152 (1~.~92)59-67 © 1992 Elsevier Science Publishers B.V. All rights reserved ()1122-175tt/92/$05.(10
Journal ofhnmunoh~gical Methods.
59
JIM 0fi355
Determination of monoclonal antibody specificity by immunoadsorption and Western blotting R o l f S c h u h , E l i s a b e t h K r e m m e r , Evelyn Ego, M i c h a c l Wasiliu a n d S t e f a n T h i e r f e l d c r (;SF-hzstitut .fiir hnmunologie, 8000 Munich, Germany
(Received 311(.)cipher 1991, revised received 6 January 1992,accepted 21 February 19921
A rapid and simple method has been developed for identifying the specificity of monoclonal antibodies at an early stage in the production of hybridomas. The technique is a micro-method utilizing biotinylated crude antigen and the surface of microtiter plates as an immunoaffinity matrix. The monoclonal antibodies to be tested are adsorbed to the microtiter wells and incubated with the labeled antigen preparation. Non-specific binding can be reduced by blocking and repeated washing steps. The specific antigen is then eluted by SDS-containing buffers, subjected to S D S - P A G E blotted onto nitrocellulose and detected by enzyme-labeled avidin in a Western blot assay. The amount of bound and removed antigen can be quantitated by developing eluted and non-eluted control wells by EL1SA techniques. Since this ELISA can be performed rapidly, only samples which contain sufficient specific material can be selected tor eleetrophoresis and blotting. The major advantages of the technique are (i) the use of a non-radioactive label resulting in an easy and time-saving procedure, (ii) the possibility of quantitating the amount of captured and detached antigen by ELISA, (iii) the need for only a minimal amount of antigen, (iv) the use of unpurified antibodies of all isotypes, (v) a high signal-to-noise ratio, and (vi) as with all immunoprecipitation techniques, the possibility of detecting SDS-sensitive epitopes and of using crude antigen preparations. Using this method we were able to characterize monoclonal antibodies against both soluble proteir"; (mouse and human Clq) and membrane determinants (human pan T ccll CD5 and CD7), Key words:
Monoclonal antibody; Biotinylation:ELISA; Immunoadsorption:Western blotting
Correspomh,nce to: R. Schuh. GSF-Forschungszentrumfiir Umwelt und Gesundheit GmbH, lnstitut fiir Immunoh)gie, Marchionin',raBe 25, 8111111Munich 711,Germany. Ahbreciations: AP, alkaline phosphatase: ATCC. American Type Culture Collection; DMF, dimethyllk~rmamide: EDTA, ethylenediaminetctraacetic acid: ELISA. enzyme-linked immunosorbent assay: FCA, flow cytometric analysis: HRP, horseradish pcroxidase; MAb, monocional antibody; MP, skimmed milk powder: MW, molecular weight; PAGE, polyacrylamide gel elcctrophoresis; PBS. phosphatc-buffi:red saline: SDS, sodium d~idecylsulfate.
Introduction
Immunization, fusion and cell culture no longer rose particular problems in the production of monoclonal antibodies. However, depending on the availability of purified antigen, it is often difficult to determine the molecular weight of the corresponding antigen and, consequently, to define the specificity of the antibodies. The simpler
this test the sooner appropriate hybridomas can be selected. Western blot techniques using electrophoretically separated unlabeled antigen and detection by the appropriate antibodies are often limited by the SDS sensitivity of the epitope, by nonspecificity of the polyclonal detection system and by the low concentration of the relevant antigen in the crude preparation. On the other hand, standard immunoprecipitation techniques using S. aureus, Sepharose conjugated protein A or G or other immunoaffinity matrices arc dependent on the isotypc of the antibodies used and accompanied by high non-specific binding. Above all, these common techniques are often too complex and time-consuming for easy screening of culture supernatants at an early stage of hybridoma establishment. We have therefore developed a micro-method based on a solid phase immunoisolation technique described by Tamura et al. (1984) using biotinylation instead of radiolabeling. This modification makes it possible to quantitate the amount of bound and removed antigen by developing the eluted microtiter wells with an ELISA using peroxidase-conjugated avidin. Non-eluted wells serve as a positive control. Two applications of this technique are given: the characterization of monoclonal antibodies recognizing SDS-sensitive epitopes on human and murine Clq, as well as the classification of monoclonal antibodies against human T cells according to the CD nomenclature.
Materials
and methods
Animals C57BL/6 and B A L B / c mice, originaqy from the Jackson Laboratory (Bar Harbor, ME), and L o u / c rats were raised and maintained in our breeding facilities. Initial breeding pairs of inbred strain L o u / c rats were a generous gift from Professor. Bazin (University of Louvain, Brussels) (Bazin, 1982). Antibodtes Rat or mouse Mab were established by fusion
of hyperimmune spleen cells with the mouse myeloma celt line P3X63 Ag 8.653 (K6hler and Milstein, 1975). For the production of antibodies against human T cells, rats were immunized with phytohemagglutinin-stimulated Ficoll-separated human peripheral mononucleated cells or the T cell line Molt 3 (CRL 1552, ATCC). Culture supernatants were screened for lgG2b and the established antibodies (21/61, 25/11/3, 23/5, 26/1II/17, 3 8 / I / 3 8 1 were further characterized as specific for human T cells by means of ELISA and FCA techniques. RmTI (rat IgG2b anti-murine Thy-1) (Kummer et al., 1987) was used as a non-specific control and HB90 (rat IgG2a anti-mouse lgG2, ATCC) as capture antibody for the reference antibodies Leul (mouse lgG2a anti-human CDS) and Leu9 (mouse IgG2,, anti-human CD7), both purchased from Becton Dickinson (Germany). The monoclonal antibody (RmC7H8. rat lgGl) against mouse C l q was raised and screened as previously described for RmC4B9 (rat IgM antimouse Clq) (Wasiliu et al., 19901. In brief, rats were immunized with mouse thymocytes which had been preincubated with RmTI (sec above), a MAb with high affinity for Clq, and C57BL/6 serum as a source for Clq. Screening was performed on microtiter plates coated with mouse lgG2a between which C l q was intercalated by incubation with fresh mouse serum containing 5 mM EDTA (Ziccardi, 19831. Mouse MAb against human C l q (MhC5Bg) was produced by immunizing B A L B / c mice with syngeneic thymocytes incubated with MmTI (mouse lgG2a anti-murine Thy-l.2) (Kremmer et al., 1989) and human serum. Screening was performed on rat lgG2b coated microtiter plates incubated with human s e r u m / E D T A . The following antibodies served as non-specific isotype controls: E6 (mouse IgGl, unpublished), RmT4 (rat IgM) (Kummer et al., 19871 and DNP2 (rat IgGl, kindly provided by Prof. Bazin). Antibodies were used either as culture supernatant containing 10% fetal calf serum or purified on protein G-Sepharose (Pharmacia, Germany) using the FPLC-system (Pharmacia, Germany), eluted with 0.1 M citrate pH 2.7. The eluate was dialysed against PBS and stored at - 2(1oc.
