Immunolog, 1975, 28, 199.
Proportional Absorption A METHOD FOR DETERMINATION OF THE RELATIVE SPECIFICITY OF ANTISERA PREPARED AGAINST CELLS A. E. REIF AND CYNTHIA M. ROBINSON
Mallory Institute of Pathology, Boston City Hospital, and the Department of Surgery, Tufts University School of Medicine, Boston City Hospital, Boston, Massachusetts, U.S.A.
(Received 6th March 1974; accepted for publication 11th April 1974) Summary. Antisera prepared against a complex of antigens such as a tissue cell may produce a mixture of antibodies of different specificities, affinities and types. Proportional absorption permits determination of the comparative specificity of such antisera. It is performed by absorbing the antisera with an amount of absorbent proportional to the initial content of (usually undesired) antibody to this absorbent; the potencies of desired and undesired antibodies are then separately determined. The method has been used to determine the specificity of a conventionally raised rabbit anti-mouse leukaemia serum, relative to one prepared with leukaemia cells admixed with rabbit antiserum against normal mouse lymphocytes (to block normal antigen sites on the leukaemia cell inoculum). The latter antiserum was more specific against leukaemia cells, as judged by immune cytolysis of leukaemia cells as compared to normal splenic lymphocytes. INTRODUCTION A great deal of work has been done in recent years with antisera prepared against allogeneic or xenogeneic cells and tissues (Reif and Kim, 1971; Klein, 1973). Such materials may be considered 'complex antigens', which give rise to a mixture of antibodies in the immunized host. While precise methods for the determination of antibodies against purified antigens have been developed (Kabat and Mayer, 1964), the theoretical basis for the analysis of the specificities of mixtures of antibodies obtained following immunization with complex antigens is still in a relatively rudimentary state (Reif, Allen and McVety, 1965). This paper describes a simple absorption procedure named 'proportional absorption', which is designed to reveal the comparative specificities ofallogeneic or xenogeneic antisera. The method is utilized here to determine the relative specificities of two types of antileukaemia sera against two cell types (normal splenic lymphocytes and leukaemia L1210MTX). However, the method has general application to the reaction of allosera or xenosera with complex antigens of any type. Immunization with a mixture of (a) a complex antigen, plus (b) antibody directed Correspondence: Dr A. E. Reif, Boston City Hospital, 818 Harrison Avenue, Boston, Massachusetts 02118, U.S.A.
199
A. E. Reif and Cynthia M. Robinson 200 against one of the antigenic determinants of this complex antigen produces reduced antibody formation against that determinant, and increased antibody formation against non-suppressed determinants (Pincus, Lamm and Nussenzweig, 1971). This approach has been used to prepare antileukaemia sera: rabbits are immunized with a complex antigen (mouse leukaemia cells) plus rabbit antiserum to normal mouse lymphocytes (Motta, 1970; Weiner, Hubbard and Mardiney, 1972; Smith, Robinson and Reif, 1974). Here, such an antileukaemia serum is compared with a conventional one.
MATERIALS AND METHODS Animals and cells Leukaemia L1210-MTX, a methotrexate-resistant antigenic form of leukaemia L1210 (Nicolin, Vadlamudi and Goldin, 1972), was transplanted in DBA/2J mice. Single cell suspensions of thymic, lymph node, splenic and leukaemic lymphocytes of DBA/2J mice were prepared in dilute Locke's buffer at 40 as previously described (Reif and Allen, 1964) and used within 2 hours.
Preparation of antileukaemia sera Two types of antileukaemia sera were produced: the conventional reagent antileukaemia serum (ALKS) and the reagent antileukaemia normal antigen-blocked serum (ALK-NABS). Each reagent was prepared by the intravenous (i.v.) injection of each of three rabbits with 2-5 x 107 L1210-MTX cells admixed for 30 minutes at room temperature either with 1-5 ml of normal rabbit serum (NRS) (for production of ALKS) or with 15 ml of normal antigen-blocking serum (NABS) (for production of ALK-NABS). All rabbits received three courses, each of four, four and five injections respectively, spaced over 2 weeks, and were bled 9 days after the last injection. The three sera for each group were pooled.
NABS The above reagent (NABS) was prepared by subcutaneous (s.c.) injection of Freund's complete adjuvant containing 4 x I07 thymic and 1 x 107 lymph node lymphocytes of DBA/2J mice into each of three rabbits, followed 3 weeks later by three daily i.v. injections of the same cell inoculum suspended in Locke's buffer. Cycles of three i.v. injections, followed by bleeding 7-10 days after the last injection, were repeated several times. The potency of different batches of NABS was determined against DBA/2J splenic lymphocytes. Each batch contained sera pooled from three rabbits. Cytolysis assay The in vitro assay was performed in 0-3-ml volumes (Reif and Allen, 1964). Each assay tube contained I05 viable lymphocytes or leukaemia cells, absorbed 10 per cent guinea-pig complement, and serial doubling dilutions of antiserum. After incubation for 1 hour at 370, a hundred cells per tube were classified as stained or unstained. Results are expressed in terms of the cytolytic potency, defined as the final dilution at which an antiserum produces 50 per cent cell lysis in the assay system; for instance, an antiserum that reaches this end-point at a final dilution of 1:100 has a cytolytic potency of 100 (Reif and Allen, 1964).
