enzyme, we originally referred to the mixture as SSAP. More recently, for euphemistic reasons, we have
chosen the term ZZAP.
Materials and Methods
Serologic
Studies
Standard serologic technics were used.13 Direct antiglobulin tests were performed with licensed monospecific anti-IgG (Gamma Biologicals, Houston, TX; Ortho Diagnostics, Raritan, NJ) or our own anti-IgG
reagent prepared in rabbits and standardized as previously described." Anti-C3 (C3c + C3d) antisera was
prepared in rabbits and standardized as previously described.11 Anti-kappa antisera was obtained from Calbiochem (La Jolla, CA) and appropriately standardized
for use in the antiglobulin test. S-alkylation of reduced
disulfide bonds was achieved using 0.02 M iodoacetamide (Sigma Chemical Co., St. Louis, MO) in phosphate buffered saline (PBS) at pH 7.4. S-alkylation was
allowed to proceed for 30 min.
DAT titrations were performed using serial doubling
0002-9173/82/0800/0161 $01.15 © American Society of Clinical Pathologists
161
BRANCH AND PETZ
162
dilutions of anti-IgG, anti-C3 or anti-kappa antisera as
previously described." End points were determined by
microscopic examination. The method of grading and
scoring the agglutination reactions was that of Petz and
Garratty." Red blood cell antigen typings were performed using commercially licensed antisera or antisera
obtained from a single donor and shown to be specific
in tests using panels of reagent red blood cells.
In Vivo Sensitized Red Blood Cells
Red blood cells drawn into EDTA or ACD were obtained from patients having acquired immune hemolytic
anemias and stored for up to 2 weeks at 4°C before
testing. The DAT ranged from weakly positive to 4+
positive.
Sulfhydryl-Proteolytic
Enzyme (ZZAP)
Reagent
Stock 1% papain (Difco Laboratories, Detroit, MI)
with added L-cysteine hydrochloride (Eastman Organic
Chemicals, Rochester, NY) was prepared as described
by Issitt and Issitt,5 aliquoted and stored at —20°C;
stock 0.2 Mdithiothreitol (DTT) (Calbiochem, La Jolla,
CA) was prepared and aliquots stored at —80°C; 0.15
M PBS, pH 7.3, was prepared and stored at 4°C.
In order to determine the optimal conditions for
ZZAP preparation and use, the concentration of papain
was varied over a wide range (0.05-0.6%); the greatest
degree of IgG dissociation occurred at a concentration
of 0.1%. The concentration of DTT was also varied
(0.01 -0.12 M), keeping the concentration of papain constant at 0.1%. Maximum dissociation of IgG from red
blood cells occurred at a concentration of 0.1 M. Using
these concentrations of papain and DTT, the pH was
varied (from pH 4 to pH 8), and a pH range of 5-6.5
was found optimal. Finally, the length of incubation at
37°C was varied (from 10-45 min) and a 30-45 min
incubation time produced maximal IgG dissociation
from RBC. Based upon these data, we developed the
following formulation for ZZAP: 2.5 ml of 0.2 M DTT
is mixed with 0.5 ml of 1% cysteine-activated papain
and 2 ml of 0.15 M PBS, pH 7.3; the final concentration
of DTT is 0.1 M and that of papain is 0.1%. The pH
of the final mixture should be in the range of 6.0-6.5.
This mixture is stable up to 5 days when stored at 4°C.
Treatment of DAT Positive RBC with
ZZAP
Two volumes of ZZAP reagent are added to one volume of DAT positive packed red blood cells and incubated at 37°C for 30 min with occasional mixing.
After incubation, the red blood cells are washed three
times using large volumes of isotonic saline before
testing.
Warm Autoabsorption
A.J.C.P. • August 1982
Technic
Red blood cells from patients having a positive DAT
were collected into EDTA and packed by centrifugation
at 1000 X g for 5 min. The plasma was removed and
two volumes of ZZAP reagent was added to one volume
of packed cells. The red blood cells were not usually
washed prior to the addition of ZZAP. After repeated
inversion to mix, the sample was incubated at 37°C for
30 min, with periodic mixing throughout the incubation.
The mixture was divided into at least two aliquots and
washed three times at room temperature using large
volumes of isotonic saline. The last saline wash was
removed as completely as possible.