61
Antigen labeling Membrane atttigens.
Phytohemagglutininstimulated lymphocytes from surgically removed human tonsils were biotinylated with 50 #tool NHS-LC-biotin per 10" cells (Pierce, Germany). After incubation for 1 h at 4°C the cells were washed twice with 5(1 ml phosphate-buffercd saline (PBS) and finally with Tris-buffered saline (0.01 M Tris/HCl, pH 7.4, 11.15 M NaCI). 2 × 10 '~ cells were lysed with 1 ml Tris buffer containing I% NP-40, 2.5 mM iodoacetamide and 2 mM phenylmethylsulfonylfluoridc (PMSF). The lysatc was shaken for 15 min on ice and centrifuged for 15 min at 20011 × g . The supernatant was precleared by ultracentrifugation 115 min, 100,000 × g) and stored in aliquots at -80°C. Serum antigens. In order to dissociate the CI complex, 5 mM E D T A was added to crude C57BL/6 or human serum. Up to 4.2 /.tmol biotin-X-NHS (Calbiochem, Germany), dissolved in 40 /zl dimethylformamide, were added to I ml s e r u m / E D T A . After incubation for 2 h at 20°C the sample was dialysed twice against PBS containing 5 mM EDTA and centrifuged for 10 rain at 10,000 × g . Purified human C l q (Calbiochem, Germany) was treated with 11.7 ,,ttmol biotin-XN H S / m g protein in a similar way without addition of EDTA.
lmmunoadsorption and ELISA Membrane antigens. MaxiSorb
microtiter plates (Nunc, Germany) were coated (six wells/sample) with either T cell specific purified rat MAbs or HB90 as capture antibody for the reference antibodies Leul and Lcu9, which could not be bound directly to the plates because of the presence, of stabilizing proteins. For coating, each well was incubated with 50/xl PBS containing !0 # g / m l purified antibody. After 16 h at 4°C the wells were blocked with PBS containing 1% skimmed milk powder (MP, Fink, Germany). Wells coated with the capture antibody HBg0 were subsequently incubated with the mouse reference antibodies Leul and Leu9. The plates were washed twice with P B S / M P / I % NP-40, incubated with 511/×l/well of the biotinylated cell lysate, diluted 1 / 2 with P B S / M P / I % NP-40 (corresponding to 5 × I11~' cells/well), for 45 min at 20°C and extensively
washed twicc with P B S / M P / I % NP-40, twice with PBS and finally with 0.15 M NaCI. Bound biotinylatcd antigens were clt, ted by successively incubating five of the six pretreated wells per sample with 511 /zl 0.08 M sodium citrate (pH 2.7) containing 2% SDS. Samples were neutralized with 5 ~1 saturated Tris and equilibrated for elcctrophoresis by :;dding 12.5/~1 of a 0.125 M Tris/HCI buffer pH 6.8 containing 2% SDS, 311% glycerol and 11.11111%bromphenol blue. In order to check the efficiency of the binding and the detachment of the biotinylated antigens, all six wells per sample, including the non-eluted ones, were extensively washed and developed with peroxidase (HRP)-Iabeled avidin (Cammon, Gcrmany). Serum antigens. Polystyrene microtiter plates (Grcincr, Germany) were coated with either purified MAbs or diffcrcnt F(ab'), fragments of polyclonal antibodies as capture antibodies (goat anti-mouse IgG for binding of MhC5B9 or goat anti-rat lgM for the binding or RmC4B9; Dianova, Germany). lmmunoprecipitation and ELISA were performed as described above with the exception that PBS was used for washing and normal electrophoresis sample buffer (0.025 M Tris/HCI, pH 6.8, 111%glycerol, 2% SDS, 0.1)01% bromphenol blue) was used for elution of the antigens.
Immunoprecipitation Immunoprecipitation was performed according to the procedure of Harlow and Lane (1988) using protein G-Scpharose (Pharmacia, Germany). For cach sample 25 /~1 of packed beads were washed five times with 400 pA PBS by centrifugation through a 0.45 tzm filter unit (Ultrafree-MC, Millipore, Germany) to remove the ethanol. The beads were then incubated with I00 /.tg purified antibody in 3(111/zl PBS. After shaking for 45 min at 20°C the beads were washed three times with P B S / I % NP-40 and incubated with 150 tzl of the biotinylated T eel[ lysate (corresponding to 3 × 107 cells/sample). After shaking for an additional 45 min at 20°C the lysate was separated from the beads by centrifugation through the filter unit. In order to reduce non-specific binding some samples were precleared by incubation with pro-
rein G-Sepharose linked to an irrelevant rat IgG2b MAb ( R m T I ) prior to treatment with the protein G-linked specific MAb. After incubation with the lysate all samples were extensively washed with P B S / I % NP-41) and PBS. The antigen was eluted by adding 25/.tl electrophoresis sample buffer, heating for 5 min at 10fi°C and centrifugation through the 0.45/~m filter unit.
151ectrophoresis atul #mmmohlottblg Samples (25/11) were used either non-reduced or reduced with (I.1 M dithiothreitol (DTT). All samples were hca~cd at 100°C for 5 rain and subjected to gradient SDS-PAGE according to Laemmli (1970) in a minigel apparatus (Hocfer Scientific Instruments, USA). Low molecular weight standard (Bio-Rad, Germany) was diluted 1 / 4 with sample buffer. For electrophorcsis of sanlplcs containing citrate the standards were adjusted to the same conccntration by adding 5 htl 0.3 M citrate pH 6.0;. The separated proteins wcre transferred at 60 V and 4°C for 60 rain onto nitrocellulose in a transfer chamber with blotting buffer (0.025 M Tris, 0.192 M glycine, 211% methanol). The nitrocellulose mcmbrane was blocked with P B S / M P . For the detection of biotinylated antigens blots were incubated tor 45 min in 30 ml P B S / M P containing 30 /.tl alkaline phosphatase (AP)-conjugated avidin (Diamwa. Germany) or HRP-conjugated avidin (Common, Germany). After three additional washing steps, blots labeled with alkaline phosphatase were developed with Fast Red (10 mg in 50 ml 50 mM Tris/HCI, pH 8.3) and naphthoI-AS-MX-phosph;ne (2 mg in 2111) /zl dimethylsulfoxidc). The reagents were mixed just before use and squeezed through a 0.22/zm filter (Schr6dcr ct al., 1989; Kunz, personal communication). Blots labeled with peroxidase were developed with diaminobenzidinetetrahydrochloridc (DAB) and H 2 0 , (Harlow and Lane, It)88).
