- Email: [email protected]
The reversed BDB technique
THE
REVERSED
BDB TECHNIQUE
The Agglutination of Antibody-Coated Homologous Antigen Cua-Lim,”
F.
M. Richter,
and B. Rose,
Red Blood Cells by Quebec, Camada
Montreal,
1942, Pressman and his associates1 described a new technique for the detection immunological reactions between soluble antigens and antibodies which has proved to be far more sensitive than the conventional interfacial ring and precipitin tests. This method involved the chemical coupling of the protein antigen to red blood cells by means of bisdiazotized benzidine (BDB) and the subsequent agglutination of such “antigen-sensitized cells” by the homologous antiserum (BDB technique). This technique was later modified by Stavitsky and Arquilla* and further developed by Gordon and co-workers.3 A technique utilizing the absorption of antigen to tannic acid-treated red blood has been described by Boyden (tanned-cell technique). Using both of these techniques, it has been possible to demonstrate, unequivocally, the presence of small quantities of precipitating antibodies in solution3, 5 and the detection of antibodies in the sera of allergic individuals.“, fi However, the preparation of “antibodysensitized red blood cells” for the detection of small quantities of antigen has not as yet been reported (reversed BDB technique). In a previous communication7 a brief description of a procedure for the coupling of antibodies to red cells which are subsequently capable of being agglutinated by the specific antigen was presented. The present paper further elaborates on the reversed BDB technique.
I
N
of
MATERIALS
AND
METHODS
1. Preparution of Antisera.-Rabbits, weighing 3 to 5 kilograms, were used throughout. The antigens used were (a) BSA (bovine serum albumin), t (b) OA (egg albumin),$ and (c) HGG (human gamma globulin Fr. II).f All injections of antigens were made intravenously through the marginal ear veins of the rabbits. The dose of antigen varied from 10 to 50 mg. per injection. The animals were given only 2 to 3 injections of antigen at intervals of 2 to 3 days. From the Divieion of Immunochemistry and Allergy, McGill University Hospital. Montreal, Quebec, Canada. This investigation was supported by grants from the Department of Welfare, Ottawa, Canada, and the National Institutes of Health, Institute fectious Diseases, Bethesda, Md., U. S. A. (Grant No. E-1322). Received for publication Nov. 2, 1962. *Submitted in partial fulfillment for the degree of Master of Science, perimental Medicine, McGill University, Montreal. Quebec. Canada. TPentex Inc., Kankakee, Illinois. *Nutritional Biochemical Co., Cleveland, Ohio. toria
142
Clinic,
Royal
Vic-
National Health and of Allergy and InDepartment
of
Ex-
Volume
Number
34 ?
REVERSED
BDB
TECHNIQUE
143
These animals are considered to have produced a primary response. In order to elicit a secondary response, some of the rabbits were re-injected several months after the initial ibmunization with a single dose of 20 to 50 mg. of the homologous antigen. The animals which were tertiarily immunized months after the secondary response are referred to as having been hyperimmunizcld. The animals were bled from the marginal ear veins at appropriate intervals of time after antigen injection. The blood was allowed to clot at room temperature and placed in the cold room at 4’ C. for 21 hours. The serum was thcII separated from the clot by centrifugation and stored at -20’ until used. 2. Immunologic Methods.-(A) Precipitin test: Precipitating antibodies were detected by the interfacial ring test. Antigen solutions (0.05-0.5 pot cent) were layered over undiluted sera in small culture tubes and the intcrfacc was observed over a period of 60 minutes. The appearance of a white ring at the interface was considered indicative of the presence of precipitating antibodies if appropriate control tubes were negative. B. Detection of antibody by the BDB technique: The bisdiazotized benzidine (BDB) technique was used as modified by (Gordon and his co-workers.Z These antibodies are hereafter referred to as hemagglutinat,ing antibodies. The titers of the antisera are expressed as the reciprocals of the highest dilutions of the antisera which gave positive agglutination patt,erns of antigen-coated red blood cells. As a control, an antiserum of known titer was always titrated along with the sera under investigation. Sera with BDB titers lower than 10 were considenbd negative. C. Purification of antibodies: The protein antigens were coupled to diazotized8 as well as to nondiazotizedg polyaminostyrene.* Purificabion and isolation of antibodies was performed by incubating the antisera with the respective insoluble immunosorbent-antigen conjugates* for 30 minutes at room temperature with constant stirring. Each suspension was then centrifuged and the antibodydepleted supernatant (referred to as “supernatant” in the text) was dialyzed overnight against saline at 4’ C. and stored at -20° C. The antibodies were isolated by incubation of the antibody-antigen-polystyrene conjugates at room temperature at pH 3.0 for 30 minutes, which resulted in the liberation ot’ antibodies into the solution. The pH of the incubated suspension was adjusted to 3.0 by the addition of O.lN HCl. The solution containing the dissociated antibodies was adjusted to pH 7.2 by the addition of phosphate buffer (pHY 7.2, ionic strength 0.1) and it was then dialyzed against distilled water overnight and lyophilized. This latter preparat,ion is referred to as “purified antibody” in the text. D. Preparation of antibody-sensitized red cells (Ab-rbc): The met,hod ascd was described briefly in a recent communication.7 The procedure followed is identical to that of the regular BDB technique3 except that antibody was used in lieu of the antigen in the coupling procedure. The optimal concentration of t.he antibody used in the coupling procedure was determined experimentally by t,rial and error. In general, it was found that 5 to 10 mg. of the lyophilized *Obtained
from
Norsk
Hydroelectric
Co., Oslo,
Norway.
144
CUA-LTM,
RTCHTER,
ROSE
J. Allerpy March-April,
I%3
purified antibody preparation in 3 ml. of saline was required for the optimal sensitization of each 0.1 ml. aliquot of rabbit red ccl,‘s. To prevent nonspecific agglutination, the purified antibody preparation was absorbed with washed, packed red cells (obtained from the same rabbit) for 15 to 30 minutes at 4’ C. in order to remove any blood group antibodies. The absorbed antibody solution was then incubated with the red cells and BDB to effect the chemical coupling. Gamma globulin preparations, obtained by ammonium sulfate fractionation of the precipit,ating antisera, werr coupled to red cells in the same manner as described above. Halving dilutions* of the antigen were prepared in twenty-eight tubes. For the BSA and OA systems, the first tube contained 200 pg per milliliter; for t,he HGG system, the first tube contained 500 pg per milliliter. E. Inhibition experiments: In these experiments, the antigen, in halving dilution, was incubated, in separate experiments, with constant, amounts of each of the following: (1) the original precipitating antiserum, (2) the purified antibody reconstituted to original volume, (3) the supernatant obtained after incubation of the antiserum with the PAS-antigen conjugates (absorbed antiserum), (4) a hcterologous antiserum, and (5) a normal rabbit serum. The incubations were carried out at room temperature for 30 minutes, following which t.he antibody-sensitized red cells (Ab-rbc) were added to all the tubes. Agglutination of the Ab-rbc by the homologous antigen was read after 16 to 20 hours. Antibody-sensitized red cells were found to be stable for at least 8 to 12 hours. 3. Ammonium Sulfa.te Fractionation.-Antisera were fractionated with ammonium sulfate (40 per cent saturation) and the resultant precipitate and supernatant were dialyzed against saline overnight and analyzed for antibody content by the conventional BDB method. 4. l,Wtmcentrifugation.-The ultracentrifugal analyses of the purified antibodies were performed using a Spinco Model E analytical ultracentrifuge. The average rotor temperature was 20.5’ (1. The sedimentation constants were not recalculated for standard conditions. 5. Electrophoresis.-The purified antibodies were subjected to paper electrophoresis using Verona1 buffer pH 8.6, ionic strength 0.05. EXPERIMENTS
AiVI
RESULTS
Determination of Antigen Uptake by Polyaminostyrene (PAS) and Diaxotized Polyaminostyrene (DPA.S).-Varying concentrations of the antigen (Ag) were incubated with constant amounts of polyaminostyrene (PAS) at room temperature (25’ (1.) for 30 minutes to an hour. Untreated, as well as diazotized, PAS was used. The supernata,nts obtained after centrifugation of the incubated mixtures were tested for the presence of unabsorbed antigen. This latter was determined by its ability to inhibit the agglutination of antigcnsensitized cells by the specific antiserum. -:;;t.in
*The technique whereby each consecutive tube
the concptration of a nerves of
of tubes
antigen cm antibody IS referred to as
is diluted by a factor “halving dilutions” in
of the
Volume 11 Number 2
REVERSED
BDR
TECHNIQUE
1 -I.-,
As can be seen in Table 1, the uptake of Ag by the immunosorbent ill)peared to be governed by the concentration of antigen with which the PAS had been incubated. Using a low concentration of antigen (1.5 mg./ml.). OYPI 90 per cent of it was absorbed, whereas at relatively high Ag concent,rat,ion (25 mg./ml.) about 75 per cent of it was a,bsorbed. The absorption of Ag 1)~ PAS and DPAS appeared to be identical. These result,s are similar to thosts reported previously from this laboratory.” TABLE
I.
AMOUNT OF ANTIGEN INCCBATED (BSA) (MG./ML.)
ADSORPTION
OF ANTIGEN
TOTAL AMOUNT OF PAS INCljBATED ( MG.)