For absorption, one volume of serum was added to
one volume of ZZAP treated red blood cells. After
mixing, the sample was placed at 37°C for 30 min with
occasional mixing during the absorption period. After
incubation, the sample was centrifuged at 1000 X g for
5 min and the supernatant serum removed. When necessary, the absorption was repeated with an additional
aliquot of ZZAP treated red blood cells.
Antibody Investigation Using
ZZAP
Commercial panels of reagent red blood cells were
treated with ZZAP in the following manner: two volumes of 3% panel cells were washed once and the supernatant saline removed. Two volumes of ZZAP reagent were added and the mixture incubated at 37°C
for 30 min. After incubation, the cells were washed
three times using large volumes of isotonic saline and
resuspended to 2-3% using 0 . 1 5 M saline or 0.03 M
LISS (0.03 M NaCl in 0.3 M glycine) 13 prior to testing.
Red Blood Cell Antigen
Typing
ZZAP treated RBC are not comparable to the red
blood cells used by commercial manufacturers of typing
reagents in their quality control testing. Therefore, each
lot of typing antiserum was tested for specificity using
phenotyped panels of red blood cells that were first
treated with ZZAP. Manufacturers' recommended procedures were utilized except when using slide/rapid
tube Rh-Hr antisera. In this case, readings were performed immediately after the addition of the antisera
to the ZZAP treated red blood cells. Rh-Hr control
reagents (the manufacturers' diluents) were employed
as negative controls. Negative reactions were incubated
for 15 min at 37°C and read again. Du testing was
performed on all samples which were still negative after
incubation at 37°C for 15 min. The appropriate manufacturers' Rh-Hr diluent was employed as a negative
control and processed in parallel. When using com-
Vol. 78 • No. 2
ZZAP: A NEW REAGENT
mercial antisera with untreated red blood cells, manufacturers' recommended procedures were followed.
Results
Effect of Papain and Dithiothreitol
Sensitized RBC
(DTT) on
A mixture of 0.1 M dithiothreitol (DTT) and 0.1%
activated papain caused marked reduction in the amount
of IgG on red blood cells. However, this effect was not
obtained using either chemical alone (Table 1). Similar
results were seen when using any mixture containing
either 2-mercaptoethanol or DTT plus any one of four
commonly used proteolytic enzymes, i.e., ficin, papain,
bromelin or trypsin. Reduction of IgG also occurred
when the sensitized red blood cells were first treated
with DTT, followed by S-alkylation and then treatment
with papain, but not when the sequence of reactions
was reversed.
Effectiveness of ZZAP in Reducing Strength of DAT
To determine the affect of ZZAP on reducing the
strength of the DAT, 67 patients having a positive DAT
ranging from 1+ to 4+ were studied (Table 2). Overall,
ZZAP treatment of their RBC was able to reduce the
strength of the DAT to microscopic or negative in 51
patients (76%). However, a number of strongly positive
antiglobulin tests were negligibly reduced. Further investigation of ten samples that were only negligibly reduced when tested with undiluted antiglobulin serum
indicated that the DAT titration score was substantially
reduced in all instances (Table 3).
Dramatic reduction in the DAT titration score after
ZZAP treatment also was seen when using anti-kappa
antisera. In three patients, the DAT titration scores
Table 1. Effect of Papain* and Dithiothreitol (DTT)
on IgG-Sensitized RBC
IgG-Coated RBC
Treated with
Saline
0.1% papain
0.1 M DTT
0.1% papain followed with
0.1 M DTT
0.1 M DTT + 0.02 M iodoacetamide followed with
0.1% papain
0.1 M DTT + 0.1% paapin
(ZZAP)
• Cysteine-activated (5).
Titer Using
Anti-IgG
163
Table 2. Reduction in DAT Strength Using ZZAP
After ZZAP Treatment
DAT
Untreated
Number of
Patients
Tested
#DAT
Negative
#DAT
Microscopic
Positive
#DAT
Macroscopic
Positive
1+
2+
3+
4+
13
12
20
22
13
10
10
5
0
2
8
3
0
0
2
14
using anti-kappa antisera were 35, 43, and 24. ZZAP
treatment reduced the score to 0, 4, and 1, respectively.