Rescflts
Bi¢)tmy&tion of serum Jor detection q[" Clq Since serum components cannot bc treated with a prccisc molar excess of biotin, the opti-
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5 6 7 8 g 10 Biolmylaled Serum [log 2 Dilution] (,
2.1pmoles
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Fig. I. Efficiency of the biotinylation of mouse serum. Mouse serum was labeled with the designated amounts of Biotin-X-
NItS per ml serum and titrated on RmC7118-coatcd nilcrotitcr plales. Plates were developed in an ELISA procedure using I IRP-conjugaled avidin.
mum concentration had to be determined. Therefore three different amounts of Biotin-X-NHS were dissolved in DMF and added to 1 ml aliquots of serum. All samples were titrated on microtiter plates coated with anti-Clq antibody and these titration curves arc shown in Fig. 1. The highest conecntration of biotin was used for further studies.
hnmtmoadsotption to microtiter plates attd ehttion of the specifically bound antigens One of the most important steps in the immunoadsorption technique is the evaluation of the efficiency of the elution of the specifically captured antigen. Fig. 2 shows the presence of biotinylatcd antigen from solubilized lymphocytes after treatment with various buffers. PBS containing I% NP-40 did not affect the amount of bound antigen. Electrophorcsis sample buW.-: (pH 6.8) containing 2% SDS was sufficient to dissociate most, but not all, antibody/antigen combinations in the wells, whereas a citrate buffer (11.08 M, pH 2.7) containing 2% SDS was able to detach a significant part of the antigen in all cases. These conditions also removed 30-50% of the coated antibody (data not shown) but since the antibodies were unlabeled this did not disturb the results of the Wcstern blot.
Comparison between the immunoadsorption atul immunoprecipitation techniques The immunoadsorption technique rcvcaled distinct bands of antigen purified by the specific antibodies ( 3 8 / ! / 3 8 , 2 3 / 5 ) and just weak background staining when the irrelevant RmTI was used (see Fig. 3). In contrast, the immunoprccipitation procedure gave rise to high background staining with just faint bands of specifically precipitated material, although approximately similar numbers of lysed cells were used per sample for both techniques. To our surprise we observed no difference whether the 2 3 / 5 sample was precleared or not.
Characterization of antibody specificity agabtst T cell atttigens The results of a representative experiment (Fig. 4) revealed identical bands for 21/61, 26/111/17, Leul (69 kDa) and for 23/5, 25/11/3, Leu9 (38 kDa) respectively. This simplc proccdure confirmed the specificity of 21/61 and 26/111/17 as CD5 and 2 3 / 5 and 2 5 / 1 1 / 3 as CD7 (Knowles, 1986). It is interesting to note that, in contrast to the results shown in Fig. 4, the immunoadsorption with 2 3 / 5 shown Fig. 3 revealed a second band at 1 25'
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Fig, 3. Western blot after electrophorcsis (10-15c~ gradient gel) under non-reducing conditions in order to compare the immunoadsorption technique (IA)with the immunoprecipitafion technique tIP). For immunoadr~rption the wells were coated with the designated anti-humap T cell antibodies (23/5. 38/1/38) or with an irrelevant conlrol antibody (RmTI) and incubated with solubilizcd biotinylated human lymphocytes. For immunoprecipitation protein G-Sepharose was loaded with the designated specific (23/51 or non-specific control antibody (RmTI) and incubated with solubilized biotinylated human lymphocytcs. The preclcared 23/5 sample was preincubated with RmTI-saturated protein G-Sepharose. Blots were stained by AP-conjugated avidin with Fast Red. LMW: prcstaincd low molecular weight marker.
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Fig. 2. Effect o f different p H and detergents on the removal o f bound biofinylatcd antigen. T h e wells were coated with the
anti-human T cell antibodies (26/111/17, 21/61. 23/5. 25/11/3), with a non-specific control antihudy (RmTI), with a capture antibody for mouse IgG2 (ItBO0) or not coated at all
(-). The microtiter plat,.., were incubated with solubilized biotinylated human lymphocytes and washed with the designated buffers (PBS; PBS/V;; NP-4II;Tris/H('l/2(f SDS. p[I 6.8:0.(18 M citrate/2C; SDS, pit 2.7). PhltcSwere developed in an ELISA procedure using tlRP-conjugated avidin.
80 kDa. This band was also present under reducing conditions (data not shown). The difference between these two results cannot be explained by any experimental change apart from the fact that different T cell lysates were used. Nevertheless, 3 8 / ! / 3 8 recognized the same antigen as 2 3 / 5 and therefore can be classified to the CD5 cluster. Fig. 5 shows the results obtained with ELISA for quantitation of the bound and detached biotinylatcd antigens. Wells with and without SOS incubation were compared. The second layer system for Leu9 showed that less antigen was bound and finally released by SDS than in the RmTIcoated control. Interestingly, the small amount of specific antigen in the Leu9 sample gave rise to
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Fig. 4. Western blot after electrophorcsis (10-15¢.~ gradient gel) under non-reducing conditions o f immunoadsorbed samples.
Microtiler plates were L~,.!ed with the designated anti-human T cell antibodies (26/111/17, 21/61, 23/5, 25/11/3) and incubated with solubilized biotinylated human lymphocytes; Leul and Leu9 were captured by coated HBgO. RrnTl-coated and uncoated wells (PBS) served as controls. Blots were stained by AP-conjugated avidin with Fast red. LMW: prestained low molecular weight marker. s u f f i c i e n t m a t e r i a l for W e s t e r n b l o t t i n g a n d s t a i n ing (Fig. 4).
s h o w n in Fig. 6. A s C l q m a y i n t e r a c t w i t h t h e Fc parts of the ,immunoglobulins, antibodies with identical subclasses but irrelevant specificities
Characterization of antibody specificity against mouse attd human Clq The results of immunoadsorption of biotinyl a t e d h u m a n o r m o u s e s e r u m to a n t i b o d i e s w i t h a p r e s u m e d s p e c i f i c i t y for h u m a n o r m o u s e C l q a r e
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Fig. 5. Q u a n l i t a t i o n
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the m i c r o t i t e r p l a t e s used f o r i m m u n o a d s o r p t i o n
of hiotiny-
lated human T cell lysate. The wells were coated as described in Fig. 3, Individual wells, before and after incubation with the citrate buffer (pit 2.7) containing 29;: SDS, were extensively washed and developed in an ELISA procedure using HRP-conjugated avidin.