201
Proportional Absorption
Absorption of antisera Antisera were inactivated by addition of 3 per cent of the serum volume of 0 5 M Na2EDTA. Antisera were gently mixed with cell suspensions for 1 hour at room temperature, then centrifuged at 3000 g for 15 minutes at 5°. Supernatants were stored frozen after the addition of a mixture containing I 0 M MgCl2.6H20 and 0 3 M CaCl2 (1 per cent of the volume of the serum), to restore the divalent cation content. Further details are given in table or figure captions.
RESULTS A conventional antileukaemia serum (ALKS) and one prepared with leukaemia cells admixed with normal antigen-blocking serum (ALK-NABS) were absorbed with increasing quantities of normal mouse lymphocytes (Table 1). This absorption reduced the cytolytic potency against leukaemia cells to a greater degree for ALKS than for ALKNABS; the inverse was true for the reduction in cytolytic potency against normal lymphocytes. Thus, the specificity for reaction against leukaemia cells relative to reaction against normal lymphocytes, as expressed by the specificity ratio (Reif, Allen and McVety, 1965), rose more rapidly for ALK-NABS than for ALKS (columns 4 and 7, Table 1). TABLE 1 SPECIFICITIES OF ALKS AND ALK-NABS REAGENTS AFTER ABSORPTION WITH NORMAL MOUSE LYMPHOCYTES
ALK-NABS
ALKS
Absorption of reagents with normal mouse lymphocytes*
(x 106/ml) 0 10 30 90
Cytolytic Specificity ratio potency vs
Cytolytic Specificity ratio potency vs
Lt
Nt
L/N
L
1060 826 456 176
1190 900 625 104
0-89
215 170 153 111
0*92 0*73
169
N
L/N
0-98 216 3-3 52 7-3 21 65-4
* The stated number of a 4: 1 mixture of DBA/2J splenic and thymic lymphocytes was used per millilitre of reagent. Fresh antiserum was used for each absorption. t L = leukaemia L1210-MTX; N = normal DBA/2J splenic lymphocytes.
The above analysis is misleading because it omits consideration of the cytolytic potencies of the two antisera prior to absorption. Since ALKS was 5*5 times as potent against normal lymphocytes as ALK-NABS, it must have contained 5-5 times as many cytolytically effective antibody molecules, or their equivalents in molecules of different types and affinities (see Discussion section). Thus, if results obtained after absorption of the two antisera are to be compared, 5-5 times as many normal lymphocytes should have been used for absorption of ALKS than for ALK-NABS. For instance, absorption of ALKNABS with 1 x 107 normal lymphocytes (specificity ratio 3 3, column 7, Table 1) is equivalent to absorption of ALKS by 10 x 1,190/216 or 5 5x I07 lymphocytes (specificity ratio between 0 73 and 1 69, column 4, Table 1). Stated in general terms, when comparing the specificity of two antisera, one must absorb each antiserum with an amount of absorbent proportional to its content of antibody reactive to that absorbent.
202 A. E. Reif and Cynthia M. Robinson An illustration of contravening or conforming to this principle is given (Fig. 1). Before absorption, a sample of ALKS had a cytolytic potency against normal lymphocytes 4-2 times higher than ALK-NABS (Fig. la, b). When both antisera were absorbed with the same quantity (30 x 106) lymphocytes, then ALK-NABS appeared to be far more specific (Fig. If, ratio 10*6) than ALKS (Fig. Ic, ratio 0 90). However, this comparison is biased in favour of ALK-NABS, because of its initially lower potency against normal lymphocytes. If such a comparison is validated by use of proportional absorption, then mild absorption (Fig. lc, d) and more extensive absorption (Fig. le and f) leave ALK(a3) 1580: 1010 (SR= 157)
698: 776 (SR = 090)
(c)
(e)
212 :184
(SR= 115)
127x 106
30x06n
m
H0
(b)
1
(d) 278: 239 (SR = 16)
99: 52 (SR = 190)
ir 71R x 06 B efore a bsorption
* n (f)
Absorption
-
m
After with stated absorption number of normal lymphocytes permillilitre of antibody
10-6:10 (SR = 106)
F Absorption After with stated absorption number of normal lymphocytes per millilitre of antibody
FIG. 1. Absorption of conventional antiserum, ALKS (a), (c) and (e), and of antileukaemia serum prepared with normal antigen-blocking serum, ALK-NABS (b), (d) and (f), with normal DBA/2J splenic and thymic lymphocytes in a 4:1 ratio (normal lymphocytes, cross-hatched columns; the number of cells per millilitre of antiserum is shown above the columns). Cytolytic potencies of the antisera were determined before (a) and (b) and after (c)-(f) absorption by titration against leukaemia L1210-MTX (-) and against DBA/2J splenic lymphocytes (L). Fresh antiserum was used for each absorption, and each millilitre of antiserum was preabsorbed with 1/10 vol DBA/2J RBC per 30 x 106 normal lymphocytes used. The figures shown above the graphs are the cytolytic potency against leukaemia: the cytolytic potency against normal lymphocytes = the specificity ratio (SR).