Reduction in detectable levels of the C3d component
of complement on red blood cells also occurred after
ZZAP treatment when using red blood cells sensitized
in vivo, where cleavage to C3d was due to serum C3b
inactivator (C3d-C3bINA). In two patients with DAT
titration scores of 24 and 23 using anti-C3 antisera, the
DAT was reduced to negative after treatment with
ZZAP. However, ZZAP had no effect on the DAT
titration scores when using red blood cells which had
been sensitized in vitro with C3b using the method of
Chaplin and associates,* and then converted to C3d
using trypsin (C3d-try). These findings suggest a difference of C3d structure on red blood cells coated in
vivo compared with those prepared in vitro. This is consistent with the findings of Freedman and Massey 4 who
reported that anti-C3d antisera made to the C3dC3bINA antigen had different specificity than antisera
made to the C3d-try antigen.
Comparison of ZZAP and Heat Elution
The effectiveness of the ZZAP reagent compared to
various heat elution methods in reducing detectable IgG
Table 3. Reduction of DAT Titration Score When
IgG is Not Completely Dissociated Using ZZAP
DAT
Titration
Score
1:32
1:32
1:32
40
43
36
1:32
35
1:4
11
1:4
6
Methods
DAT Titration Score
Patient
Untreated
RBC
ZZAP
RBC
Untreated
RBC
ZZAP
RBC
1
2
3
4
5
6
7
8
9
10
4+
4+
4+
4+
4+
4+
4+
4+
4+
4+
3+
1+
2+
2+
2+
4+
4+
2+
1+
2+
62
50
64
49
67
53
56
53
54
47
27
12
19
17
18
35
41
17
9
8
BRANCH AND PETZ
164
Table 4. Comparison of ZZAP with Heat Methods
for Reducing the Detectable Immunoglobulin
Molecules Sensitizing RBC
A.J.C.P. • August 1982
to allow for adequate autoabsorption and subsequent
identification of underlying significant anti-Fy a + E alloantibodies in one of the patient's sera.
DAT Titration Scores Using Anti •IgG
Effect of ZZAP on Red Blood Cell Antigens
No.
DAT
Untreated
1
2
3
4
5
6
7
8
9
10
11
12
13
4+
2+
4+
4+
4+
3+
3+
4+
4+
4+
3+
4+
4+
Untreated
45°C
30 min
50°C
10 min
56°C
5 min
ZZAP
59
23
54
64
47
32
24
68
52
53
40
44
67
58
17
42
51
0
20
20
68
45
45
39
33
68
45
0
33
45
0
19
14
59
38
37
32
18
62
12
0
12
19
0
6
4
50
22
20
14
12
42
8
0
9
19
0
2
4
47
19
5
6
0
18
immunoglobulin from in vivo sensitized human red
blood cells is shown in Table 4. ZZAP dissociation of
IgG, as measured by a reduction in the DAT titration
score, was similar to 56°C heat elution in 9 of 13 patients. However, in 4 of 13 patients, ZZAP technic was
superior to 56°C heat. In all but one instance, ZZAP
was considerably more efficient than 45°C heat elution
and in all but two instances, it was far better than when
using 50°C heat elution. A major disadvantage to the
use of heat elution technics is their tendency to weaken
the red blood cell membrane sufficiently to cause severe
hemojysis, especially when the red blood cells are used
in subsequent warm autoabsorption studies. ZZAP only
rarely caused very slight hemolysis, whereas the red
blood cells from patients with severe autoimmune hemolytic anemia may readily hemolyze after 56°C heat
elution for 3-5 min.
Warm Autoabsorption
Technic
In 14 patients with AIHA with a serum autoantibody
activity of 1 + or greater, ZZAP treated autologous red
blood cells were successful in removing sufficient autoantibody to allow for alloantibody assessment. In 11
of 14 (79%), only one autoabsorption was required to
completely remove autoantibody from the serum. Nine
underlying clinically significant alloantibodies were
identified in five patients: 2 Fya, 3 E, 1 C, 1 c, 1 Kell
and 1 S. In two patients, warm autoabsorption using
red blood cells that were incubated at 56°C to first
remove IgG was unsuccessful in removing the autoantibody even after multiple absorptions. Although ZZAP
did not completely dissociate IgG from the red blood
cells of these two patients, enough IgG was dissociated
Table 5 contains a listing of red blood cell antigens
which appear to be unaffected by ZZAP treatment and
those shown to be denatured. Antigens within the ABH,
Lewis, Rh-Hr, Kidd and Lutheran blood group systems
appear to be unaffected by the ZZAP reagent. In addition, various antigens having a very high frequency
in the random population have also been shown to be
unaffected by ZZAP.