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Fig. 6. Western blot after elcctrophoresis (8-15f$ gradient gel) of immunoadsorbed samples under reducing and non-reducing conditions. Microtiter plates were coated with the designated antibodies either directly (RmCTH8, DNP2) or via appropriate capture antibodies (E6, MhC5Bg, RmT4, RmC4Bg). The wells were incubated with biotinylated human (E6, MhC5Bg) or mouse serum (RmT4, RmC4Bg, RmC7tt8, DNP2); biotinylated human Clq served as reference. E6, RmT4 and DNP2 as non-specific isotype controls. Blots were stained by HRP-conjugated avidin with DAB. LMW: prestained low molecular weight marker.
were used as controls: E6, mouse lgGl; RmT4, rat lgM; DNP2, rat lgGl. These control antibodies showed only poor background staining. Electrophoretic separation of purified biotinylated human C l q has been shown to result in two major bands under reducing conditions (34 kDa for the A and B chain and 25 kDa for the C chain) and non-reducing conditions (66 kDa for the A-B dimer and 44 kDa for the C-C dimcr) (Stemmer and Loos, 19841. The same molecules were precipitated by MhC5B9, confirming its specificity for human Clq; the extra band at 62 kDa does not correspond to any known chain of the C l q macromolecule, but seems to dissociate into A, B or C monomers after reduction. The reduced C l q sample also contained some faint contamination with non-reduced dimers. I m m u n o a d s o r p t i o n of crude biotinylatcd mouse serum with RmC4B9 and RmC7H8 gave rise to identical main bands for both antibodies under reducing (34 kDa for the A chain, 30 kDa for the B chain and 25 kDa for the C chain) as well as under non-reducing conditions (62.5 kDa for the A-B dimer and 46 kDa for the C-C dimer). The additional band at 55 kDa of unknown specificity has also been described by S t e m m e r et al. (19841. F u r t h e r m o r e , the RmC7H8-adsorbed sample, particularly, showed a high molecular weight band at about 201) kDa under reducing and non-reducing conditions. For all rat antibodies contaminating bands of 80 and 55 kDa were seen under reducing conditions and were interpreted as probably non-reduced dimers. In order to control the efficiency of the immunoadsorption the amount of bound antigen before and after SDS-elution was quantitated as shown in Fig. 7.
Discussion Characterization of monoclonal antibodies by standard Western blotting of unlabeled antigens and detection via a second layer system is often limited by the SDS-sensitivity of the corresponding epitope. This was true for RmC7H8 and RmC4B9 which were thought to have the same specificity as RmCI3C9 for mouse C l q (Wasiliu et al., 1990) but failed to stain blotted serum. A
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Fig. 7. Ouantitatiorl ~1"antigen bound to ~md detached from the microtiter phltes used for immunoadsorption of biotinylated serum. The wells were coated as described in Fig. 5. Individual wells, heft,re and after incubation wilh electrophoresis buffer containing 2% SDS, were extensively washed and developed in an ELISA procedure usi~tg HRPconjugated avidin. general, simple and reliable method for determining the specificity of MAb at an early stage in the production of hybridomas wouid be extremely valuable. The micro-method described here fvlfils these demands: it combines a non-radioactive labeling tecbnique using biotin (Cole et id.. 1987) and the use of antibody-coated microtiter plates as an immunoaffinity matrix {Tamura t:t al., 19841. The advantage of biotinylation over internally or externally labeling with radioactive nuclides resides in the long storage time as well as in the simple and harmless nature of the procedure. Corwenicnt i m m u n o p r e c i p i ~ i o n techniques using S. altretts, Sephzrose-conjugated protein A / G or other immunoaffinity matrices are often limited by n~',n-specific binding, which has to be reduced by various incubations with the matrix alone or in combination with ir:elevant antibodies (Knowles, 19861. Using the mcth,-,d described here this problem can easii;.' be ove~ :ome by blocking and repeated washing steps. A e-~mparison between the two techniques is shown in Fig. 3. Although the immunoprecipitation has no, been optimized with respect to incubation and washing procedures, the figure clcarly shows the advantage ef the immunoadsorption technique ovcr a standard immunoprecipitation method. In the case of crude serum preparations the
use of protein A / G - S e p h a r o s e is not to be reco m m e n d e d , b e c a u s e the biotinylated s e r u m imm u n o g l o b u l i n s c o m p e t e with the u n l a b e l e d capture antibody for binding to the i m m u n o a f f i n i t y matrix. Alternative a p p r o a c h e s , such as the p r e p a r a t i o n o f an i m m u n o g l o b u l i n - f r c e antigen, the covalent binding o f the antibody to protein A / G or direct coupling of purified antibodies to activated matrices are r a t h e r difficult a n d therefore not applicable for rapid antibody screening. Moreover, the binding o f protein A a n d proteip, G is restricted to selected isotypes. Rat M A b s do not react with protein A at all a n d only partially with protein G ( P h a r m a c i a , G e r m a n y ; product information). O n t h e o t h e r h a n d t h e r e is hardly any isotype restriction w h e n coating to polystyrene microtitcr plates. It is t h e r e f o r e possible to use a n d c o m p a r e antibodies o f different isotype. T h e ability of protein A- or G - S e p h a r o s e to bind u n p u r i f i e d MAb, for example, from culture s u p e r n a t a n t s also holds for the i m m u n o a d sorption t e c h n i q u e described here, since t h e polystyrene surface can be coated with polyclonal a n d m o n o c l o n a l c a p t u r e antibodies or with protein A (Randall et al., 1983) or G (data not shown). T h e c o m b i n a t i o n of microtiter plates a n d biotin labeling also p e r m i t s q u a n t i t a t i o n of the a m o u n t of b o u n d a n t i g e n by d e v e l o p i n g the plates according to s t a n d a r d E L I S A m e t h o d s with H R P - c o n j u g a t e d avidin. T h e c o m p a r i s o n of appropriate wells before or after removal of the specific a n t i g e n by SDS h c l p s to establish the optimal p a r a m e t e r s for i m m u n o a d s o r p t i o n without t h e n e e d for carrying out the whole electrophoretic a n d blotting p r o c e d u r e . W e were able to optimize the system for different a n t i g e n s with regard to eh.tion conditions a n d biotin labeling. Since individual crude a n t i g e n p r e p a r a t i o n s often diffcr with respect to the c o n c e n t r a t i o n , specific label a n d biochemical p r o p e r t i e s of an individual antigen, wc routinely include as a control a p p r o p r i a t e a n t i g e n - i n c u b a t e d wells without SDS elution.
Acknowl¢dgement T h e a u t h o r s wish to acknowlege the excellent technical assistance provided by U. M e i n c k e n , M.