A.
Entwated 50 zi
25 12.5 6.2 3.1 1.5
HY POLYAMINOSTPRENE
QT'ANTITY OF BSA" StTPERNATANT (MO/ML.)
B. Diazotized z:: 50 50 50
3.1
1.5 *Iktermined antiserum.
by
inhibition
of
QUANTITY OF BSA TAKEN CI' SY PAS
(nm )
Polynminostywnr 6.0 3.0 0.75 0.375 0.092
19 9.5 5.45 2.725 1.408
Polyaminosty~cv~ 3.0 1.5 0.75 0.375 0.092
2’ 11.0 5.45 2.725 1.408
50 50
25 12.5 6.2
IX
agglutination
of
RSA-sensitized
red
cells
by
the
_--._ Hperific
Purifica.tion of Antibodies Using Diaxotized Polyaminostyrene-Antigen (‘[email protected] different precipitating antisera-anti-BSA, anti-0A, anti-I!lGC: obtained from hyperimmunized rabbits, were used in these experiments. Only the antisera which gave positive ring tests and high BDR hernagglut,ination titers were used. As can be seen in Table IT, almost 100 per cent of the anti-BSA antibodicls were recovered in the cluatc obtained from the AR-MA-SPAS conjugatc~s. TABLE
II.
PL~RIFICATION
OF ANTIBODIES
USING I~IAZOTIZEI) CONJI'CATES
POI.YAMINOSTYKE~-E-ANTIOF:S _ .~~~-~
REAGRNTS 1. Original
I
autiserum
a. Anti-BRA b. Anti-OA c. Anti-HGG 2. Purified antibodies reconstituted to original volumr~ a. Anti-BSA h. Anti-OA c. Anti-HGG 3. Supcmatant of antiserum incubated with PAS-Ag conjugates a. Anti-BSA b. Anti-OA c. Anti-HGG 4. Last
BDB TITER
washing
a. Anti-BRA 1,. Anti-OA c. Anti-HGG
of
PAS-antigen-antihody
200,000 100,000-200,rIoo 100,000-200,000 100,000-200,000 25,00050,000 100,000 100 100-200 1 nn-2on
complexes
0 0 0
146
CUA-MM,
RICHTER,
ROSE
March-April,
J. Allergy 1963
Similarly, from the results obtained with anti-OA antisera, we can conclude that at least 25 to 50 per cent of the antibodies incubated with the immunosorbent could be recovered in the eluates obtained from the Ab-OA-DPAS conjugates. The titer of the antibodies remaining in the supernatant which was obtained after the incubation of the specific antiserum with the homologous AgDPAS complex is insignificant. Only 0.1 to 0.4 per cent of the antibodies were not absorbed by the immunosorbent. The method of purification appears to be the most specific and reliable of all the purification methods available for the isolation of antibody protein. The purified ant,ibodies gave a single band by paper electrophoresis and migrated at a rate identical to that of gamma globulin. The ultracentrifugal analysis of the material revealed a single peak with a sedimentation constant of 7.14 Svedberg units, which corresponds to t,hat of the normal gamma globulin. Elution of Precipitating A.ntibodies Using Untreated PolyaminostyreneAntigen Conjugates.-It is interesting to note that while antibodies could be absorbed onto and subsequently eluted from DPAS-Ag conjugates at pH 3.0, they could only be absorbed onto, but not eluted from, PAS-Ag conjugates (Table III). The reason for this finding is not clear. However, the failure to detect ant.ibody in the eluates obtained from Ab-Ag-PAS conjugates cannot be attributed to the elution of soluble Ab-Ag complexes at pH 3.0, since soluble Ag could at no time be detected in ?%e supernatants obtained from saline suspensions of Ag-PAS conjugates (kept at pH 3.0 for up to 2 hours). Therefore, it is unlikely that Ag would be elubed from the Ag-PAS conjugate in the presence of Ab. El&ion of Precipitating and Nonprecipitating Antibodies Using Diazotized Polyaminostyrene-Antigen Conjugates.-In order to determine whether the hemagglutinating titer of the antiserum is of prime importance in the elution of antibodies, the following antisera* (anti-BSA) were analyzed (Table IV). I. Precipitating antisera obtained within 30 days after the last injection of the primary immunization. These are referred to as “early” antisera.l@l* II. Hyperimmune precipitating antisera, obtained 5 to 15 days after tertiary immunization. A. Nonprecipitating antisera obtained 90 to 120 days after the last injection of primary immunization. These are referred to as ‘ ‘ late ” antisera.10-12 B. Nonprecipitating antisera obtained 4 to 6 months after primary immunization.10-12 From t,he resu1t.s in Table IV, it appeared that the precipitability of an antibody is not the main criterion in considering its ability to be subsequently eluted from the immunosorbent. The antibodies in the antisera of high hemagglutinating titer were capable of being eluted regardless of whether they originated in precipitating or nonprecipitating antisera. Antibodies in precipitating and nonprecipitat,ing antisera having low BDB titers (< 10,000) tive
*Several sera are
antisera presented
of each clsssiflcation in Table IV.
(I,
II,
A,
B)
were
analyzed
but
only
representa-
R’EVERSED TABLE
III.
BDB
TECHNIQUE
ELUTION OF PRECIPITATING ANTIBODIES NONDIAZOTIZED POLYAMINOSTYRENE-ANTIGEN
(ANTI-BSA) USING COKJUGATES
REAGENTS 1. Original precipitating antiserum 2. Antibody-depleted supernatant of precipitating antiserum incubation with PAS-antigen conjugates 3. Eluted antibody obtained from precipitating antiserum” *Reconstituted to the original volume of the antiserum.
TABLE
IV.
ELUTION
BDB ---.._~ TITER 100,000-200,000
after
O-100
SERA TESTED ANTISERCM
PRECIPITATING I
II
I
Original antiserum 1,280 Hupernatant” 0 Eluted antibody+ 0 *Supernatant refers to antibody-depleted PAS-antigen conjugates. tR@constituted to the original volume
200,000 o-100 200,000 antiserum of
the
0 .-~~
___--
OF PRECIPITATWO ANI) NONPRE(:IPI'~ATIS(: ANTIBODIES POLYAMINOSTPRENE-AKTIGES CONJUGATES
BDB TITER OF
USTREATED
//
USING
(ANTI-BSA) NOh-PRECIPITATING * /
5,000 100 200 obtained after
.
I)~A%~TI%EI~
ANTISERUM ,z:: 40,000
“00 its
40,000 incubation
with
antiserum.
could be eluted only with difficulty from t,he immunosorbent and then only tck a minimal extent (less than 5 per cent of the original titer). El&ion of gntibodies T/sing Varying- Concentrations of the dntise,rn.-To further ascertain the relationship between the hemagglutinating titer of tht> antiserum and the elutability of its antibodies from the immunosorbent, the following experiment was performed. The antisera were diluted with normal rabbit serum and different dilutions of the same antiserum were incubated with the homologous DPAS-Ag conjugates. As shown in Table V, incubation oi’ anti-OA, anti-HGG, and anti-BSA with the homologous immunosorbent, in dilutions greater than 4, 10 and 20, respectively, result,ed in marked decreases OF the quantities of antibodies subsequently elutcd from the specific immunost )I$-
TABLE
V.
EIJJTION
OF ANTIBODIES
USING
VARYING
CON~EXTRATIOKS
BDB TITER REAGENTS
1. Whole
T;SED
ANTI-BSA
antiserum
OF AmIs~Rrm
OF EI,ITTEI) I--
ANTI-0A
ANTIBODIES* -...-.-.--~__1 ANTI-HOC:
“00,000
2n0,ooo
100,000
2. Antibodies 4-f-01(1*
purified
from
antiserum
diluted
200,000
2on,ooo
50.000
3. Antibodies lo-fold”
purified
from
antiserum
diluted
200,000
8,000
.jO,OOO
4. Antibodies 20-fold*
purified
from
antiserum
diluted
100,000
0
10,01m
5. Antibodies 50.fold”
purified
from
antiserum
diluted
2,000
0
650
0 6. Antibodies purified from antiserum diluted loo-fold* *Reconstituted to the original volume of the antiserum. fAI1 dilutions made with normal rabbit serum.
0
0
148
CUA-LlM,
MCHTEIL,
ROSE
March--April.
.I. Allergy 1963
bent. When more dilute solutions of the antisera were used, no antibody could subsequently be eluted from 6he immunosorbcnt, notwithstanding the fact that the titer of the diluted antiserum was still considerable and that all the antibodies were absorbed onto the immunosorbent. Agglutination of Antibody-Sensitized Red Cells by Homologous Antigen.Precipitating antisera (anti-BSA) with BDB hemagglutination titers of 25,000 to 200,000 were used in these experiments. A pilot experiment to determine the concentration of the purified antibodies necessary to be incubated with the red cells in order to obtain optirnally sensitized cells was performed with each system. The proportions of the washed red cells and the BDB used were identical to those used in the regular BDB technique, except that the antibody was used in lieu of the antigen in the coupling procedure. Different quantibies of the purified antibodies were used: I.5 mg., 3.0 mg., 6.0 mg., and 12 mg. per 0.1 ml. of a 50 per cent washed red cell suspension. No agglutination was observed with the cells sensitized with 1.5 mg. of purified antibody. AggluCnation was observed with the red cells sensitized with 3.0 mg. or 12 mg. of purified antibody and the range of antigen concentration through which agglutination was observed was 0.0025 pg to 0.19 pg. With the red cells sensitized with 6.0 mg. of purified antibody, the range of antigen concentration in which agglutination was observed wa.s 0.0003 to 0.75 pg therefore, 6 mg. of antibody per (Table VI). In subsequent experiments, aliquot of sensitized cells was considered optimal. TABLE
VI.