As expected, ZZAP treatment was shown to denature
the various antigens within the M N S and Duffy blood
group systems. Additionally, ZZAP partially denatures
the s antigen and, unexpectedly, denatures all Kell antigens which we tested including Kell, cellano, Kp a , Kp b ,
Js a , Js b , Ku, K:ll through K:14, K:18, K:19 and Gerbich. The antigen Yt a (Cartwright) appeared to be variable in its susceptibility to denaturation by ZZAP with
some examples of anti-Yt a nonreactive after ZZAP
treatment while other examples were unaffected.
Antibody Investigation Using
ZZAP
In vitro mixtures of anti-cellano plus anti-Jk a and
anti-cellano plus anti-D, -C, -E were prepared so that
when tested using panels of reagent red blood cells,
either untreated or enzyme pretreated, strong positive
reactions ( 2 - 3 + ) were seen with all test cells. After
pretreatment of the same panel cells with ZZAP reagent, the anti-Jk a and anti-D, -C, -E were easily identified. Additionally, one patient's serum known to contain anti-Ku, reacting 2 - 3 + with all random red blood
cells, was tested against a 16-cell panel of ZZAP treated
reagent red blood cells and revealed an underlying
anti-Le a .
Table 5. Effect of ZZAP on RBC Antigens
Antigens Denatured
Antigens Unaffected
ABH
Lewis*
Rh-Hr
Kidd (Jk a , Jk b ,
Jk:3)
Lutheran (Lu a , L u \
Lu:17)
U
Vel
Lah
Jr"
Co"
Tj a
Dib
Gya
MNS
Duffy (Fy a , Fy b )
Kell (K, k, Kp a , K p \ Js a , Js b ,
Ku, K : l l , K:12, K:13, K:14,
K:18, K:19, Gerbich)
st
Yt a t
Yt a t
* Tested using human antibody,
t Substantially but not completely denatured.
t Denaturation was seen with some examples of anti-Yt* after ZZAP treatment while others
remained reactive.
Vol. 78 • No. 2
Kidd Antigen
ZZAP: A NEW REAGENT
Typing
a
b
Commercially obtained anti-Jk and anti-Jk antisera
were shown to be specific in tests using panels of RBC
having known antigenic makeup which had been pretreated with ZZAP reagent. Additional testing using
RBC of known Kidd phenotype which had been strongly
sensitized in vitro with IgG antibody and then treated
with ZZAP reagent were shown to give reliable typing
results. Finally, seven patients having a moderately to
strongly positive DAT were typed for their Kidd antigens after ZZAP treatment of their RBC. The following
results were obtained: 2 J k ( a + b - ) ; 2 Jk(a+b+); 3
J k ( a - b + ) . The DAT was negative or only weakly positive and much stronger reactions were obtained with
the typing antisera.
Rh-Hr Antigen
Typing
Fifty-two successful Rh phenotypings in patients having a positive direct antiglobulin test were performed
using ZZAP treated red blood cells with slide/rapid
tube typing reagents. Twelve of these patient's untreated red blood cells were found to be positive with
manufacturers' diluent controls. In seven of these, there
was no spontaneous agglutination in isotonic saline or
6% albumin and therefore, they could be successfully
Rho(D) typed using chemically modified antisera, saline reactive antisera or by using ZZAP treated red
blood cells with slide/ rapid tube reagents. However, in
five examples, Rho(D) antigen typings were unreliable
using chemically modified or saline reactive antisera
due to spontaneous agglutination in isotonic saline and
6% albumin. However, after these red blood cells were
treated with ZZAP, strongly positive results with slide/
rapid tube anti-Rho(D) and negative results using
matching Rh-Hr diluent control demonstrated these
cells to be clearly Rho(D) positive and, in fact, these
red blood cells were successfully phenotyped for the Rh
antigens D, C, E, c and e.
Additionally, nine of ten red blood cell samples having a positive DAT and which were initially typed as
Rho(D) negative, using either chemically modified or
saline reactive antisera, were successfully typed for their
Du antigen status using slide/rapid tube anti-Rho(D)
after ZZAP treatment. Three samples were shown to
be Du positive. In one instance, the DAT remained positive as evidenced by a weakly positive Rh-Hr control
and therefore, Du typing was indeterminate.