Griel3er, U. Erler a n d V. H u n k e . W e t h a n k Drs. N. S h e a r m a n , R. Mocikat a n d E. L e d e r e r for helpful discussion. T h i s work was s u p p o r t e d by a g r a n t from B M F T 0702571,
References Bazin. H. (1982) Production of rat monoclonal antibodies with the Lou rat nun secreting IR 983F myeloma cell line. Prof. Biol. Fluids 29. 615. Cole. S.R., Ashman, L.K. and Ey, P.L. (1987) Biotinylation: an alternative to radioiodination for the identification of cell surface antigens in immunoprecipitates. Mol. Immunol. 24, 699. Harlow, E. and Lane. D. (1988) Antibodies - a laboratory manual, Cold Spring Itarbor Laboratory, Cold Spring Harbor. NY, p. 508. Knowles. R.W. (1986) Immunoehemical analysis of T cellspecific antigens. In: E. Reinherz (Ed.), Leucocyte Typing~ 11, Vol. 2. Springer, New York. p. 259. Kiihler. G. and Milstein. C (1975) Continuous cultures of fused cells secreting antibody of predefined specificity. Nature 256. 495. Kremmer, E., Th[erfelder, S., Kummer. U., Lederer, R. and Mysliwietz, J. (1989) Neutralization of immunosuppression by antibodies against variable as well as constant regions of monoclonal anti-Thy-I xenoantibodies and their ability to be suppressed by initial T cell depletion. Transplantation 47, 641. Kummer. U., Thierfelder. S.. Hoffman-Fezer, G. and Sehuh, R. (1987) In vivo immunosuppression by pan-T cell antibodies rehaes to their isotype and their Chl uptake. J. lmmunol. 138. 4(169. Laemmli, U.K. (1971)) Cleavage of s!ructural proteins during the assembly of the head of bacteriophage T4. Nature 227, 68(1. Randall, R.E. (1983) Preparation and use of immunoabsorbent monolayers in purificatiun of virus proteins and separation of cells on the basis of their cell surface antigens. J. Immunol. Methods 611. 147. Schr(ider, J., Symmons, P. and Kunz, W. (1989) Immunenzym~tische Farbkodierung zur Erkennung yon DNA-Klonen in Expressionsvektor-Bibliotheken. BioEngineering 89, 24. Stemmer, F. and Lops. M. (1984) Purification and characterization of human, guinea pig and mouse Clq by fast protein liquid chromatography (FPLCI. J. lmmunol. Methods 74, 9. Tamura. G.S., Daily, M.O., Gallatin, W.M., McGrath, M.S., Weissman, J.L. and Pillemer, E.A. (1984) Isolation of molecules recognized by monoclunal antibodies and antisera: the solid phase immunoisolation technique. Anal. Biochem. 136, 458. Wasiliu, M.. Kremmer. E., Thierfelder, S., Felber, E,. Hoffmann-Fezer, G. and Kummer, U. (19891 Monoclonal anti-
bodies to complc'~ent components without the nccd of their prior purification. 1. Antibodies to mouse Clq. tlybridoma 8, 165.
Ziccardi, R.J. { 1983) Nature of the metal ion requirement Ik)r assembly and function of the first component of comp|cmont. J. Biol. ('hem. 258. 6187.
Journal ofhnmunoh~gical Methods.
59
JIM 0fi355
Determination of monoclonal antibody specificity by immunoadsorption and Western blotting R o l f S c h u h , E l i s a b e t h K r e m m e r , Evelyn Ego, M i c h a c l Wasiliu a n d S t e f a n T h i e r f e l d c r (;SF-hzstitut .fiir hnmunologie, 8000 Munich, Germany
(Received 311(.)cipher 1991, revised received 6 January 1992,accepted 21 February 19921
A rapid and simple method has been developed for identifying the specificity of monoclonal antibodies at an early stage in the production of hybridomas. The technique is a micro-method utilizing biotinylated crude antigen and the surface of microtiter plates as an immunoaffinity matrix. The monoclonal antibodies to be tested are adsorbed to the microtiter wells and incubated with the labeled antigen preparation. Non-specific binding can be reduced by blocking and repeated washing steps. The specific antigen is then eluted by SDS-containing buffers, subjected to S D S - P A G E blotted onto nitrocellulose and detected by enzyme-labeled avidin in a Western blot assay. The amount of bound and removed antigen can be quantitated by developing eluted and non-eluted control wells by EL1SA techniques. Since this ELISA can be performed rapidly, only samples which contain sufficient specific material can be selected tor eleetrophoresis and blotting. The major advantages of the technique are (i) the use of a non-radioactive label resulting in an easy and time-saving procedure, (ii) the possibility of quantitating the amount of captured and detached antigen by ELISA, (iii) the need for only a minimal amount of antigen, (iv) the use of unpurified antibodies of all isotypes, (v) a high signal-to-noise ratio, and (vi) as with all immunoprecipitation techniques, the possibility of detecting SDS-sensitive epitopes and of using crude antigen preparations. Using this method we were able to characterize monoclonal antibodies against both soluble proteir"; (mouse and human Clq) and membrane determinants (human pan T ccll CD5 and CD7), Key words:
Monoclonal antibody; Biotinylation:ELISA; Immunoadsorption:Western blotting
Correspomh,nce to: R. Schuh. GSF-Forschungszentrumfiir Umwelt und Gesundheit GmbH, lnstitut fiir Immunoh)gie, Marchionin',raBe 25, 8111111Munich 711,Germany. Ahbreciations: AP, alkaline phosphatase: ATCC. American Type Culture Collection; DMF, dimethyllk~rmamide: EDTA, ethylenediaminetctraacetic acid: ELISA. enzyme-linked immunosorbent assay: FCA, flow cytometric analysis: HRP, horseradish pcroxidase; MAb, monocional antibody; MP, skimmed milk powder: MW, molecular weight; PAGE, polyacrylamide gel elcctrophoresis; PBS. phosphatc-buffi:red saline: SDS, sodium d~idecylsulfate.
Introduction
Immunization, fusion and cell culture no longer rose particular problems in the production of monoclonal antibodies. However, depending on the availability of purified antigen, it is often difficult to determine the molecular weight of the corresponding antigen and, consequently, to define the specificity of the antibodies. The simpler
this test the sooner appropriate hybridomas can be selected. Western blot techniques using electrophoretically separated unlabeled antigen and detection by the appropriate antibodies are often limited by the SDS sensitivity of the epitope, by nonspecificity of the polyclonal detection system and by the low concentration of the relevant antigen in the crude preparation. On the other hand, standard immunoprecipitation techniques using S. aureus, Sepharose conjugated protein A or G or other immunoaffinity matrices arc dependent on the isotypc of the antibodies used and accompanied by high non-specific binding. Above all, these common techniques are often too complex and time-consuming for easy screening of culture supernatants at an early stage of hybridoma establishment. We have therefore developed a micro-method based on a solid phase immunoisolation technique described by Tamura et al. (1984) using biotinylation instead of radiolabeling. This modification makes it possible to quantitate the amount of bound and removed antigen by developing the eluted microtiter wells with an ELISA using peroxidase-conjugated avidin. Non-eluted wells serve as a positive control. Two applications of this technique are given: the characterization of monoclonal antibodies recognizing SDS-sensitive epitopes on human and murine Clq, as well as the classification of monoclonal antibodies against human T cells according to the CD nomenclature.