NABS more specific against leukaemia cells than ALKS by respectively 1 90/0-90 = 2 1 times and 10-6/1 15 = 9-2 times. The latter comparisons show that ALK-NABS possesses greater specificity against leukaemia cells than does ALKS, irrespective of the absolute potency of these antisera. Essentially identical results were obtained when individual rabbit antisera (three for each group) were titred before and after proportional
absorption.
Normally,
an
DISCUSSION absorption is performed by addition of undesired antigens to
a
mixture
Proportional Absorption
203 of antibodies of different specificities, to remove undesired antibodies. The method by which ALK-NABS is produced consists of immunization with antigen incubated with absorption, antibody prepared against the undesired antigen. This represents an inverse rather in the sense that antigen is absorbed with antibody prior to immunization, that antibody is absorbed with antigen following immunization. Undesired antigens the absorbing material in conventional absorption, while antibodies against undesired connection 'undesired antigens are the absorbed reagent in inverse absorption. In this antibody components. antigens' are defined as antigens capable of removing undesired sparing more is The results obtained (Fig. 1) also suggest that inverse absorption conventional absorption, in terms of the quantity of absorbing material required prepare purified antibody against the desired specificity. than
are
than
to
Several explanations are possible for differences in specificity
(Reif et al., 1965) against
leukaemia cells relative to normal lymphocytes. First, we may be dealing with of antibodies of different potencies that react with antigenic specificities present in different mixtures
is concenconcentrations on each cell type. Although absorption of antibodies by antigens stoichiometric and Allen, 1964), a simple model that noassumes (Reif tration-dependent antigenic contains A If antigen can be complex (Tableto2).removal all reactivity against complex A in ourspecificity helpful suffice binding example; 2, then two absorptions
complex activity against will not further reduce antibody thereafter, exhaustive absorption 2 specificity of B (example a, Table 2). If complex A contains insufficient determinants sites (e.g. such antigen (5 per cent in example b) to be detected in the assay for antibody three then 1962)), the aband M6ller, (Mollercomplex may be too sparse to permit immune cytolysisagainst point, will this at A; will detectable remove reactivity absorptions times) (forty sorbed serum will contain anti-2 antibodies, but exhaustive absorption per 2 (10 will specificity remove these (example b). If complex A contains sufficient antigenic absorption extensive cent in example c) for detection in the antibody assay, then remove all reactivity against both complex A and B. While many more than twotheantigenic are involved in the present situation, example b (Table 2) best fits experispecificities mental data (Table 1, Fig. 1). and those Two sets of antigens are considered above: those associated with leukaemia, quantities
associated with normal splenic lymphocytes. Each antiserum contains different is the ratio of these two sets of antibodies, and its relative specificity corresponding quantities. Exhaustive absorption is not helpful, since (a) it cannot be used to determine the relative content of the two sets of antibodies, (b) the quantities of the absorbing may be a
of the two
antigen required may be impracticably high, and (c) dilution of antibody A second explanation is that ALKS and ALK-NABS contain antibodies of different between affinities. Antibody binding is a dynamic process which involves an equilibrium1964). At and Mayer,lysed (Reif (Kabat and desorption (escape into solution) (binding) adsorption are the endpoint of the immune cytolysis assay when 50 per cent of the cells and Allen, 1964), the lytically effective residence time for antibody molecules of the same concentration must be identical for two sera of equal cytolytic potencies. Thus, a lowconcentration type of high a as cytolysis of high affinity antibody can be equally effective in absorpduring binding antibody for low affinity antibody. In contrast, the requirements tions are far less rigid than for lytically effective binding (Asakuma and Reif, 1968), and terms therefore both high and low affinity antibody are likely to compete on more equal achieve to required be would absorbent more relatively during absorptions. Therefore, problem.