AGOLTJTINATION AGGLUTINATION
(‘ONC. OF ANTIGEN (Bs-4) (/%/ML.) 200 100
50 25 12.5 6.2 3.1 1.5
ANTIGEN ONLY
OR ANTIBODY-~~ENSITIZEI) OF ANTIBODY-SENSlTIZED ANTIGEN PLUS ORIGINAL ANTI-BSA ANTISERUM
-
-
.-
-.
ANTIGEN PLUS SUPERNATANTt
HEIJ
CELLS’
RED CELL
ANTIGEN PLUS NRS
BY
IIOMOLOGOUS
CONJlJGATES
ANTIGEN
INCUBATED
ANTIGEN PLUS PURIFIED ANTI-BSA ANTIBODY
WITH
ANTIGEN PLUS ANTI-OA ANTISERUM
-
-
+ 0.38 + 0.19 + 0.00 4 0.045 0.022 r -+0.011 + 0.005 0.0025 + 0.0012 -+ 0.0006 + + 0.0003 0.0001 Legend: + signifies agglutination; *Red cells sensitized with purified tAntiserum depleted OP antibody antigen conjugate.
-
.-
O.i5
+ + + f -L + + + + -
* T A + i + i + + + + -
- signifles no agglutination. antibodies obtained from precipitating after incubation with homologous
-
T + + + T + + + + + + +
antiserum. polyaminostyrene-
REVERSED
Volume 34
Number
?
RDR
TECHNIQUE
I49
It is interesting to note (Table VI) that the quantity of antigen capabk of eliciting agglutination was exceedingly small. Higher concentration of the antigen inhibited the agglutination of the antibody-sensitized cells, thus prclsenting the classical picture of a “prozone” phenomenon. It is cvidcnt, that agglutination was specific since the reaction could be inhibited only by then original antiserum and the reconstituted purified ant,ibody while it was not inhibited by the antibody-depleted supernatant or heterologons antiscrnm 01. normal rabbit serum. Similar experiments were carried out with the OA-anti-CIA (Table 1~111 a,nd HOG-anti-HGG (Table VIII) systems. The results wrrc identical to thosch obtained for the BSA-anti-BRA system describetl ahovc. TABLE
~11.
AGGLUTINATION
OF ANTIBODY-SENSITIZED
AGGLUTINATION
CONC. OF ANTIGEN (‘JAI (P~/~~I~.)
ANTIGEN ONLY
RED CELLS*
OF ANTIBODY-SENSITIZED ANTIGEN PLUS ORIGINAL ANTI-OA ANTISERIJM
ANTIGEN PLUS SUPERNATANT+
-
-
100
-
-
-
50
-
-
25
-
-
6.2
-
-
3.1
-
200
RED CELL
ANTIGF,N PJ,US IL’RS
BY HOMOLOGOUS COR’JUGATES
ANTIGEP~
INCUBATED
ANTIGEN FLUS PURIFIED ANTI-OA ANTIBODY
WITII
ANTIGKX PLCR ANTI-BSA ANTISXRI’M -__... -~
12.5
-
1.5 O.iS
+
+
0.38
+
0.19
+
t
0.09
+
+
0.045
+
+
0.022
+
+
0.011
+
0.005 0.0025
+
+
1
t
+
l
4
+
+
+
+
+
+
+
+
+
-
0.0012 0.0006
\ --
0.0003
-
0.00015
-
0.00008 Legend: + signifies *Red cells sensitized 1Antiserum depleted
._ -
-
~ .~_ agglutination : - signifies no agglutination. with puriAed antibodies obtained from precipitating antiserum. of antibody after incubation with homologous PAS-antigen conjugates.
150
CUA-LIM,
TABLE VIII.
AGGLUTINATION
RICHTER,
OF ANTIBODY-SENSITIZED
lAGGLUTlNATION
ANTIGEN ONLY
RED
OF ANTIBODY-SENSITIZED ANTIGEN PLUS ORIGINAL ANTI-HGG ANTISERUM
CONC. OF ANTIGEN
ROSE
March-April,
CELLS*
BY HOMOLOGOUS
RED CELL CONJUGATES
ANTIGEN PLUS SUPERNATANT?
ANTIGEN PLUS NRS
J. Allergy 1963
ANTIGEN
INCUBATED
ANTIGEN PLUS PURIFIED ANTI-HGG ANTIBODY
WITH
ANTIGEN PLUS ANTI-BSA ANTISERUM
500
-
-
-
-
-
250 125
-
-
-
-
-
-
-
-
62.5 31.2
-
-
-
-
-
-
-
15.6
-
-
7.8
-
-
3.9
-
-
-
1.8
f
-
t
0.9
+
-
0.45
+
-
0.225 0.112
-
-
-
t
-
t
f
+
-
t
+
t
-
t
t
t
+
+
+
t
-
+
-
+ f f
+
+
+
0.028
+
-
+
t
0.014
+
+
t
-
0.007
+
f
+
-
0.0035
+
-
+
+
-
t t
0.0017
+
-
+
+
-
+
0.0008
+
-
+
+
-
+
0.0004
+ -
-
t -
+ -
-
+
-
-
-
-
0.0001
Legend: + signifies agglutination: *Red cells sensitized with purified iAntiserum depleted of antibody
conjugates.
IX.
DEMONSTRATION
OF ANTIBODIES
AFTER FRACTIONATION CENT SATURATION)
REAGENTS
1. Original
precipitating
2. Supernatant
after
WITH
antiserum.
polyaminostyrene-
A~~MONIUM
SULFATE
BDB TITER
200,000
antiserum ammonium
-
- signifles no agglutination. antibodies obtained from precipitating after incubation with homologous
(40 PER
ing
-
-
0.0002
TABLE
-
+
0.056
antigen
-
sulfate
precipitation
of precipitat-
200
antiserum
3. Lyophilized precipitate tating antibody)
reconstituted
to original
volume
(precipi-
200,000
Volume 34 Nllmber 2
REVERSED
RDR
TECHNIQUE
1 r,l
Demonstration of Antibodies After Fractionation With Ammonium Sulfate. -Precipitating and nonprecipitating antisera were fractionated with ammonium sulfate (40 per cent saturation). The quantities of the antibodies remaining in the supernatants after precipitation were insignificant. The HDR titers of the reconstituted lyophilized ammonium sulfate precipitates were identical to those of the original antisera. These results are in accorfl with those obtained by Richter and associates” and are shown in Table IX. Agglutination of Red Cells Xensitized With &mma Globulin I’reycl&iorrs of Precipitating Antiserum.-Varying quantities of immune gamma globulin obtained aft,er ammonium sulfate fractionation of strongly precipitating antiscra (rabbit) were used in the coupling procedure. As a control for this esl)eriment, purified antibodies were also coupled to red cells. As shown in Table .X. none of the red cells sensitized with the antibody-containing gamma globulin preparations gave positive reactions, regardless of t,he antigen dilution usc(l. The control red cells, which were sensit.izcd with purified antibody, were agglutinatcd by the homologous antigen.
TABLE
X.
AGGLUTINATION
OF RED CELLS SENSITIZED WITH OF PRECIPITATING ANTISERUM
AMOIJNT OF GAMMA GLOBULIN INCUBATED WITH RED CELLS (MG.)
GAMMA
GLOBULIN
AGGLUTINATION HOMOLOGOUS
1.5
negative
3.0
ncgativc
6.0
negative
12.0
negative
18.0
nes2ative
PREPBRATIONS
WITH ANTIGEN
In order to ascertain unequivocally that the gamma globulin preparations were coupled onto the red cells, these cells were subsequently incubated with varying dilutions of goat antirabbit gamma globulin (RGG) serum. Marked agglutination of these cells was observed (Table XI).
TABLE
XI.