We have encountered one instance in which ZZAP
treated red blood cells appeared to acquire an unusual
form of polyagglutination. This was characterized by
weakly positive reactions with approximately 10% of
random sera tested and strongly positive reactions using
the lectin Salvia horminum. However, negative reac-
165
tions with the lectins Arachis hypogea and Salvia sclarea were inconsistent with either T, Tk, Tn or Cad
polyagglutination. This unusual polyagglutination occurred with the red blood cells of a patient who had a
very strongly positive DAT and who had severe warm
antibody AIHA. Nonetheless, accurate Rh phenotyping
was accomplished using selected slide/rapid tube reagents.
Discussion
We have investigated the use of a mixture containing
a sulfhydryl reagent plus a proteolytic enzyme as a
means of dissociating immunoglobulin molecules from
sensitized red blood cells. This reagent which we have
named ZZAP is effective and rapid and has multiple
applications in immunohematology.
One such use involves the typing of red blood cells
that have a strongly positive DAT. ZZAP treated red
blood cells can be used for ABH, Rh-Hr, Kidd and
certain other antigen typings in those patients whose
red blood cells are strongly sensitized with IgG. Previously advocated methods of typing DAT positive red
blood cells," for antigens other than Rh, require heating
the patient's red blood cells at 45°C, 50°C or 51°C56°C to dissociate red blood cell bound antibody. A
disadvantage of these heat elution methods is that red
blood cell antigens can become denatured and possibly
lose their antigenicity when heated above 37°C. Moreover, gross hemolysis of red blood cells occasionally
occurs when they are heated above 50°C.
Another method, previously described by Mantel and
Holtz 6 as an elution technic, and more recently by Edwards and associates3 for red blood cell antigen typing
of immunoglobulin sensitized cells, uses the quinoline
derivative chloroquine diphosphate. Chloroquine shows
promise as an effective technic for the dissociation of
sufficient IgG antibody to allow for various antigen determinations and has not been shown to denature any
red blood cell antigens. However, the chloroquine technic may require as long as 2 hours to be effective, and
even then complete dissociation of IgG may not occur.
Also, preliminary experiments in our laboratory indicate that marked hemolysis of red blood cells may occur
after treatment with chloroquine.
An additional technic for RBC typing when the DAT
is positive is a differential adsorption procedure.' 1 Aliquots of typing sera are absorbed with RBC of the
patient, RBC positive for the antigen(s) in question,
and RBC negative for the antigen(s). The antibody
activity of the absorbed sera are compared as a means
of determining whether or not the patient's RBC contain the antigen(s). This technic, although useful, is
cumbersome.
166
BRANCH AND PETZ
In all 52 instances in which ZZAP was used to dissociate IgG from RBC having a positive DAT, sufficient
IgG was removed to allow for accurate Rh typing using
slide/rapid tube reagents including nine often examples
where the Du status could be determined. It must be
emphasized that appropriate controls are mandatory.
In particular, commercial typing reagents are not approved for use with ZZAP treated red blood cells, and
must be standardized against known antigen positive
and antigen negative cells.
In those instances in which the red biood cells of
patients with warm antibody autoimmune hemolytic
anemia spontaneously agglutinate in isotonic saline or
6% albumin, we have found that this agglutination is
no longer present after ZZAP treatment. As a result,
we have been able to obtain accurate Rh typings of
these RBC although such typing would not have been
possible using saline reactive or chemically modified Rh
antisera. Thus, all red blood cell typing of Rh-Hr antigens was accomplished using slide/rapid tube reagents
making unnecessary the use of saline reactive or chemically modified antisera. Kidd antigen typing is also
possible using indirect antiglobulin test reactive antisera
and ZZAP treated RBC and can be much faster than
the chloroquine technic.
In addition to antigen typings, removal of IgG from
red blood cells of patients with autoimmune hemolytic
anemia is useful in compatibility testing. This is because
these patients may have both autoantibody and clinically significant red blood cell alloantibodies in their
sera. A technic to absorb out all autoantibody is valuable to detect or exclude the presence of alloantibody.