A. E. Reif and Cynthia M. Robinson
204
TABLE 2 THEORETICAL
RESULTS FOR THE ABSORPTION WITH COMPLEX ANTIGEN
A
OF AN
ANTISERUM PREPARED AGAINST COMPLEX ANTIGEN B, WHICH CONTAINS DIFFERENT PROPORTIONS OF THE SAME TWO SPECIFICITIES, 1 AND 2, PRESENT ON COMPLEX A
Complex antigen A (antigenic specificity) 1
Total
1
2
Total
100
75
25
100
Potency of antiserum* after absorptiont with complex antigen A Unabsorbed 0 200 200 200 200 1 x absorbed 100 0 100 100 200 0 0 2 x absorbed 0 0 200 100 x absorbed 0 0 0 0 200
400 300 200 200
(b) Antigen content (units): 95
25
100
Potency of antiserum* after absorptiont with complex antigen A Unabsorbed 0 200 200 200 200 1 x absorbed 105 0 105 105 195 2 x absorbed 10 0 10 10 190 3 x absorbed 0 0 0 0 185 0 0 10 x absorbed 0 0 150 0 0 0 0 25 x absorbed 75 40 x absorbed 0 0 0 0 0
400 300 200 185 150 75 0
(c) Antigen content (units): 90
25
100
Potency of antiserum* after absorptiont with complex antigen A Unabsorbed 200 200 400 200 200 1 x absorbed 110 190 110 300 190 20 180 200 20 180 2 x absorbed 0 0 100 100 10 x absorbed 100 0 0 0 0 0 20 x absorbed
400 300 200 100 0
(a) Antigen content (units): 100
2
Complex antigen B (antigenic specificity)
0
51
10
100
100
75
75
* The antiserum contains 200 units of antibody activity per millilitre against each of the two antigenic specificities, 1 and 2. t One millilitre of antiserum is absorbed with the stated number of antigen units of complex A, which bind the same number of the corresponding antibody units. t Antigen concentration was too sparse to permit detection in assay of antibody.
comparable absorption of low than of high affinity antibody. Since absorptions were relatively more effective for ALK-NABS than for ALKS (Table 1, Fig. 1), this suggests that ALK-NABS contained antibodies with higher affinities for leukaemia cells than did ALKS. A third explanation is that different antisera contain different classes of antibodies. If antiserum A contained only C-fixing antibodies, while B contained a 1: 2 proportion of C-fixing: non-C-fixing antibodies, the same result would be obtained as if A contained antibody with three-fold higher affinity than B. This merits consideration, since the efficiency of the binding of all C components necessary for lysis to cell antigen site-2yG-Cl can be 1000-fold greater than for binding to cell antigen site-yM-Cl (Reif and Kim, 1971). While future work is required to determine which of the above three explanations is mainly responsible for the differences in specificity observed here (Fig. 1), the present considerations may be relevant in many other situations in which antisera against antigen complexes such as cells or tissues are employed.
Proportional Absorption
205
ACKNOWLEDGMENT This work was supported by Public Health Service research grant number CA 15952 from the National Cancer Institute. REFERENCES
ASAKUMA, R. and REIF, A. E. (1968). 'The specificity of antilymphocyte sera to thymic, splenic, and leukemic lymphocytes.' Cancer Res., 28, 707. KABAT, E. A. and MAYER, M. M. (1964). Experimental Immunochemistry, 2nd edn (ed. by E. A. Kabat), p. 22, 326. Thomas, Springfield. KLEIN, J. (1973). 'The H-2 system: past and present.' Transplant. Proc., 5, 1 1. M6LLER, E. and MOLLER, G. (1962). 'Quantitative studies of the sensitivity of normal and neoplastic mouse cells to the cytotoxic action of isoantibodies.' J. exp. Med., 115, 527. MOTTA, R. (1970). 'The passive immunotherapy of murine leukaemia. I. The production of antisera against leukaemic antigens.' Europ. Rev. clin. biol. Res., 15, 161. NICOLIN, A., VADLAMUDI, S. and GOLDIN, A. (1972). 'Antigenicity of L12 10 leukemic sublines induced by drugs.' Cancer Res., 32, 653. PINCUS, C. S., LAMm, M. E. and NuSSENZWEIG, V.
(1971). 'Regulation of the immune response: suppressive and enhancing effects of passively administered antibody.'_J. exp. Med., 133, 987. REIF, A. E. and ALLEN, J. M. (1964). 'The AKR thymic antigen and its distribution in leukemias and nervous tissues.' 7. exp. Med., 120, 413. REIF, A. E., ALLEN, J. M. V. and MCVETY, L. M. (1965). 'A general method for the calculation of serological specificity.' Immunology, 8, 384. REIF, A. E. and KIM, C.-A. H. (1971). 'Specificity of rabbit antisera against mouse leukaemias in vitro.' Immunology, 20, 1087. SMITH, P.J., ROBINSON, C. M. and REIF, A. E. (1974). 'Specificity of antileukemia sera prepared by immunization with leukemia cells admixed with normal antigen-blocking sera.' Cancer Res., 34, 169. WEINER, R. S., HUBBARD,J. D. and MARDINEY, M. R., JR (1972). 'Production of tumor-specific antibody in the xenogeneic host: use of blocking antibody.' J. nat. Cancer Inst., 49, 1063.