AGGLUTINATION OF RED CELLS SENSITIZED PREPARATIONS BY GOAT ANTIRABBIT
CONCENTRATION
OF GOAT
ANTI-RGG
I
WITH GAMMA
RABBIT-JMMUNE GAMMA GLOBULIN SERUM AGGWJTINATION
1:lO 1:20 1:40 1:80 1:160 1:320 1:640
+ + + + + + -
1:129A
-
BLOBUI~IN
152
CUAZ-LIM,
RlCHTEII,
ROSE
March--April,
J. Allergy 1963
DISCUSSION
The aim of this study was twofold. The primary objective was to evolve a system whereby it could be demonstrated whether the precipitating and nonprecipitating antibodies which can be det,ected in the same animal at varying intervals of time subsequent to immunization are directed against the identical antigenic sites of the antigen system injected (BSA, HGG, or OA). It was felt that the ultimate experiment to achieve this aim would be the inhibition of agglutinat,ion of antibody-sensitized red cells (precipitating antibodies) by the respective antigen by prior incubation of the antigen with nonprecipitating antiserum obtained from the same animal. I3 However, before such an experiment could be undertaken, it was necessary to determine whether antibodysensitized red cells could indeed be prepared, since it has not been reported previously. Sehon14 claimed to have used the “reversed” hemagglutination test to detect very small quantities of antigen, but no publication followed subsequent to this statement. The initial problem was to determine the optimal technique for the isolation of antibodies in pure form. The method described by Gyenes and Sehons* I5 using antigen-diazopolyaminostyrene conjugates, appeared to be the most suitable. Recently, it has been reported from our laboratory that nondiazotized polyaminostyrene was capable of binding antigen more efficiently t’han the diazonium derivative9 and that such antigen-polystyrene (nondiazotized) conjugates would specifically take up the homologous antibody from so1ution.‘G This finding was corroborated in this study using BSA, HGG, and OA as antigens. However, it is of more than academic interest to note t,hat while antibodies could be easily eluted from the diazotized polyaminostyrene-antigenantibody complex, they could not be eluted from the nondiazot.ized polyaminostyrene-antigen-antibody complex. The reason for this finding is not obvious and has not been investigated further. Undoubtedly, the diazotization of the polyaminostyrene affects the resin in a manner totally unrelated to the known conversion of amino groups to diazonium groups. The purified antibodies were furthermore shown t,o migrate as a pure gamma globulin band, both by paper and starch gel electrophoresis, and to sediment at the same rate as gamma globulin. Since only small quantities of protein were recovered as the eluted antibody preparation and since the latter possessed essentially all the ant,ibodies present in the original antiserum, it may be assumed that the preparation consists of only antibody molecules, although no attempt was made to determine the percentage of antibody in the eluates. It may be that some contamination with nonantibody gamma globulin may have occurred during the purification procedure. It was shown that the recovery of antibodies from the immunosorbknt is directly proportional to the hemagglutinating titer of the original antiserum. Antibodies from several strongly precipitating antisera, but with low BDB hemagglutinating titers of 640 and 1,280, could not be eluted; on the other hand, antibodies from several nonprecipitating antisera with high hemagglutinating titers of 20,000 t,o 50,000 were capable of being eluted. Thus, it appears
that a minimum hemagglutinating titer is required in order that antibodies can be elutcd from a particular antiserurn. This finding may explain why previous attempts to elute antibodies from allergic sera gave insignificant yields.‘” since these antisera usually possessed titers lower than 10,000. The findings that “antibody-sensitized ret1 cells, ” prepared by coupling Imrifietl antibody to red blood cells by means of rSI)R, can he agglutinated by the respective antigen, attests to the innocuous nature of the coupling procedurc~ although some denaturation of the antibody may have taken place. The antibody-reactive sites on the antibody molcc~tlc arc not affected. It is intcresting to not,c t,hat even the maximum amount of antigen capable of eliciting agglutination was exceedingly srnall (BSA-0.73 pg/ml., HGG-1.8 ~~g./ml. ; OA-1.5 pg’ml.). Higher concentrations of the homologous antigen prercntctI the agglutination of the antibody-sensitized cells, thus presenting the classical ‘ ’ prozon e ’ ’ phenomenon. I’urthermorc, as little as 0.0003 to 0.0004 ,q per nlilliliter of the antigen could agglutinate the homologous antibody-sensitized ~11s. It was also established that the agglutination of the ant.ibody-scIusitizcci ~11s by the homologous antigen is specific in that it coultl he inhibitctl l)y t h(h original antiserum or the eluted, purified antibody preparation but, not by the supcrnatant (antibody-depleted antiserum ), other hctcrologous antisera, 01’ normal rabbit serum. Sehon” stated that the amount of antigen which still 1~~1 to the aggregation of antibody-sensit,ized red cells was of the order of lo-’ pg of the antigen. Hc did not state whcthcr, in his experiments, purified antibod,v or the gamma globulin fraction of rabbit antiscrurn was usctl. In our esl~ri mcnts, it was shown that the red cells coated with antibotly-rich gamma glol)ulin did not agglutinate with the homologous antigen. However, these red eelIs were agglutinated by goat antirabbit gamma globulin serum, t,hus drmon~trating that gamma globulin exhibiting it,s original antigenic specificity had been coupled to these red cells. Red cells to which purified antibody was CWU~IWI gave- a positive agglutination reaction. This finding suggests that the ant,ibod~ content of the gamma globulin fraction obtained from strongly precipitating rabbit, antiserum is insufficient to produce optimally sensitized red cells. This stat,e of affairs is due probably to competition between ant,ibody gamma globnlin molecules and the much greater number of non-antibody gamma globulin molecules for sites on the red cell.
1. The use of immunosorbent-antigen conjugates for the isolation and puri fication of antibodies has been discussed. 2. Both the diazotized as well as the nondiazotized polpaminostyrene-antigen conjugates are capable of taking up the specific antibody; however, antibody can be eluted only from the former, and not, from the latter, immunosorbent. 3. The degree of recovery of the antibodies from an antiserum is directly IVlated to the hemagglutinating titer of the ant,iscrum. Only ant,isera with hamagglutinating titers of over 20,000 can be used for antibody elution experiments. 4. The method of using antibody-sensitized cells for the detection of antigen
ih
Mad-April,
1963
is described. The agglutination of antibody-sensitized red cells by the homologous antigen is specific as it could only be inhibited by the original antiserum and the eluted antibody but not by the antibody-depleted supernatant or heterologous antiserum. 5. Red blood cells conjugated with gamma globulin preparations of these antisera failed to be agglutinated b&ythe homologous antigen. REFERENCES
1. Pressman, D., Campbell, D. H., and Pauling, L.: The Agglutination of Intact AZOErythrocytes by Antisera Homologous to the Attached Groups, J. Immunol. 44: 101, 1942. 2. Stavitsky, A. B., and Arquilla, E. R.: Micromethods For the Study of Proteins and Antibodies. III. Procedure and Application of Hemagglutination and Hemagglutination-Inhibition Reactions With Bis-Diazotized Benzidine and Protein-Conjugated Red Blood Cells, J. Immunol. 74: 306, 1955. 3. Gordon, J., Rose,, B., and Sehon, A. H.: Detection of “Non-Precipitating” Antibodies in Sera of Indrviduals to Ragweed Pollen by an In Vitro Method, J. Exper. Med. 108: 37, 1958. 4. Boyden, S. V.: The Absorption of Proteins on Erythrocytes Treated With Tannic Acid and Subsequent Hemagglutination by Antiprotein Sera, J. Exper. Med. 93: 107, 1951. 5. Sherwin, A. L:, Richter, M., Cosgrove, J. B. R., and Rose, B.: Studies of the BloodCerebrosprnal Fluid Barrier to Antibodies and Ot,her Proteins, J. Neurology. (Submitted for publication.) 6. Arbesman, C. E., Rose, N. R., Kantor, S. Z., and Beede, R. B.: Immunologic Studies of Ragweed-Sensitive Patients. I. Specificity and Sensit.ivity of Hemagglutination Reactions, J. ALLERGY 31: 317, 1960. 7. Richter, M., Cua-Lim, F., and Rose, B.: The Agglutination of Antibody-Coated Red Blood Cells by Antigen, Biochem. & Biophys. Res. Commun. 7: 241, 1962. 8. Gyenes, L., and Sehon, A. H.: Preparation and Evaluation of Polystyrene-Antigen Conjugates for the Isolation of Antibodies, Canad. J. Biochem. & Physiol. 38: 1235,
1960. 9. Richter,
M.,
Delorme, P., Grant, S., and Rose, B.: Studies on the Uptake of Ragweed Allergens by Polyaminostyrene, Canad. J. Biochem. & Physiol. 40: 471, 1962. Richter, M., and Haurowitz, F.: Continuous Synthesis of Antibody After Primary Immunization With Protein Antigens, J. Immunol. 84: 420, 1960. Richter, M.! Blumer, H., Cua-Lim, F., and Rose, B.: Physiochemical Studies on Precipitatrng and Non-Precipitating Antibodies After Primary Immunization, Canad. J. Biochem. & Physiol. 40: 105, 1962. and Non-Precipitating Blumer, H., Richter, M., Cua-Lim, F., and Rose, B.: Precipitating Antibodies in the Primary and Secondary Immune Responses, J. Immunol. 88: 669, 1962. Richter, M., Cua-Lim, F.! and Rose, B.: The Demonstration of Identical Immunological Specificity of Preclprtating and Non-Precipitating Antibodies. Internat. Arch. Allergy. Pollen
10. 11. 12. 13.
14. Seho,‘l~.p~s’) .: In. Mechanisms of Hypersensitivity, edited by J. H. Shaffer, C. A. Lo drippo, and M. W. Chase, Toronto, Canada, 1959, Little, Brown, & Company, p. 118. 15. Gyenes, L., and Sehon, A. H.: The Use of Polystyrene-Allergen Conjugates for the Removal of Antibodies From Sera of Allergic Individuals, Canad. J. Biochem. & Physiol. 38: 1249, 1960. Studies on the Uptake of Reagin by 16. Delorme, P., Richter, M., Grant, S., and Rose, B.: Polyaminostyrene-Ragweed Complexes, Canad. J. Biochem. & Physiol. 40: 519, 1962.