The warm autoabsorption technic, i.e., absorption of
the patient's autoantibody from the patient's serum
using autologous red blood cells after first removing
some of the cell bound autoantibody by heat elution,
has been recommended as the best technic for this purpose.8" However, heat elution is a relative ineffective
means of removing IgG from red blood cells, and gross
hemolysis of the eluted cells occasionally occurs when
they are subsequently used for absorption.
The fact that ZZAP quickly reduces the number of
molecules of immunoglobulin sensitizing red blood cells
and simultaneously enzyme modifies the cells makes this
reagent valuable for use in the warm autoabsorption
technic. Even though ZZAP treatment does not reduce
the DAT to negative in all cases, sufficient IgG was removed to allow for adequate autoabsorption in all cases
tested and marked hemolysis did not occur. Although
one absorption was sufficient to remove all autoantibody
in 79% of the samples we studied, we would recommend
at least two absorptions as a general routine.
Other applications for ZZAP arise from its effects
A.J.C.P. • August 1982
on Kell antigens. Although Morton in 19629 reported
the denaturation of the Kell antigen using trypsin, this
was not reproducible using other sources of trypsin.
Bove and Baird1 reported on the inhibition of reactions
between anti-Kell and RBC treated with sulfhydryi
blocking reagents {i.e., p-chloromercuribenzoic acid or
N-ethyl maleimide) and suggested that sulfhydryi
groups may be important in Kell antigen structure.
ZZAP, without exception, denatured all Kell antigens
tested. This extraordinary property of ZZAP affords
a number of exciting uses: (1) red blood cells of any
ABO blood group can be prepared which react similarly
to Ko red blood cells; (2) ZZAP treated red blood cells
may be used to identify red blood cell alloantibodies
occurring in association with Kell antibodies, for example anti-cellano with anti-Jka; (3) Kell alloantibodies
may be detected using the warm autoabsorption technic
in patients who have recently been transfused with Kell
positive red blood cells; (4) Kell autoantibodies {i.e.,
auto-ariti-Kpb) may be detected in patients with AIHA.
That is, if warm autoabsorption does not remove autoantibody, the specificity may be within the Kell system, as has been reported to occur in approximately 1
in 250 patients with warm autoantibody7; and finally,
(5) ZZAP may allow for better characterization of Kell
antigens.
Although the mechanism by which ZZAP effects IgG
dissociation from red blood cells is not entirely clear,
Venyaminov and colleagues14 and Romans and associates'2 demonstrated that reduction of the disulfide
linkages of IgG immunoglobulin results in increased
segmental flexibility of the molecule and that this increases the extent to which the IgG polypeptides are
exposed to the surrounding medium. Additionally, Venyaminov and colleagues14 showed that the lack of interchain disulfide bridges results in protein destabilization affecting about 90% of the peptide groups. This
resulted in increased sensitivity to digestion by papain.
Based upon these reports, we believe the mechanism by
which the ZZAP reagent dissociates IgG from red blood
cells may involve the initial reduction of the interchain
disulfide linkages. This would result in increased exposure of the IgG polypeptides to the surrounding medium allowing the proteolytic enzyme increased accessibility to the peptide groups. The IgG molecule may
lose integrity and dissociate completely from the red
blood cell antigen with which it had reacted. This proposed mechanism is supported by our results using sequential treatment of IgG sensitized red blood cells by
sulfhydryi reduction, S-alkylation and papain treatment. Although we were able to effect dissociation using
this particular sequence, we could not demonstrate significant dissociation when the sequence was reversed.
Vol. 78 • No. 2
ZZAP: A NEW REAGENT
Additionally, negative results obtained with anti-kappa
antisera after ZZAP treatment is suggestive that the
entire immunoglobulin may be dissociating from the
red blood cells and that the reduction in the detectable
IgG is not solely a result of cleavage of the Fc portion
of the immunoglobulin molecule. This proposed mechanism may provide an explanation for the effectiveness
of ZZAP technic compared with other methods for
warm autoabsorption in AIHA.
Acknowledgment. We wish to thank Lucy Brooks for her generous
supply of DAT positive RBC and W. L. Marsh for kindly confirming
our results with anti-Kell, k, Kpa, Kpb, Js", Js b and Ku versus ZZAP
treated RBC and extending these findings to K: 11 through K: 14, K: 18,
K:19 as well as testing numerous other high-frequency antigens.
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