Proportional Absorption A METHOD FOR DETERMINATION OF THE RELATIVE SPECIFICITY OF ANTISERA PREPARED AGAINST CELLS A. E. REIF AND CYNTHIA M. ROBINSON
Mallory Institute of Pathology, Boston City Hospital, and the Department of Surgery, Tufts University School of Medicine, Boston City Hospital, Boston, Massachusetts, U.S.A.
(Received 6th March 1974; accepted for publication 11th April 1974) Summary. Antisera prepared against a complex of antigens such as a tissue cell may produce a mixture of antibodies of different specificities, affinities and types. Proportional absorption permits determination of the comparative specificity of such antisera. It is performed by absorbing the antisera with an amount of absorbent proportional to the initial content of (usually undesired) antibody to this absorbent; the potencies of desired and undesired antibodies are then separately determined. The method has been used to determine the specificity of a conventionally raised rabbit anti-mouse leukaemia serum, relative to one prepared with leukaemia cells admixed with rabbit antiserum against normal mouse lymphocytes (to block normal antigen sites on the leukaemia cell inoculum). The latter antiserum was more specific against leukaemia cells, as judged by immune cytolysis of leukaemia cells as compared to normal splenic lymphocytes. INTRODUCTION A great deal of work has been done in recent years with antisera prepared against allogeneic or xenogeneic cells and tissues (Reif and Kim, 1971; Klein, 1973). Such materials may be considered 'complex antigens', which give rise to a mixture of antibodies in the immunized host. While precise methods for the determination of antibodies against purified antigens have been developed (Kabat and Mayer, 1964), the theoretical basis for the analysis of the specificities of mixtures of antibodies obtained following immunization with complex antigens is still in a relatively rudimentary state (Reif, Allen and McVety, 1965). This paper describes a simple absorption procedure named 'proportional absorption', which is designed to reveal the comparative specificities ofallogeneic or xenogeneic antisera. The method is utilized here to determine the relative specificities of two types of antileukaemia sera against two cell types (normal splenic lymphocytes and leukaemia L1210MTX). However, the method has general application to the reaction of allosera or xenosera with complex antigens of any type. Immunization with a mixture of (a) a complex antigen, plus (b) antibody directed Correspondence: Dr A. E. Reif, Boston City Hospital, 818 Harrison Avenue, Boston, Massachusetts 02118, U.S.A.
199
A. E. Reif and Cynthia M. Robinson 200 against one of the antigenic determinants of this complex antigen produces reduced antibody formation against that determinant, and increased antibody formation against non-suppressed determinants (Pincus, Lamm and Nussenzweig, 1971). This approach has been used to prepare antileukaemia sera: rabbits are immunized with a complex antigen (mouse leukaemia cells) plus rabbit antiserum to normal mouse lymphocytes (Motta, 1970; Weiner, Hubbard and Mardiney, 1972; Smith, Robinson and Reif, 1974). Here, such an antileukaemia serum is compared with a conventional one.
MATERIALS AND METHODS Animals and cells Leukaemia L1210-MTX, a methotrexate-resistant antigenic form of leukaemia L1210 (Nicolin, Vadlamudi and Goldin, 1972), was transplanted in DBA/2J mice. Single cell suspensions of thymic, lymph node, splenic and leukaemic lymphocytes of DBA/2J mice were prepared in dilute Locke's buffer at 40 as previously described (Reif and Allen, 1964) and used within 2 hours.
Preparation of antileukaemia sera Two types of antileukaemia sera were produced: the conventional reagent antileukaemia serum (ALKS) and the reagent antileukaemia normal antigen-blocked serum (ALK-NABS). Each reagent was prepared by the intravenous (i.v.) injection of each of three rabbits with 2-5 x 107 L1210-MTX cells admixed for 30 minutes at room temperature either with 1-5 ml of normal rabbit serum (NRS) (for production of ALKS) or with 15 ml of normal antigen-blocking serum (NABS) (for production of ALK-NABS). All rabbits received three courses, each of four, four and five injections respectively, spaced over 2 weeks, and were bled 9 days after the last injection. The three sera for each group were pooled.
NABS The above reagent (NABS) was prepared by subcutaneous (s.c.) injection of Freund's complete adjuvant containing 4 x I07 thymic and 1 x 107 lymph node lymphocytes of DBA/2J mice into each of three rabbits, followed 3 weeks later by three daily i.v. injections of the same cell inoculum suspended in Locke's buffer. Cycles of three i.v. injections, followed by bleeding 7-10 days after the last injection, were repeated several times. The potency of different batches of NABS was determined against DBA/2J splenic lymphocytes. Each batch contained sera pooled from three rabbits. Cytolysis assay The in vitro assay was performed in 0-3-ml volumes (Reif and Allen, 1964). Each assay tube contained I05 viable lymphocytes or leukaemia cells, absorbed 10 per cent guinea-pig complement, and serial doubling dilutions of antiserum. After incubation for 1 hour at 370, a hundred cells per tube were classified as stained or unstained. Results are expressed in terms of the cytolytic potency, defined as the final dilution at which an antiserum produces 50 per cent cell lysis in the assay system; for instance, an antiserum that reaches this end-point at a final dilution of 1:100 has a cytolytic potency of 100 (Reif and Allen, 1964).