REVERSED
BDB TECHNIQUE
The Agglutination of Antibody-Coated Homologous Antigen Cua-Lim,”
F.
M. Richter,
and B. Rose,
Red Blood Cells by Quebec, Camada
Montreal,
1942, Pressman and his associates1 described a new technique for the detection immunological reactions between soluble antigens and antibodies which has proved to be far more sensitive than the conventional interfacial ring and precipitin tests. This method involved the chemical coupling of the protein antigen to red blood cells by means of bisdiazotized benzidine (BDB) and the subsequent agglutination of such “antigen-sensitized cells” by the homologous antiserum (BDB technique). This technique was later modified by Stavitsky and Arquilla* and further developed by Gordon and co-workers.3 A technique utilizing the absorption of antigen to tannic acid-treated red blood has been described by Boyden (tanned-cell technique). Using both of these techniques, it has been possible to demonstrate, unequivocally, the presence of small quantities of precipitating antibodies in solution3, 5 and the detection of antibodies in the sera of allergic individuals.“, fi However, the preparation of “antibodysensitized red blood cells” for the detection of small quantities of antigen has not as yet been reported (reversed BDB technique). In a previous communication7 a brief description of a procedure for the coupling of antibodies to red cells which are subsequently capable of being agglutinated by the specific antigen was presented. The present paper further elaborates on the reversed BDB technique.
I
N
of
MATERIALS
AND
METHODS
1. Preparution of Antisera.-Rabbits, weighing 3 to 5 kilograms, were used throughout. The antigens used were (a) BSA (bovine serum albumin), t (b) OA (egg albumin),$ and (c) HGG (human gamma globulin Fr. II).f All injections of antigens were made intravenously through the marginal ear veins of the rabbits. The dose of antigen varied from 10 to 50 mg. per injection. The animals were given only 2 to 3 injections of antigen at intervals of 2 to 3 days. From the Divieion of Immunochemistry and Allergy, McGill University Hospital. Montreal, Quebec, Canada. This investigation was supported by grants from the Department of Welfare, Ottawa, Canada, and the National Institutes of Health, Institute fectious Diseases, Bethesda, Md., U. S. A. (Grant No. E-1322). Received for publication Nov. 2, 1962. *Submitted in partial fulfillment for the degree of Master of Science, perimental Medicine, McGill University, Montreal. Quebec. Canada. TPentex Inc., Kankakee, Illinois. *Nutritional Biochemical Co., Cleveland, Ohio. toria
142
Clinic,
Royal
Vic-
National Health and of Allergy and InDepartment
of
Ex-
Volume
Number
34 ?
REVERSED
BDB
TECHNIQUE
143
These animals are considered to have produced a primary response. In order to elicit a secondary response, some of the rabbits were re-injected several months after the initial ibmunization with a single dose of 20 to 50 mg. of the homologous antigen. The animals which were tertiarily immunized months after the secondary response are referred to as having been hyperimmunizcld. The animals were bled from the marginal ear veins at appropriate intervals of time after antigen injection. The blood was allowed to clot at room temperature and placed in the cold room at 4’ C. for 21 hours. The serum was thcII separated from the clot by centrifugation and stored at -20’ until used. 2. Immunologic Methods.-(A) Precipitin test: Precipitating antibodies were detected by the interfacial ring test. Antigen solutions (0.05-0.5 pot cent) were layered over undiluted sera in small culture tubes and the intcrfacc was observed over a period of 60 minutes. The appearance of a white ring at the interface was considered indicative of the presence of precipitating antibodies if appropriate control tubes were negative. B. Detection of antibody by the BDB technique: The bisdiazotized benzidine (BDB) technique was used as modified by (Gordon and his co-workers.Z These antibodies are hereafter referred to as hemagglutinat,ing antibodies. The titers of the antisera are expressed as the reciprocals of the highest dilutions of the antisera which gave positive agglutination patt,erns of antigen-coated red blood cells. As a control, an antiserum of known titer was always titrated along with the sera under investigation. Sera with BDB titers lower than 10 were considenbd negative. C. Purification of antibodies: The protein antigens were coupled to diazotized8 as well as to nondiazotizedg polyaminostyrene.* Purificabion and isolation of antibodies was performed by incubating the antisera with the respective insoluble immunosorbent-antigen conjugates* for 30 minutes at room temperature with constant stirring. Each suspension was then centrifuged and the antibodydepleted supernatant (referred to as “supernatant” in the text) was dialyzed overnight against saline at 4’ C. and stored at -20° C. The antibodies were isolated by incubation of the antibody-antigen-polystyrene conjugates at room temperature at pH 3.0 for 30 minutes, which resulted in the liberation ot’ antibodies into the solution. The pH of the incubated suspension was adjusted to 3.0 by the addition of O.lN HCl. The solution containing the dissociated antibodies was adjusted to pH 7.2 by the addition of phosphate buffer (pHY 7.2, ionic strength 0.1) and it was then dialyzed against distilled water overnight and lyophilized. This latter preparat,ion is referred to as “purified antibody” in the text. D. Preparation of antibody-sensitized red cells (Ab-rbc): The met,hod ascd was described briefly in a recent communication.7 The procedure followed is identical to that of the regular BDB technique3 except that antibody was used in lieu of the antigen in the coupling procedure. The optimal concentration of t.he antibody used in the coupling procedure was determined experimentally by t,rial and error. In general, it was found that 5 to 10 mg. of the lyophilized *Obtained
from
Norsk
Hydroelectric
Co., Oslo,
Norway.
144
CUA-LTM,
RTCHTER,
ROSE
J. Allerpy March-April,
I%3
purified antibody preparation in 3 ml. of saline was required for the optimal sensitization of each 0.1 ml. aliquot of rabbit red ccl,‘s. To prevent nonspecific agglutination, the purified antibody preparation was absorbed with washed, packed red cells (obtained from the same rabbit) for 15 to 30 minutes at 4’ C. in order to remove any blood group antibodies. The absorbed antibody solution was then incubated with the red cells and BDB to effect the chemical coupling. Gamma globulin preparations, obtained by ammonium sulfate fractionation of the precipit,ating antisera, werr coupled to red cells in the same manner as described above. Halving dilutions* of the antigen were prepared in twenty-eight tubes. For the BSA and OA systems, the first tube contained 200 pg per milliliter; for t,he HGG system, the first tube contained 500 pg per milliliter. E. Inhibition experiments: In these experiments, the antigen, in halving dilution, was incubated, in separate experiments, with constant, amounts of each of the following: (1) the original precipitating antiserum, (2) the purified antibody reconstituted to original volume, (3) the supernatant obtained after incubation of the antiserum with the PAS-antigen conjugates (absorbed antiserum), (4) a hcterologous antiserum, and (5) a normal rabbit serum. The incubations were carried out at room temperature for 30 minutes, following which t.he antibody-sensitized red cells (Ab-rbc) were added to all the tubes. Agglutination of the Ab-rbc by the homologous antigen was read after 16 to 20 hours. Antibody-sensitized red cells were found to be stable for at least 8 to 12 hours. 3. Ammonium Sulfa.te Fractionation.-Antisera were fractionated with ammonium sulfate (40 per cent saturation) and the resultant precipitate and supernatant were dialyzed against saline overnight and analyzed for antibody content by the conventional BDB method. 4. l,Wtmcentrifugation.-The ultracentrifugal analyses of the purified antibodies were performed using a Spinco Model E analytical ultracentrifuge. The average rotor temperature was 20.5’ (1. The sedimentation constants were not recalculated for standard conditions. 5. Electrophoresis.-The purified antibodies were subjected to paper electrophoresis using Verona1 buffer pH 8.6, ionic strength 0.05. EXPERIMENTS
AiVI
RESULTS
Determination of Antigen Uptake by Polyaminostyrene (PAS) and Diaxotized Polyaminostyrene (DPA.S).-Varying concentrations of the antigen (Ag) were incubated with constant amounts of polyaminostyrene (PAS) at room temperature (25’ (1.) for 30 minutes to an hour. Untreated, as well as diazotized, PAS was used. The supernata,nts obtained after centrifugation of the incubated mixtures were tested for the presence of unabsorbed antigen. This latter was determined by its ability to inhibit the agglutination of antigcnsensitized cells by the specific antiserum. -:;;t.in
*The technique whereby each consecutive tube
the concptration of a nerves of
of tubes
antigen cm antibody IS referred to as
is diluted by a factor “halving dilutions” in
of the
Volume 11 Number 2
REVERSED
BDR
TECHNIQUE
1 -I.-,
As can be seen in Table 1, the uptake of Ag by the immunosorbent ill)peared to be governed by the concentration of antigen with which the PAS had been incubated. Using a low concentration of antigen (1.5 mg./ml.). OYPI 90 per cent of it was absorbed, whereas at relatively high Ag concent,rat,ion (25 mg./ml.) about 75 per cent of it was a,bsorbed. The absorption of Ag 1)~ PAS and DPAS appeared to be identical. These result,s are similar to thosts reported previously from this laboratory.” TABLE
I.
AMOUNT OF ANTIGEN INCCBATED (BSA) (MG./ML.)
ADSORPTION
OF ANTIGEN
TOTAL AMOUNT OF PAS INCljBATED ( MG.)