Materials
and methods
Animals C57BL/6 and B A L B / c mice, originaqy from the Jackson Laboratory (Bar Harbor, ME), and L o u / c rats were raised and maintained in our breeding facilities. Initial breeding pairs of inbred strain L o u / c rats were a generous gift from Professor. Bazin (University of Louvain, Brussels) (Bazin, 1982). Antibodtes Rat or mouse Mab were established by fusion
of hyperimmune spleen cells with the mouse myeloma celt line P3X63 Ag 8.653 (K6hler and Milstein, 1975). For the production of antibodies against human T cells, rats were immunized with phytohemagglutinin-stimulated Ficoll-separated human peripheral mononucleated cells or the T cell line Molt 3 (CRL 1552, ATCC). Culture supernatants were screened for lgG2b and the established antibodies (21/61, 25/11/3, 23/5, 26/1II/17, 3 8 / I / 3 8 1 were further characterized as specific for human T cells by means of ELISA and FCA techniques. RmTI (rat IgG2b anti-murine Thy-1) (Kummer et al., 1987) was used as a non-specific control and HB90 (rat IgG2a anti-mouse lgG2, ATCC) as capture antibody for the reference antibodies Leul (mouse lgG2a anti-human CDS) and Leu9 (mouse IgG2,, anti-human CD7), both purchased from Becton Dickinson (Germany). The monoclonal antibody (RmC7H8. rat lgGl) against mouse C l q was raised and screened as previously described for RmC4B9 (rat IgM antimouse Clq) (Wasiliu et al., 19901. In brief, rats were immunized with mouse thymocytes which had been preincubated with RmTI (sec above), a MAb with high affinity for Clq, and C57BL/6 serum as a source for Clq. Screening was performed on microtiter plates coated with mouse lgG2a between which C l q was intercalated by incubation with fresh mouse serum containing 5 mM EDTA (Ziccardi, 19831. Mouse MAb against human C l q (MhC5Bg) was produced by immunizing B A L B / c mice with syngeneic thymocytes incubated with MmTI (mouse lgG2a anti-murine Thy-l.2) (Kremmer et al., 1989) and human serum. Screening was performed on rat lgG2b coated microtiter plates incubated with human s e r u m / E D T A . The following antibodies served as non-specific isotype controls: E6 (mouse IgGl, unpublished), RmT4 (rat IgM) (Kummer et al., 19871 and DNP2 (rat IgGl, kindly provided by Prof. Bazin). Antibodies were used either as culture supernatant containing 10% fetal calf serum or purified on protein G-Sepharose (Pharmacia, Germany) using the FPLC-system (Pharmacia, Germany), eluted with 0.1 M citrate pH 2.7. The eluate was dialysed against PBS and stored at - 2(1oc.
61
Antigen labeling Membrane atttigens.
Phytohemagglutininstimulated lymphocytes from surgically removed human tonsils were biotinylated with 50 #tool NHS-LC-biotin per 10" cells (Pierce, Germany). After incubation for 1 h at 4°C the cells were washed twice with 5(1 ml phosphate-buffercd saline (PBS) and finally with Tris-buffered saline (0.01 M Tris/HCl, pH 7.4, 11.15 M NaCI). 2 × 10 '~ cells were lysed with 1 ml Tris buffer containing I% NP-40, 2.5 mM iodoacetamide and 2 mM phenylmethylsulfonylfluoridc (PMSF). The lysatc was shaken for 15 min on ice and centrifuged for 15 min at 20011 × g . The supernatant was precleared by ultracentrifugation 115 min, 100,000 × g) and stored in aliquots at -80°C. Serum antigens. In order to dissociate the CI complex, 5 mM E D T A was added to crude C57BL/6 or human serum. Up to 4.2 /.tmol biotin-X-NHS (Calbiochem, Germany), dissolved in 40 /zl dimethylformamide, were added to I ml s e r u m / E D T A . After incubation for 2 h at 20°C the sample was dialysed twice against PBS containing 5 mM EDTA and centrifuged for 10 rain at 10,000 × g . Purified human C l q (Calbiochem, Germany) was treated with 11.7 ,,ttmol biotin-XN H S / m g protein in a similar way without addition of EDTA.
lmmunoadsorption and ELISA Membrane antigens. MaxiSorb
microtiter plates (Nunc, Germany) were coated (six wells/sample) with either T cell specific purified rat MAbs or HB90 as capture antibody for the reference antibodies Leul and Lcu9, which could not be bound directly to the plates because of the presence, of stabilizing proteins. For coating, each well was incubated with 50/xl PBS containing !0 # g / m l purified antibody. After 16 h at 4°C the wells were blocked with PBS containing 1% skimmed milk powder (MP, Fink, Germany). Wells coated with the capture antibody HBg0 were subsequently incubated with the mouse reference antibodies Leul and Leu9. The plates were washed twice with P B S / M P / I % NP-40, incubated with 511/×l/well of the biotinylated cell lysate, diluted 1 / 2 with P B S / M P / I % NP-40 (corresponding to 5 × I11~' cells/well), for 45 min at 20°C and extensively
washed twicc with P B S / M P / I % NP-40, twice with PBS and finally with 0.15 M NaCI. Bound biotinylatcd antigens were clt, ted by successively incubating five of the six pretreated wells per sample with 511 /zl 0.08 M sodium citrate (pH 2.7) containing 2% SDS. Samples were neutralized with 5 ~1 saturated Tris and equilibrated for elcctrophoresis by :;dding 12.5/~1 of a 0.125 M Tris/HCI buffer pH 6.8 containing 2% SDS, 311% glycerol and 11.11111%bromphenol blue. In order to check the efficiency of the binding and the detachment of the biotinylated antigens, all six wells per sample, including the non-eluted ones, were extensively washed and developed with peroxidase (HRP)-Iabeled avidin (Cammon, Gcrmany). Serum antigens. Polystyrene microtiter plates (Grcincr, Germany) were coated with either purified MAbs or diffcrcnt F(ab'), fragments of polyclonal antibodies as capture antibodies (goat anti-mouse IgG for binding of MhC5B9 or goat anti-rat lgM for the binding or RmC4B9; Dianova, Germany). lmmunoprecipitation and ELISA were performed as described above with the exception that PBS was used for washing and normal electrophoresis sample buffer (0.025 M Tris/HCI, pH 6.8, 111%glycerol, 2% SDS, 0.1)01% bromphenol blue) was used for elution of the antigens.
Immunoprecipitation Immunoprecipitation was performed according to the procedure of Harlow and Lane (1988) using protein G-Scpharose (Pharmacia, Germany). For cach sample 25 /~1 of packed beads were washed five times with 400 pA PBS by centrifugation through a 0.45 tzm filter unit (Ultrafree-MC, Millipore, Germany) to remove the ethanol. The beads were then incubated with I00 /.tg purified antibody in 3(111/zl PBS. After shaking for 45 min at 20°C the beads were washed three times with P B S / I % NP-40 and incubated with 150 tzl of the biotinylated T eel[ lysate (corresponding to 3 × 107 cells/sample). After shaking for an additional 45 min at 20°C the lysate was separated from the beads by centrifugation through the filter unit. In order to reduce non-specific binding some samples were precleared by incubation with pro-
rein G-Sepharose linked to an irrelevant rat IgG2b MAb ( R m T I ) prior to treatment with the protein G-linked specific MAb. After incubation with the lysate all samples were extensively washed with P B S / I % NP-41) and PBS. The antigen was eluted by adding 25/.tl electrophoresis sample buffer, heating for 5 min at 10fi°C and centrifugation through the 0.45/~m filter unit.