201
Proportional Absorption
Absorption of antisera Antisera were inactivated by addition of 3 per cent of the serum volume of 0 5 M Na2EDTA. Antisera were gently mixed with cell suspensions for 1 hour at room temperature, then centrifuged at 3000 g for 15 minutes at 5°. Supernatants were stored frozen after the addition of a mixture containing I 0 M MgCl2.6H20 and 0 3 M CaCl2 (1 per cent of the volume of the serum), to restore the divalent cation content. Further details are given in table or figure captions.
RESULTS A conventional antileukaemia serum (ALKS) and one prepared with leukaemia cells admixed with normal antigen-blocking serum (ALK-NABS) were absorbed with increasing quantities of normal mouse lymphocytes (Table 1). This absorption reduced the cytolytic potency against leukaemia cells to a greater degree for ALKS than for ALKNABS; the inverse was true for the reduction in cytolytic potency against normal lymphocytes. Thus, the specificity for reaction against leukaemia cells relative to reaction against normal lymphocytes, as expressed by the specificity ratio (Reif, Allen and McVety, 1965), rose more rapidly for ALK-NABS than for ALKS (columns 4 and 7, Table 1). TABLE 1 SPECIFICITIES OF ALKS AND ALK-NABS REAGENTS AFTER ABSORPTION WITH NORMAL MOUSE LYMPHOCYTES
ALK-NABS
ALKS
Absorption of reagents with normal mouse lymphocytes*
(x 106/ml) 0 10 30 90
Cytolytic Specificity ratio potency vs
Cytolytic Specificity ratio potency vs
Lt
Nt
L/N
L
1060 826 456 176
1190 900 625 104
0-89
215 170 153 111
0*92 0*73
169
N
L/N
0-98 216 3-3 52 7-3 21 65-4
* The stated number of a 4: 1 mixture of DBA/2J splenic and thymic lymphocytes was used per millilitre of reagent. Fresh antiserum was used for each absorption. t L = leukaemia L1210-MTX; N = normal DBA/2J splenic lymphocytes.
The above analysis is misleading because it omits consideration of the cytolytic potencies of the two antisera prior to absorption. Since ALKS was 5*5 times as potent against normal lymphocytes as ALK-NABS, it must have contained 5-5 times as many cytolytically effective antibody molecules, or their equivalents in molecules of different types and affinities (see Discussion section). Thus, if results obtained after absorption of the two antisera are to be compared, 5-5 times as many normal lymphocytes should have been used for absorption of ALKS than for ALK-NABS. For instance, absorption of ALKNABS with 1 x 107 normal lymphocytes (specificity ratio 3 3, column 7, Table 1) is equivalent to absorption of ALKS by 10 x 1,190/216 or 5 5x I07 lymphocytes (specificity ratio between 0 73 and 1 69, column 4, Table 1). Stated in general terms, when comparing the specificity of two antisera, one must absorb each antiserum with an amount of absorbent proportional to its content of antibody reactive to that absorbent.
202 A. E. Reif and Cynthia M. Robinson An illustration of contravening or conforming to this principle is given (Fig. 1). Before absorption, a sample of ALKS had a cytolytic potency against normal lymphocytes 4-2 times higher than ALK-NABS (Fig. la, b). When both antisera were absorbed with the same quantity (30 x 106) lymphocytes, then ALK-NABS appeared to be far more specific (Fig. If, ratio 10*6) than ALKS (Fig. Ic, ratio 0 90). However, this comparison is biased in favour of ALK-NABS, because of its initially lower potency against normal lymphocytes. If such a comparison is validated by use of proportional absorption, then mild absorption (Fig. lc, d) and more extensive absorption (Fig. le and f) leave ALK(a3) 1580: 1010 (SR= 157)
698: 776 (SR = 090)
(c)
(e)
212 :184
(SR= 115)
127x 106
30x06n
m
H0
(b)
1
(d) 278: 239 (SR = 16)
99: 52 (SR = 190)
ir 71R x 06 B efore a bsorption
* n (f)
Absorption
-
m
After with stated absorption number of normal lymphocytes permillilitre of antibody
10-6:10 (SR = 106)
F Absorption After with stated absorption number of normal lymphocytes per millilitre of antibody
FIG. 1. Absorption of conventional antiserum, ALKS (a), (c) and (e), and of antileukaemia serum prepared with normal antigen-blocking serum, ALK-NABS (b), (d) and (f), with normal DBA/2J splenic and thymic lymphocytes in a 4:1 ratio (normal lymphocytes, cross-hatched columns; the number of cells per millilitre of antiserum is shown above the columns). Cytolytic potencies of the antisera were determined before (a) and (b) and after (c)-(f) absorption by titration against leukaemia L1210-MTX (-) and against DBA/2J splenic lymphocytes (L). Fresh antiserum was used for each absorption, and each millilitre of antiserum was preabsorbed with 1/10 vol DBA/2J RBC per 30 x 106 normal lymphocytes used. The figures shown above the graphs are the cytolytic potency against leukaemia: the cytolytic potency against normal lymphocytes = the specificity ratio (SR).