A.
Entwated 50 zi
25 12.5 6.2 3.1 1.5
HY POLYAMINOSTPRENE
QT'ANTITY OF BSA" StTPERNATANT (MO/ML.)
B. Diazotized z:: 50 50 50
3.1
1.5 *Iktermined antiserum.
by
inhibition
of
QUANTITY OF BSA TAKEN CI' SY PAS
(nm )
Polynminostywnr 6.0 3.0 0.75 0.375 0.092
19 9.5 5.45 2.725 1.408
Polyaminosty~cv~ 3.0 1.5 0.75 0.375 0.092
2’ 11.0 5.45 2.725 1.408
50 50
25 12.5 6.2
IX
agglutination
of
RSA-sensitized
red
cells
by
the
_--._ Hperific
Purifica.tion of Antibodies Using Diaxotized Polyaminostyrene-Antigen (‘[email protected] different precipitating antisera-anti-BSA, anti-0A, anti-I!lGC: obtained from hyperimmunized rabbits, were used in these experiments. Only the antisera which gave positive ring tests and high BDR hernagglut,ination titers were used. As can be seen in Table IT, almost 100 per cent of the anti-BSA antibodicls were recovered in the cluatc obtained from the AR-MA-SPAS conjugatc~s. TABLE
II.
PL~RIFICATION
OF ANTIBODIES
USING I~IAZOTIZEI) CONJI'CATES
POI.YAMINOSTYKE~-E-ANTIOF:S _ .~~~-~
REAGRNTS 1. Original
I
autiserum
a. Anti-BRA b. Anti-OA c. Anti-HGG 2. Purified antibodies reconstituted to original volumr~ a. Anti-BSA h. Anti-OA c. Anti-HGG 3. Supcmatant of antiserum incubated with PAS-Ag conjugates a. Anti-BSA b. Anti-OA c. Anti-HGG 4. Last
BDB TITER
washing
a. Anti-BRA 1,. Anti-OA c. Anti-HGG
of
PAS-antigen-antihody
200,000 100,000-200,rIoo 100,000-200,000 100,000-200,000 25,00050,000 100,000 100 100-200 1 nn-2on
complexes
0 0 0
146
CUA-MM,
RICHTER,
ROSE
March-April,
J. Allergy 1963
Similarly, from the results obtained with anti-OA antisera, we can conclude that at least 25 to 50 per cent of the antibodies incubated with the immunosorbent could be recovered in the eluates obtained from the Ab-OA-DPAS conjugates. The titer of the antibodies remaining in the supernatant which was obtained after the incubation of the specific antiserum with the homologous AgDPAS complex is insignificant. Only 0.1 to 0.4 per cent of the antibodies were not absorbed by the immunosorbent. The method of purification appears to be the most specific and reliable of all the purification methods available for the isolation of antibody protein. The purified ant,ibodies gave a single band by paper electrophoresis and migrated at a rate identical to that of gamma globulin. The ultracentrifugal analysis of the material revealed a single peak with a sedimentation constant of 7.14 Svedberg units, which corresponds to t,hat of the normal gamma globulin. Elution of Precipitating A.ntibodies Using Untreated PolyaminostyreneAntigen Conjugates.-It is interesting to note that while antibodies could be absorbed onto and subsequently eluted from DPAS-Ag conjugates at pH 3.0, they could only be absorbed onto, but not eluted from, PAS-Ag conjugates (Table III). The reason for this finding is not clear. However, the failure to detect ant.ibody in the eluates obtained from Ab-Ag-PAS conjugates cannot be attributed to the elution of soluble Ab-Ag complexes at pH 3.0, since soluble Ag could at no time be detected in ?%e supernatants obtained from saline suspensions of Ag-PAS conjugates (kept at pH 3.0 for up to 2 hours). Therefore, it is unlikely that Ag would be elubed from the Ag-PAS conjugate in the presence of Ab. El&ion of Precipitating and Nonprecipitating Antibodies Using Diazotized Polyaminostyrene-Antigen Conjugates.-In order to determine whether the hemagglutinating titer of the antiserum is of prime importance in the elution of antibodies, the following antisera* (anti-BSA) were analyzed (Table IV). I. Precipitating antisera obtained within 30 days after the last injection of the primary immunization. These are referred to as “early” antisera.l@l* II. Hyperimmune precipitating antisera, obtained 5 to 15 days after tertiary immunization. A. Nonprecipitating antisera obtained 90 to 120 days after the last injection of primary immunization. These are referred to as ‘ ‘ late ” antisera.10-12 B. Nonprecipitating antisera obtained 4 to 6 months after primary immunization.10-12 From t,he resu1t.s in Table IV, it appeared that the precipitability of an antibody is not the main criterion in considering its ability to be subsequently eluted from the immunosorbent. The antibodies in the antisera of high hemagglutinating titer were capable of being eluted regardless of whether they originated in precipitating or nonprecipitating antisera. Antibodies in precipitating and nonprecipitat,ing antisera having low BDB titers (< 10,000) tive
*Several sera are
antisera presented
of each clsssiflcation in Table IV.
(I,
II,
A,
B)
were
analyzed
but
only
representa-
R’EVERSED TABLE
III.
BDB
TECHNIQUE
ELUTION OF PRECIPITATING ANTIBODIES NONDIAZOTIZED POLYAMINOSTYRENE-ANTIGEN
(ANTI-BSA) USING COKJUGATES
REAGENTS 1. Original precipitating antiserum 2. Antibody-depleted supernatant of precipitating antiserum incubation with PAS-antigen conjugates 3. Eluted antibody obtained from precipitating antiserum” *Reconstituted to the original volume of the antiserum.
TABLE
IV.
ELUTION
BDB ---.._~ TITER 100,000-200,000
after
O-100
SERA TESTED ANTISERCM
PRECIPITATING I
II
I
Original antiserum 1,280 Hupernatant” 0 Eluted antibody+ 0 *Supernatant refers to antibody-depleted PAS-antigen conjugates. tR@constituted to the original volume
200,000 o-100 200,000 antiserum of
the
0 .-~~
___--
OF PRECIPITATWO ANI) NONPRE(:IPI'~ATIS(: ANTIBODIES POLYAMINOSTPRENE-AKTIGES CONJUGATES
BDB TITER OF
USTREATED
//
USING
(ANTI-BSA) NOh-PRECIPITATING * /
5,000 100 200 obtained after
.
I)~A%~TI%EI~
ANTISERUM ,z:: 40,000
“00 its
40,000 incubation
with
antiserum.
could be eluted only with difficulty from t,he immunosorbent and then only tck a minimal extent (less than 5 per cent of the original titer). El&ion of gntibodies T/sing Varying- Concentrations of the dntise,rn.-To further ascertain the relationship between the hemagglutinating titer of tht> antiserum and the elutability of its antibodies from the immunosorbent, the following experiment was performed. The antisera were diluted with normal rabbit serum and different dilutions of the same antiserum were incubated with the homologous DPAS-Ag conjugates. As shown in Table V, incubation oi’ anti-OA, anti-HGG, and anti-BSA with the homologous immunosorbent, in dilutions greater than 4, 10 and 20, respectively, result,ed in marked decreases OF the quantities of antibodies subsequently elutcd from the specific immunost )I$-
TABLE
V.
EIJJTION
OF ANTIBODIES
USING
VARYING
CON~EXTRATIOKS
BDB TITER REAGENTS
1. Whole
T;SED
ANTI-BSA
antiserum
OF AmIs~Rrm
OF EI,ITTEI) I--
ANTI-0A
ANTIBODIES* -...-.-.--~__1 ANTI-HOC:
“00,000
2n0,ooo
100,000
2. Antibodies 4-f-01(1*
purified
from
antiserum
diluted
200,000
2on,ooo
50.000
3. Antibodies lo-fold”
purified
from
antiserum
diluted
200,000
8,000
.jO,OOO
4. Antibodies 20-fold*
purified
from
antiserum
diluted
100,000
0
10,01m
5. Antibodies 50.fold”
purified
from
antiserum
diluted
2,000
0
650
0 6. Antibodies purified from antiserum diluted loo-fold* *Reconstituted to the original volume of the antiserum. fAI1 dilutions made with normal rabbit serum.
0
0
148
CUA-LlM,
MCHTEIL,
ROSE
March--April.
.I. Allergy 1963
bent. When more dilute solutions of the antisera were used, no antibody could subsequently be eluted from 6he immunosorbcnt, notwithstanding the fact that the titer of the diluted antiserum was still considerable and that all the antibodies were absorbed onto the immunosorbent. Agglutination of Antibody-Sensitized Red Cells by Homologous Antigen.Precipitating antisera (anti-BSA) with BDB hemagglutination titers of 25,000 to 200,000 were used in these experiments. A pilot experiment to determine the concentration of the purified antibodies necessary to be incubated with the red cells in order to obtain optirnally sensitized cells was performed with each system. The proportions of the washed red cells and the BDB used were identical to those used in the regular BDB technique, except that the antibody was used in lieu of the antigen in the coupling procedure. Different quantibies of the purified antibodies were used: I.5 mg., 3.0 mg., 6.0 mg., and 12 mg. per 0.1 ml. of a 50 per cent washed red cell suspension. No agglutination was observed with the cells sensitized with 1.5 mg. of purified antibody. AggluCnation was observed with the red cells sensitized with 3.0 mg. or 12 mg. of purified antibody and the range of antigen concentration through which agglutination was observed was 0.0025 pg to 0.19 pg. With the red cells sensitized with 6.0 mg. of purified antibody, the range of antigen concentration in which agglutination was observed wa.s 0.0003 to 0.75 pg therefore, 6 mg. of antibody per (Table VI). In subsequent experiments, aliquot of sensitized cells was considered optimal. TABLE
VI.