151ectrophoresis atul #mmmohlottblg Samples (25/11) were used either non-reduced or reduced with (I.1 M dithiothreitol (DTT). All samples were hca~cd at 100°C for 5 rain and subjected to gradient SDS-PAGE according to Laemmli (1970) in a minigel apparatus (Hocfer Scientific Instruments, USA). Low molecular weight standard (Bio-Rad, Germany) was diluted 1 / 4 with sample buffer. For electrophorcsis of sanlplcs containing citrate the standards were adjusted to the same conccntration by adding 5 htl 0.3 M citrate pH 6.0;. The separated proteins wcre transferred at 60 V and 4°C for 60 rain onto nitrocellulose in a transfer chamber with blotting buffer (0.025 M Tris, 0.192 M glycine, 211% methanol). The nitrocellulose mcmbrane was blocked with P B S / M P . For the detection of biotinylated antigens blots were incubated tor 45 min in 30 ml P B S / M P containing 30 /.tl alkaline phosphatase (AP)-conjugated avidin (Diamwa. Germany) or HRP-conjugated avidin (Common, Germany). After three additional washing steps, blots labeled with alkaline phosphatase were developed with Fast Red (10 mg in 50 ml 50 mM Tris/HCI, pH 8.3) and naphthoI-AS-MX-phosph;ne (2 mg in 2111) /zl dimethylsulfoxidc). The reagents were mixed just before use and squeezed through a 0.22/zm filter (Schr6dcr ct al., 1989; Kunz, personal communication). Blots labeled with peroxidase were developed with diaminobenzidinetetrahydrochloridc (DAB) and H 2 0 , (Harlow and Lane, It)88).
Rescflts
Bi¢)tmy&tion of serum Jor detection q[" Clq Since serum components cannot bc treated with a prccisc molar excess of biotin, the opti-
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Fig. I. Efficiency of the biotinylation of mouse serum. Mouse serum was labeled with the designated amounts of Biotin-X-
NItS per ml serum and titrated on RmC7118-coatcd nilcrotitcr plales. Plates were developed in an ELISA procedure using I IRP-conjugaled avidin.
mum concentration had to be determined. Therefore three different amounts of Biotin-X-NHS were dissolved in DMF and added to 1 ml aliquots of serum. All samples were titrated on microtiter plates coated with anti-Clq antibody and these titration curves arc shown in Fig. 1. The highest conecntration of biotin was used for further studies.
hnmtmoadsotption to microtiter plates attd ehttion of the specifically bound antigens One of the most important steps in the immunoadsorption technique is the evaluation of the efficiency of the elution of the specifically captured antigen. Fig. 2 shows the presence of biotinylatcd antigen from solubilized lymphocytes after treatment with various buffers. PBS containing I% NP-40 did not affect the amount of bound antigen. Electrophorcsis sample buW.-: (pH 6.8) containing 2% SDS was sufficient to dissociate most, but not all, antibody/antigen combinations in the wells, whereas a citrate buffer (11.08 M, pH 2.7) containing 2% SDS was able to detach a significant part of the antigen in all cases. These conditions also removed 30-50% of the coated antibody (data not shown) but since the antibodies were unlabeled this did not disturb the results of the Wcstern blot.
Comparison between the immunoadsorption atul immunoprecipitation techniques The immunoadsorption technique rcvcaled distinct bands of antigen purified by the specific antibodies ( 3 8 / ! / 3 8 , 2 3 / 5 ) and just weak background staining when the irrelevant RmTI was used (see Fig. 3). In contrast, the immunoprccipitation procedure gave rise to high background staining with just faint bands of specifically precipitated material, although approximately similar numbers of lysed cells were used per sample for both techniques. To our surprise we observed no difference whether the 2 3 / 5 sample was precleared or not.
Characterization of antibody specificity agabtst T cell atttigens The results of a representative experiment (Fig. 4) revealed identical bands for 21/61, 26/111/17, Leul (69 kDa) and for 23/5, 25/11/3, Leu9 (38 kDa) respectively. This simplc proccdure confirmed the specificity of 21/61 and 26/111/17 as CD5 and 2 3 / 5 and 2 5 / 1 1 / 3 as CD7 (Knowles, 1986). It is interesting to note that, in contrast to the results shown in Fig. 4, the immunoadsorption with 2 3 / 5 shown Fig. 3 revealed a second band at 1 25'
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anti-human T cell antibodies (26/111/17, 21/61. 23/5. 25/11/3), with a non-specific control antihudy (RmTI), with a capture antibody for mouse IgG2 (ItBO0) or not coated at all
(-). The microtiter plat,.., were incubated with solubilized biotinylated human lymphocytes and washed with the designated buffers (PBS; PBS/V;; NP-4II;Tris/H('l/2(f SDS. p[I 6.8:0.(18 M citrate/2C; SDS, pit 2.7). PhltcSwere developed in an ELISA procedure using tlRP-conjugated avidin.
80 kDa. This band was also present under reducing conditions (data not shown). The difference between these two results cannot be explained by any experimental change apart from the fact that different T cell lysates were used. Nevertheless, 3 8 / ! / 3 8 recognized the same antigen as 2 3 / 5 and therefore can be classified to the CD5 cluster. Fig. 5 shows the results obtained with ELISA for quantitation of the bound and detached biotinylatcd antigens. Wells with and without SOS incubation were compared. The second layer system for Leu9 showed that less antigen was bound and finally released by SDS than in the RmTIcoated control. Interestingly, the small amount of specific antigen in the Leu9 sample gave rise to
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Microtiler plates were L~,.!ed with the designated anti-human T cell antibodies (26/111/17, 21/61, 23/5, 25/11/3) and incubated with solubilized biotinylated human lymphocytes; Leul and Leu9 were captured by coated HBgO. RrnTl-coated and uncoated wells (PBS) served as controls. Blots were stained by AP-conjugated avidin with Fast red. LMW: prestained low molecular weight marker. s u f f i c i e n t m a t e r i a l for W e s t e r n b l o t t i n g a n d s t a i n ing (Fig. 4).
s h o w n in Fig. 6. A s C l q m a y i n t e r a c t w i t h t h e Fc parts of the ,immunoglobulins, antibodies with identical subclasses but irrelevant specificities
Characterization of antibody specificity against mouse attd human Clq The results of immunoadsorption of biotinyl a t e d h u m a n o r m o u s e s e r u m to a n t i b o d i e s w i t h a p r e s u m e d s p e c i f i c i t y for h u m a n o r m o u s e C l q a r e
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of hiotiny-
lated human T cell lysate. The wells were coated as described in Fig. 3, Individual wells, before and after incubation with the citrate buffer (pit 2.7) containing 29;: SDS, were extensively washed and developed in an ELISA procedure using HRP-conjugated avidin.