NABS more specific against leukaemia cells than ALKS by respectively 1 90/0-90 = 2 1 times and 10-6/1 15 = 9-2 times. The latter comparisons show that ALK-NABS possesses greater specificity against leukaemia cells than does ALKS, irrespective of the absolute potency of these antisera. Essentially identical results were obtained when individual rabbit antisera (three for each group) were titred before and after proportional
absorption.
Normally,
an
DISCUSSION absorption is performed by addition of undesired antigens to
a
mixture
Proportional Absorption
203 of antibodies of different specificities, to remove undesired antibodies. The method by which ALK-NABS is produced consists of immunization with antigen incubated with absorption, antibody prepared against the undesired antigen. This represents an inverse rather in the sense that antigen is absorbed with antibody prior to immunization, that antibody is absorbed with antigen following immunization. Undesired antigens the absorbing material in conventional absorption, while antibodies against undesired connection 'undesired antigens are the absorbed reagent in inverse absorption. In this antibody components. antigens' are defined as antigens capable of removing undesired sparing more is The results obtained (Fig. 1) also suggest that inverse absorption conventional absorption, in terms of the quantity of absorbing material required prepare purified antibody against the desired specificity. than
are
than
to
Several explanations are possible for differences in specificity
(Reif et al., 1965) against
leukaemia cells relative to normal lymphocytes. First, we may be dealing with of antibodies of different potencies that react with antigenic specificities present in different mixtures
is concenconcentrations on each cell type. Although absorption of antibodies by antigens stoichiometric and Allen, 1964), a simple model that noassumes (Reif tration-dependent antigenic contains A If antigen can be complex (Tableto2).removal all reactivity against complex A in ourspecificity helpful suffice binding example; 2, then two absorptions
complex activity against will not further reduce antibody thereafter, exhaustive absorption 2 specificity of B (example a, Table 2). If complex A contains insufficient determinants sites (e.g. such antigen (5 per cent in example b) to be detected in the assay for antibody three then 1962)), the aband M6ller, (Mollercomplex may be too sparse to permit immune cytolysisagainst point, will this at A; will detectable remove reactivity absorptions times) (forty sorbed serum will contain anti-2 antibodies, but exhaustive absorption per 2 (10 will specificity remove these (example b). If complex A contains sufficient antigenic absorption extensive cent in example c) for detection in the antibody assay, then remove all reactivity against both complex A and B. While many more than twotheantigenic are involved in the present situation, example b (Table 2) best fits experispecificities mental data (Table 1, Fig. 1). and those Two sets of antigens are considered above: those associated with leukaemia, quantities
associated with normal splenic lymphocytes. Each antiserum contains different is the ratio of these two sets of antibodies, and its relative specificity corresponding quantities. Exhaustive absorption is not helpful, since (a) it cannot be used to determine the relative content of the two sets of antibodies, (b) the quantities of the absorbing may be a
of the two
antigen required may be impracticably high, and (c) dilution of antibody A second explanation is that ALKS and ALK-NABS contain antibodies of different between affinities. Antibody binding is a dynamic process which involves an equilibrium1964). At and Mayer,lysed (Reif (Kabat and desorption (escape into solution) (binding) adsorption are the endpoint of the immune cytolysis assay when 50 per cent of the cells and Allen, 1964), the lytically effective residence time for antibody molecules of the same concentration must be identical for two sera of equal cytolytic potencies. Thus, a lowconcentration type of high a as cytolysis of high affinity antibody can be equally effective in absorpduring binding antibody for low affinity antibody. In contrast, the requirements tions are far less rigid than for lytically effective binding (Asakuma and Reif, 1968), and terms therefore both high and low affinity antibody are likely to compete on more equal achieve to required be would absorbent more relatively during absorptions. Therefore, problem.