AGOLTJTINATION AGGLUTINATION
(‘ONC. OF ANTIGEN (Bs-4) (/%/ML.) 200 100
50 25 12.5 6.2 3.1 1.5
ANTIGEN ONLY
OR ANTIBODY-~~ENSITIZEI) OF ANTIBODY-SENSlTIZED ANTIGEN PLUS ORIGINAL ANTI-BSA ANTISERUM
-
-
.-
-.
ANTIGEN PLUS SUPERNATANTt
HEIJ
CELLS’
RED CELL
ANTIGEN PLUS NRS
BY
IIOMOLOGOUS
CONJlJGATES
ANTIGEN
INCUBATED
ANTIGEN PLUS PURIFIED ANTI-BSA ANTIBODY
WITH
ANTIGEN PLUS ANTI-OA ANTISERUM
-
-
+ 0.38 + 0.19 + 0.00 4 0.045 0.022 r -+0.011 + 0.005 0.0025 + 0.0012 -+ 0.0006 + + 0.0003 0.0001 Legend: + signifies agglutination; *Red cells sensitized with purified tAntiserum depleted OP antibody antigen conjugate.
-
.-
O.i5
+ + + f -L + + + + -
* T A + i + i + + + + -
- signifles no agglutination. antibodies obtained from precipitating after incubation with homologous
-
T + + + T + + + + + + +
antiserum. polyaminostyrene-
REVERSED
Volume 34
Number
?
RDR
TECHNIQUE
I49
It is interesting to note (Table VI) that the quantity of antigen capabk of eliciting agglutination was exceedingly small. Higher concentration of the antigen inhibited the agglutination of the antibody-sensitized cells, thus prclsenting the classical picture of a “prozone” phenomenon. It is cvidcnt, that agglutination was specific since the reaction could be inhibited only by then original antiserum and the reconstituted purified ant,ibody while it was not inhibited by the antibody-depleted supernatant or heterologons antiscrnm 01. normal rabbit serum. Similar experiments were carried out with the OA-anti-CIA (Table 1~111 a,nd HOG-anti-HGG (Table VIII) systems. The results wrrc identical to thosch obtained for the BSA-anti-BRA system describetl ahovc. TABLE
~11.
AGGLUTINATION
OF ANTIBODY-SENSITIZED
AGGLUTINATION
CONC. OF ANTIGEN (‘JAI (P~/~~I~.)
ANTIGEN ONLY
RED CELLS*
OF ANTIBODY-SENSITIZED ANTIGEN PLUS ORIGINAL ANTI-OA ANTISERIJM
ANTIGEN PLUS SUPERNATANT+
-
-
100
-
-
-
50
-
-
25
-
-
6.2
-
-
3.1
-
200
RED CELL
ANTIGF,N PJ,US IL’RS
BY HOMOLOGOUS COR’JUGATES
ANTIGEP~
INCUBATED
ANTIGEN FLUS PURIFIED ANTI-OA ANTIBODY
WITII
ANTIGKX PLCR ANTI-BSA ANTISXRI’M -__... -~
12.5
-
1.5 O.iS
+
+
0.38
+
0.19
+
t
0.09
+
+
0.045
+
+
0.022
+
+
0.011
+
0.005 0.0025
+
+
1
t
+
l
4
+
+
+
+
+
+
+
+
+
-
0.0012 0.0006
\ --
0.0003
-
0.00015
-
0.00008 Legend: + signifies *Red cells sensitized 1Antiserum depleted
._ -
-
~ .~_ agglutination : - signifies no agglutination. with puriAed antibodies obtained from precipitating antiserum. of antibody after incubation with homologous PAS-antigen conjugates.
150
CUA-LIM,
TABLE VIII.
AGGLUTINATION
RICHTER,
OF ANTIBODY-SENSITIZED
lAGGLUTlNATION
ANTIGEN ONLY
RED
OF ANTIBODY-SENSITIZED ANTIGEN PLUS ORIGINAL ANTI-HGG ANTISERUM
CONC. OF ANTIGEN
ROSE
March-April,
CELLS*
BY HOMOLOGOUS
RED CELL CONJUGATES
ANTIGEN PLUS SUPERNATANT?
ANTIGEN PLUS NRS
J. Allergy 1963
ANTIGEN
INCUBATED
ANTIGEN PLUS PURIFIED ANTI-HGG ANTIBODY
WITH
ANTIGEN PLUS ANTI-BSA ANTISERUM
500
-
-
-
-
-
250 125
-
-
-
-
-
-
-
-
62.5 31.2
-
-
-
-
-
-
-
15.6
-
-
7.8
-
-
3.9
-
-
-
1.8
f
-
t
0.9
+
-
0.45
+
-
0.225 0.112
-
-
-
t
-
t
f
+
-
t
+
t
-
t
t
t
+
+
+
t
-
+
-
+ f f
+
+
+
0.028
+
-
+
t
0.014
+
+
t
-
0.007
+
f
+
-
0.0035
+
-
+
+
-
t t
0.0017
+
-
+
+
-
+
0.0008
+
-
+
+
-
+
0.0004
+ -
-
t -
+ -
-
+
-
-
-
-
0.0001
Legend: + signifies agglutination: *Red cells sensitized with purified iAntiserum depleted of antibody
conjugates.
IX.
DEMONSTRATION
OF ANTIBODIES
AFTER FRACTIONATION CENT SATURATION)
REAGENTS
1. Original
precipitating
2. Supernatant
after
WITH
antiserum.
polyaminostyrene-
A~~MONIUM
SULFATE
BDB TITER
200,000
antiserum ammonium
-
- signifles no agglutination. antibodies obtained from precipitating after incubation with homologous
(40 PER
ing
-
-
0.0002
TABLE
-
+
0.056
antigen
-
sulfate
precipitation
of precipitat-
200
antiserum
3. Lyophilized precipitate tating antibody)
reconstituted
to original
volume
(precipi-
200,000
Volume 34 Nllmber 2
REVERSED
RDR
TECHNIQUE
1 r,l
Demonstration of Antibodies After Fractionation With Ammonium Sulfate. -Precipitating and nonprecipitating antisera were fractionated with ammonium sulfate (40 per cent saturation). The quantities of the antibodies remaining in the supernatants after precipitation were insignificant. The HDR titers of the reconstituted lyophilized ammonium sulfate precipitates were identical to those of the original antisera. These results are in accorfl with those obtained by Richter and associates” and are shown in Table IX. Agglutination of Red Cells Xensitized With &mma Globulin I’reycl&iorrs of Precipitating Antiserum.-Varying quantities of immune gamma globulin obtained aft,er ammonium sulfate fractionation of strongly precipitating antiscra (rabbit) were used in the coupling procedure. As a control for this esl)eriment, purified antibodies were also coupled to red cells. As shown in Table .X. none of the red cells sensitized with the antibody-containing gamma globulin preparations gave positive reactions, regardless of t,he antigen dilution usc(l. The control red cells, which were sensit.izcd with purified antibody, were agglutinatcd by the homologous antigen.
TABLE
X.
AGGLUTINATION
OF RED CELLS SENSITIZED WITH OF PRECIPITATING ANTISERUM
AMOIJNT OF GAMMA GLOBULIN INCUBATED WITH RED CELLS (MG.)
GAMMA
GLOBULIN
AGGLUTINATION HOMOLOGOUS
1.5
negative
3.0
ncgativc
6.0
negative
12.0
negative
18.0
nes2ative
PREPBRATIONS
WITH ANTIGEN
In order to ascertain unequivocally that the gamma globulin preparations were coupled onto the red cells, these cells were subsequently incubated with varying dilutions of goat antirabbit gamma globulin (RGG) serum. Marked agglutination of these cells was observed (Table XI).
TABLE
XI.