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Fig. 6. Western blot after elcctrophoresis (8-15f$ gradient gel) of immunoadsorbed samples under reducing and non-reducing conditions. Microtiter plates were coated with the designated antibodies either directly (RmCTH8, DNP2) or via appropriate capture antibodies (E6, MhC5Bg, RmT4, RmC4Bg). The wells were incubated with biotinylated human (E6, MhC5Bg) or mouse serum (RmT4, RmC4Bg, RmC7tt8, DNP2); biotinylated human Clq served as reference. E6, RmT4 and DNP2 as non-specific isotype controls. Blots were stained by HRP-conjugated avidin with DAB. LMW: prestained low molecular weight marker.
were used as controls: E6, mouse lgGl; RmT4, rat lgM; DNP2, rat lgGl. These control antibodies showed only poor background staining. Electrophoretic separation of purified biotinylated human C l q has been shown to result in two major bands under reducing conditions (34 kDa for the A and B chain and 25 kDa for the C chain) and non-reducing conditions (66 kDa for the A-B dimer and 44 kDa for the C-C dimcr) (Stemmer and Loos, 19841. The same molecules were precipitated by MhC5B9, confirming its specificity for human Clq; the extra band at 62 kDa does not correspond to any known chain of the C l q macromolecule, but seems to dissociate into A, B or C monomers after reduction. The reduced C l q sample also contained some faint contamination with non-reduced dimers. I m m u n o a d s o r p t i o n of crude biotinylatcd mouse serum with RmC4B9 and RmC7H8 gave rise to identical main bands for both antibodies under reducing (34 kDa for the A chain, 30 kDa for the B chain and 25 kDa for the C chain) as well as under non-reducing conditions (62.5 kDa for the A-B dimer and 46 kDa for the C-C dimer). The additional band at 55 kDa of unknown specificity has also been described by S t e m m e r et al. (19841. F u r t h e r m o r e , the RmC7H8-adsorbed sample, particularly, showed a high molecular weight band at about 201) kDa under reducing and non-reducing conditions. For all rat antibodies contaminating bands of 80 and 55 kDa were seen under reducing conditions and were interpreted as probably non-reduced dimers. In order to control the efficiency of the immunoadsorption the amount of bound antigen before and after SDS-elution was quantitated as shown in Fig. 7.
Discussion Characterization of monoclonal antibodies by standard Western blotting of unlabeled antigens and detection via a second layer system is often limited by the SDS-sensitivity of the corresponding epitope. This was true for RmC7H8 and RmC4B9 which were thought to have the same specificity as RmCI3C9 for mouse C l q (Wasiliu et al., 1990) but failed to stain blotted serum. A
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. sos
Fig. 7. Ouantitatiorl ~1"antigen bound to ~md detached from the microtiter phltes used for immunoadsorption of biotinylated serum. The wells were coated as described in Fig. 5. Individual wells, heft,re and after incubation wilh electrophoresis buffer containing 2% SDS, were extensively washed and developed in an ELISA procedure usi~tg HRPconjugated avidin. general, simple and reliable method for determining the specificity of MAb at an early stage in the production of hybridomas wouid be extremely valuable. The micro-method described here fvlfils these demands: it combines a non-radioactive labeling tecbnique using biotin (Cole et id.. 1987) and the use of antibody-coated microtiter plates as an immunoaffinity matrix {Tamura t:t al., 19841. The advantage of biotinylation over internally or externally labeling with radioactive nuclides resides in the long storage time as well as in the simple and harmless nature of the procedure. Corwenicnt i m m u n o p r e c i p i ~ i o n techniques using S. altretts, Sephzrose-conjugated protein A / G or other immunoaffinity matrices are often limited by n~',n-specific binding, which has to be reduced by various incubations with the matrix alone or in combination with ir:elevant antibodies (Knowles, 19861. Using the mcth,-,d described here this problem can easii;.' be ove~ :ome by blocking and repeated washing steps. A e-~mparison between the two techniques is shown in Fig. 3. Although the immunoprecipitation has no, been optimized with respect to incubation and washing procedures, the figure clcarly shows the advantage ef the immunoadsorption technique ovcr a standard immunoprecipitation method. In the case of crude serum preparations the
use of protein A / G - S e p h a r o s e is not to be reco m m e n d e d , b e c a u s e the biotinylated s e r u m imm u n o g l o b u l i n s c o m p e t e with the u n l a b e l e d capture antibody for binding to the i m m u n o a f f i n i t y matrix. Alternative a p p r o a c h e s , such as the p r e p a r a t i o n o f an i m m u n o g l o b u l i n - f r c e antigen, the covalent binding o f the antibody to protein A / G or direct coupling of purified antibodies to activated matrices are r a t h e r difficult a n d therefore not applicable for rapid antibody screening. Moreover, the binding o f protein A a n d proteip, G is restricted to selected isotypes. Rat M A b s do not react with protein A at all a n d only partially with protein G ( P h a r m a c i a , G e r m a n y ; product information). O n t h e o t h e r h a n d t h e r e is hardly any isotype restriction w h e n coating to polystyrene microtitcr plates. It is t h e r e f o r e possible to use a n d c o m p a r e antibodies o f different isotype. T h e ability of protein A- or G - S e p h a r o s e to bind u n p u r i f i e d MAb, for example, from culture s u p e r n a t a n t s also holds for the i m m u n o a d sorption t e c h n i q u e described here, since t h e polystyrene surface can be coated with polyclonal a n d m o n o c l o n a l c a p t u r e antibodies or with protein A (Randall et al., 1983) or G (data not shown). T h e c o m b i n a t i o n of microtiter plates a n d biotin labeling also p e r m i t s q u a n t i t a t i o n of the a m o u n t of b o u n d a n t i g e n by d e v e l o p i n g the plates according to s t a n d a r d E L I S A m e t h o d s with H R P - c o n j u g a t e d avidin. T h e c o m p a r i s o n of appropriate wells before or after removal of the specific a n t i g e n by SDS h c l p s to establish the optimal p a r a m e t e r s for i m m u n o a d s o r p t i o n without t h e n e e d for carrying out the whole electrophoretic a n d blotting p r o c e d u r e . W e were able to optimize the system for different a n t i g e n s with regard to eh.tion conditions a n d biotin labeling. Since individual crude a n t i g e n p r e p a r a t i o n s often diffcr with respect to the c o n c e n t r a t i o n , specific label a n d biochemical p r o p e r t i e s of an individual antigen, wc routinely include as a control a p p r o p r i a t e a n t i g e n - i n c u b a t e d wells without SDS elution.
Acknowl¢dgement T h e a u t h o r s wish to acknowlege the excellent technical assistance provided by U. M e i n c k e n , M.
Griel3er, U. Erler a n d V. H u n k e . W e t h a n k Drs. N. S h e a r m a n , R. Mocikat a n d E. L e d e r e r for helpful discussion. T h i s work was s u p p o r t e d by a g r a n t from B M F T 0702571,
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