A. E. Reif and Cynthia M. Robinson
204
TABLE 2 THEORETICAL
RESULTS FOR THE ABSORPTION WITH COMPLEX ANTIGEN
A
OF AN
ANTISERUM PREPARED AGAINST COMPLEX ANTIGEN B, WHICH CONTAINS DIFFERENT PROPORTIONS OF THE SAME TWO SPECIFICITIES, 1 AND 2, PRESENT ON COMPLEX A
Complex antigen A (antigenic specificity) 1
Total
1
2
Total
100
75
25
100
Potency of antiserum* after absorptiont with complex antigen A Unabsorbed 0 200 200 200 200 1 x absorbed 100 0 100 100 200 0 0 2 x absorbed 0 0 200 100 x absorbed 0 0 0 0 200
400 300 200 200
(b) Antigen content (units): 95
25
100
Potency of antiserum* after absorptiont with complex antigen A Unabsorbed 0 200 200 200 200 1 x absorbed 105 0 105 105 195 2 x absorbed 10 0 10 10 190 3 x absorbed 0 0 0 0 185 0 0 10 x absorbed 0 0 150 0 0 0 0 25 x absorbed 75 40 x absorbed 0 0 0 0 0
400 300 200 185 150 75 0
(c) Antigen content (units): 90
25
100
Potency of antiserum* after absorptiont with complex antigen A Unabsorbed 200 200 400 200 200 1 x absorbed 110 190 110 300 190 20 180 200 20 180 2 x absorbed 0 0 100 100 10 x absorbed 100 0 0 0 0 0 20 x absorbed
400 300 200 100 0
(a) Antigen content (units): 100
2
Complex antigen B (antigenic specificity)
0
51
10
100
100
75
75
* The antiserum contains 200 units of antibody activity per millilitre against each of the two antigenic specificities, 1 and 2. t One millilitre of antiserum is absorbed with the stated number of antigen units of complex A, which bind the same number of the corresponding antibody units. t Antigen concentration was too sparse to permit detection in assay of antibody.
comparable absorption of low than of high affinity antibody. Since absorptions were relatively more effective for ALK-NABS than for ALKS (Table 1, Fig. 1), this suggests that ALK-NABS contained antibodies with higher affinities for leukaemia cells than did ALKS. A third explanation is that different antisera contain different classes of antibodies. If antiserum A contained only C-fixing antibodies, while B contained a 1: 2 proportion of C-fixing: non-C-fixing antibodies, the same result would be obtained as if A contained antibody with three-fold higher affinity than B. This merits consideration, since the efficiency of the binding of all C components necessary for lysis to cell antigen site-2yG-Cl can be 1000-fold greater than for binding to cell antigen site-yM-Cl (Reif and Kim, 1971). While future work is required to determine which of the above three explanations is mainly responsible for the differences in specificity observed here (Fig. 1), the present considerations may be relevant in many other situations in which antisera against antigen complexes such as cells or tissues are employed.
Proportional Absorption
205
ACKNOWLEDGMENT This work was supported by Public Health Service research grant number CA 15952 from the National Cancer Institute. REFERENCES
ASAKUMA, R. and REIF, A. E. (1968). 'The specificity of antilymphocyte sera to thymic, splenic, and leukemic lymphocytes.' Cancer Res., 28, 707. KABAT, E. A. and MAYER, M. M. (1964). Experimental Immunochemistry, 2nd edn (ed. by E. A. Kabat), p. 22, 326. Thomas, Springfield. KLEIN, J. (1973). 'The H-2 system: past and present.' Transplant. Proc., 5, 1 1. M6LLER, E. and MOLLER, G. (1962). 'Quantitative studies of the sensitivity of normal and neoplastic mouse cells to the cytotoxic action of isoantibodies.' J. exp. Med., 115, 527. MOTTA, R. (1970). 'The passive immunotherapy of murine leukaemia. I. The production of antisera against leukaemic antigens.' Europ. Rev. clin. biol. Res., 15, 161. NICOLIN, A., VADLAMUDI, S. and GOLDIN, A. (1972). 'Antigenicity of L12 10 leukemic sublines induced by drugs.' Cancer Res., 32, 653. PINCUS, C. S., LAMm, M. E. and NuSSENZWEIG, V.
(1971). 'Regulation of the immune response: suppressive and enhancing effects of passively administered antibody.'_J. exp. Med., 133, 987. REIF, A. E. and ALLEN, J. M. (1964). 'The AKR thymic antigen and its distribution in leukemias and nervous tissues.' 7. exp. Med., 120, 413. REIF, A. E., ALLEN, J. M. V. and MCVETY, L. M. (1965). 'A general method for the calculation of serological specificity.' Immunology, 8, 384. REIF, A. E. and KIM, C.-A. H. (1971). 'Specificity of rabbit antisera against mouse leukaemias in vitro.' Immunology, 20, 1087. SMITH, P.J., ROBINSON, C. M. and REIF, A. E. (1974). 'Specificity of antileukemia sera prepared by immunization with leukemia cells admixed with normal antigen-blocking sera.' Cancer Res., 34, 169. WEINER, R. S., HUBBARD,J. D. and MARDINEY, M. R., JR (1972). 'Production of tumor-specific antibody in the xenogeneic host: use of blocking antibody.' J. nat. Cancer Inst., 49, 1063.