AGGLUTINATION OF RED CELLS SENSITIZED PREPARATIONS BY GOAT ANTIRABBIT
CONCENTRATION
OF GOAT
ANTI-RGG
I
WITH GAMMA
RABBIT-JMMUNE GAMMA GLOBULIN SERUM AGGWJTINATION
1:lO 1:20 1:40 1:80 1:160 1:320 1:640
+ + + + + + -
1:129A
-
BLOBUI~IN
152
CUAZ-LIM,
RlCHTEII,
ROSE
March--April,
J. Allergy 1963
DISCUSSION
The aim of this study was twofold. The primary objective was to evolve a system whereby it could be demonstrated whether the precipitating and nonprecipitating antibodies which can be det,ected in the same animal at varying intervals of time subsequent to immunization are directed against the identical antigenic sites of the antigen system injected (BSA, HGG, or OA). It was felt that the ultimate experiment to achieve this aim would be the inhibition of agglutinat,ion of antibody-sensitized red cells (precipitating antibodies) by the respective antigen by prior incubation of the antigen with nonprecipitating antiserum obtained from the same animal. I3 However, before such an experiment could be undertaken, it was necessary to determine whether antibodysensitized red cells could indeed be prepared, since it has not been reported previously. Sehon14 claimed to have used the “reversed” hemagglutination test to detect very small quantities of antigen, but no publication followed subsequent to this statement. The initial problem was to determine the optimal technique for the isolation of antibodies in pure form. The method described by Gyenes and Sehons* I5 using antigen-diazopolyaminostyrene conjugates, appeared to be the most suitable. Recently, it has been reported from our laboratory that nondiazotized polyaminostyrene was capable of binding antigen more efficiently t’han the diazonium derivative9 and that such antigen-polystyrene (nondiazotized) conjugates would specifically take up the homologous antibody from so1ution.‘G This finding was corroborated in this study using BSA, HGG, and OA as antigens. However, it is of more than academic interest to note t,hat while antibodies could be easily eluted from the diazotized polyaminostyrene-antigenantibody complex, they could not be eluted from the nondiazot.ized polyaminostyrene-antigen-antibody complex. The reason for this finding is not obvious and has not been investigated further. Undoubtedly, the diazotization of the polyaminostyrene affects the resin in a manner totally unrelated to the known conversion of amino groups to diazonium groups. The purified antibodies were furthermore shown t,o migrate as a pure gamma globulin band, both by paper and starch gel electrophoresis, and to sediment at the same rate as gamma globulin. Since only small quantities of protein were recovered as the eluted antibody preparation and since the latter possessed essentially all the ant,ibodies present in the original antiserum, it may be assumed that the preparation consists of only antibody molecules, although no attempt was made to determine the percentage of antibody in the eluates. It may be that some contamination with nonantibody gamma globulin may have occurred during the purification procedure. It was shown that the recovery of antibodies from the immunosorbknt is directly proportional to the hemagglutinating titer of the original antiserum. Antibodies from several strongly precipitating antisera, but with low BDB hemagglutinating titers of 640 and 1,280, could not be eluted; on the other hand, antibodies from several nonprecipitating antisera with high hemagglutinating titers of 20,000 t,o 50,000 were capable of being eluted. Thus, it appears
that a minimum hemagglutinating titer is required in order that antibodies can be elutcd from a particular antiserurn. This finding may explain why previous attempts to elute antibodies from allergic sera gave insignificant yields.‘” since these antisera usually possessed titers lower than 10,000. The findings that “antibody-sensitized ret1 cells, ” prepared by coupling Imrifietl antibody to red blood cells by means of rSI)R, can he agglutinated by the respective antigen, attests to the innocuous nature of the coupling procedurc~ although some denaturation of the antibody may have taken place. The antibody-reactive sites on the antibody molcc~tlc arc not affected. It is intcresting to not,c t,hat even the maximum amount of antigen capable of eliciting agglutination was exceedingly srnall (BSA-0.73 pg/ml., HGG-1.8 ~~g./ml. ; OA-1.5 pg’ml.). Higher concentrations of the homologous antigen prercntctI the agglutination of the antibody-sensitized cells, thus presenting the classical ‘ ’ prozon e ’ ’ phenomenon. I’urthermorc, as little as 0.0003 to 0.0004 ,q per nlilliliter of the antigen could agglutinate the homologous antibody-sensitized ~11s. It was also established that the agglutination of the ant.ibody-scIusitizcci ~11s by the homologous antigen is specific in that it coultl he inhibitctl l)y t h(h original antiserum or the eluted, purified antibody preparation but, not by the supcrnatant (antibody-depleted antiserum ), other hctcrologous antisera, 01’ normal rabbit serum. Sehon” stated that the amount of antigen which still 1~~1 to the aggregation of antibody-sensit,ized red cells was of the order of lo-’ pg of the antigen. Hc did not state whcthcr, in his experiments, purified antibod,v or the gamma globulin fraction of rabbit antiscrurn was usctl. In our esl~ri mcnts, it was shown that the red cells coated with antibotly-rich gamma glol)ulin did not agglutinate with the homologous antigen. However, these red eelIs were agglutinated by goat antirabbit gamma globulin serum, t,hus drmon~trating that gamma globulin exhibiting it,s original antigenic specificity had been coupled to these red cells. Red cells to which purified antibody was CWU~IWI gave- a positive agglutination reaction. This finding suggests that the ant,ibod~ content of the gamma globulin fraction obtained from strongly precipitating rabbit, antiserum is insufficient to produce optimally sensitized red cells. This stat,e of affairs is due probably to competition between ant,ibody gamma globnlin molecules and the much greater number of non-antibody gamma globulin molecules for sites on the red cell.
1. The use of immunosorbent-antigen conjugates for the isolation and puri fication of antibodies has been discussed. 2. Both the diazotized as well as the nondiazotized polpaminostyrene-antigen conjugates are capable of taking up the specific antibody; however, antibody can be eluted only from the former, and not, from the latter, immunosorbent. 3. The degree of recovery of the antibodies from an antiserum is directly IVlated to the hemagglutinating titer of the ant,iscrum. Only ant,isera with hamagglutinating titers of over 20,000 can be used for antibody elution experiments. 4. The method of using antibody-sensitized cells for the detection of antigen
ih
Mad-April,
1963
is described. The agglutination of antibody-sensitized red cells by the homologous antigen is specific as it could only be inhibited by the original antiserum and the eluted antibody but not by the antibody-depleted supernatant or heterologous antiserum. 5. Red blood cells conjugated with gamma globulin preparations of these antisera failed to be agglutinated b&ythe homologous antigen. REFERENCES
1. Pressman, D., Campbell, D. H., and Pauling, L.: The Agglutination of Intact AZOErythrocytes by Antisera Homologous to the Attached Groups, J. Immunol. 44: 101, 1942. 2. Stavitsky, A. B., and Arquilla, E. R.: Micromethods For the Study of Proteins and Antibodies. III. Procedure and Application of Hemagglutination and Hemagglutination-Inhibition Reactions With Bis-Diazotized Benzidine and Protein-Conjugated Red Blood Cells, J. Immunol. 74: 306, 1955. 3. Gordon, J., Rose,, B., and Sehon, A. H.: Detection of “Non-Precipitating” Antibodies in Sera of Indrviduals to Ragweed Pollen by an In Vitro Method, J. Exper. Med. 108: 37, 1958. 4. Boyden, S. V.: The Absorption of Proteins on Erythrocytes Treated With Tannic Acid and Subsequent Hemagglutination by Antiprotein Sera, J. Exper. Med. 93: 107, 1951. 5. Sherwin, A. L:, Richter, M., Cosgrove, J. B. R., and Rose, B.: Studies of the BloodCerebrosprnal Fluid Barrier to Antibodies and Ot,her Proteins, J. Neurology. (Submitted for publication.) 6. Arbesman, C. E., Rose, N. R., Kantor, S. Z., and Beede, R. B.: Immunologic Studies of Ragweed-Sensitive Patients. I. Specificity and Sensit.ivity of Hemagglutination Reactions, J. ALLERGY 31: 317, 1960. 7. Richter, M., Cua-Lim, F., and Rose, B.: The Agglutination of Antibody-Coated Red Blood Cells by Antigen, Biochem. & Biophys. Res. Commun. 7: 241, 1962. 8. Gyenes, L., and Sehon, A. H.: Preparation and Evaluation of Polystyrene-Antigen Conjugates for the Isolation of Antibodies, Canad. J. Biochem. & Physiol. 38: 1235,
1960. 9. Richter,
M.,
Delorme, P., Grant, S., and Rose, B.: Studies on the Uptake of Ragweed Allergens by Polyaminostyrene, Canad. J. Biochem. & Physiol. 40: 471, 1962. Richter, M., and Haurowitz, F.: Continuous Synthesis of Antibody After Primary Immunization With Protein Antigens, J. Immunol. 84: 420, 1960. Richter, M.! Blumer, H., Cua-Lim, F., and Rose, B.: Physiochemical Studies on Precipitatrng and Non-Precipitating Antibodies After Primary Immunization, Canad. J. Biochem. & Physiol. 40: 105, 1962. and Non-Precipitating Blumer, H., Richter, M., Cua-Lim, F., and Rose, B.: Precipitating Antibodies in the Primary and Secondary Immune Responses, J. Immunol. 88: 669, 1962. Richter, M., Cua-Lim, F.! and Rose, B.: The Demonstration of Identical Immunological Specificity of Preclprtating and Non-Precipitating Antibodies. Internat. Arch. Allergy. Pollen
10. 11. 12. 13.
14. Seho,‘l~.p~s’) .: In. Mechanisms of Hypersensitivity, edited by J. H. Shaffer, C. A. Lo drippo, and M. W. Chase, Toronto, Canada, 1959, Little, Brown, & Company, p. 118. 15. Gyenes, L., and Sehon, A. H.: The Use of Polystyrene-Allergen Conjugates for the Removal of Antibodies From Sera of Allergic Individuals, Canad. J. Biochem. & Physiol. 38: 1249, 1960. Studies on the Uptake of Reagin by 16. Delorme, P., Richter, M., Grant, S., and Rose, B.: Polyaminostyrene-Ragweed Complexes, Canad. J. Biochem. & Physiol. 40: 519, 1962.