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Isoelectric focusing of IgA and IgM in composite acrylamide-agarose gels

Isoelectric focusing of IgA and IgM in composite acrylamide-agarose gels

Journal of Immunological Methods, 36 (1980) 315--324 © Elsevier/North-Holland Biomedical Press 315 I S O E L E C T R I C F O C U S I N G O F IgA A N...

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Journal of Immunological Methods, 36 (1980) 315--324 © Elsevier/North-Holland Biomedical Press

315

I S O E L E C T R I C F O C U S I N G O F IgA A N D IgM I N C O M P O S I T E ACRYLAMIDE-AGAROSE GELS

D.E. JACKSON, C.A. SKANDERA, J. OWEN, E.T. LALLY and P.C. MONTGOMERY Departments of Microbiology and Pathology, Center for Oral Health Research, School of Dental Medicine, University of Pennsylvania, Philadelphia, PA 19104, U.S.A.

(Received 6 March 1980, accepted 10 April 1980)

A convenient method for isoelectric focusing of intact polymeric IgA and IgM is described. This technique employed composite gels containing 1.0% acrylamide and 0.75% agarose which exhibited minimal electroendosmotic properties. The spectrotypes obtained with mouse IgA myeloma proteins, a human IgA myeloma and rabbit secretory IgA preparations were compared in three gel systems: 5% acrylamide, 0.8% agarose and the composite gel. With respect to resolution of component bands, the composite gel was superior to the other two systems. Hapten binding studies with MOPC-315 IgA and a rabbit secretory IgA anti-DNP antibody indicated that the focused IgA molecules retained their binding site integrity in the composite gel. The pI ranges obtained with microscale sucrose isoelectric focusing and composite gel system showed good correspondence, with the latter exhibiting enhanced resolution. Studies with MOPC-104E IgM revealed improved resolution in the composite gel when compared to the agarose system. Comparison of pI ranges for IgA and IgM immunoglobulins obtained in the present study with those reported previously suggest that IgA spectrotypes are confined to an acidic pI range (3.4--6.4), whereas IgM spectrotypes are not (4.3--8.8).

INTRODUCTION A n a l y t i c a l isoelectric f o c u s i n g ( I E F ) in thin l a y e r 5% p o l y a c r y l a m i d e gels has p r o v i d e d a p o w e r f u l w a y o f assessing a n t i b o d y h e t e r o g e n e i t y ( A w d e h et al., 1 9 6 9 ; Williamson, 1 9 7 1 ) . F o r t h e m o s t p a r t this t e c h n i q u e has b e e n l i m i t e d to the analysis o f I g G s p e c t r o t y p e s b e c a u s e o f t h e sieving e f f e c t o f t h e a c r y l a m i d e o n large p r o t e i n s ( m o l . w t . > 2 0 0 , 0 0 0 ) in general a n d on p o l y m e r i c i m m u n o g l o b u l i n s ( s o m e s e r u m IgAs, s e c r e t o r y IgA a n d I g M ) i n p a r t i c u l a r . A p p r o a c h e s such as r e d u c t i o n o f p o l y m e r i c IgM m o l e c u l e s t o m o n o m e r s ( B o u m a n et al., 1 9 7 6 ) m a y give rise to a r t i f a c t s in t h e IgM isoelectric f o c u s i n g p a t t e r n . An a l t e r n a t i v e a p p r o a c h , t h e m i c r o s c a l e sucrose d e n s i t y g r a d i e n t isoelectric f o c u s i n g s y s t e m (Press a n d K l i n m a n , 1 9 7 3 ) , has b e e n used m o r e e x t e n s i v e l y t o s t u d y t h e h e t e r o g e n e i t y o f i n t a c t IgM (Press a n d K l i n m a n , 1 9 7 3 ; G e a r h a r t et al., 1 9 7 5 ) a n d s e c r e t o r y IgA a n t i b o d i e s ( M o n t g o m e r y a n d S k a n d e r a , 1 9 7 8 ; M o n t g o m e r y et al., 1 9 7 9 ) . T h e sucrose s y s t e m r e q u i r e s a sensitive r a d i o i m m u n o a s s a y f o r d e t e c t i o n o f f o c u s e d c o m p o n e n t s a n d in c o m b i n a t i o n w i t h such an assay is well suited f o r ana-

316 lytical studies. However, the m e t h o d is generally tedious and does not permit rapid screening of large numbers of samples. The development of agarose with low electroendosmosis (Laas, 1972; Johansson and Hjerten, 1974) has provided a medium that is capable of maintaining stable pH gradients while remaining free of molecular sieving effects noted in 5% acrylamide gels. Most recently, Rosen et al. (1979) have described an agarose isoelectric focusing system able to resolve native 19S IgM and a2-macroglobulin. In addition, Saravis and Zamcheck (1979) have shown that a similar agarose system will permit focusing of macromolecular proteins of molecular weight greater than 1 × 106. In an a t t e m p t to apply these systems to intact secretory IgA we found that the resolution of component bands within a spectrotype was often poor. The present study was undertaken to develop an improved flat plate isoelectric system which would be particularly suitable for studying the molecular heterogeneity of intact secretory IgA immunoglobulins and antibodies. MATERIALS AND METHODS

Reagents The following purified mouse m y e l o m a proteins were obtained from Litton Bionetics: MOPC-315 IgA (a, X2), TEPC-15 IgA (a, ~) and MOPC104E IgM (p, Xl). MOPC-315 IgA was also purified from ascites fluid by procedures that are detailed elsewhere (Montgomery et al., 1975). The purification of human IgA (Vu, IgA1) m y e l o m a protein has been reported previously (Dorrington and R o c k e y , 1968). For the present study no a t t e m p t was made to separate monomeric and polymeric fractions. Secretory IgA was purified from the milk of an unimmunized rabbit (BM93) or a rabbit (PR62) immunized by intramammary injections of dinitrophenylated t y p e III pneumococcus (DNP-Pn). An ammonium sulfate (50%, w/v) cut was prepared from the milk of an additional rabbit (C262) immunized in an identical fashion. The rabbit immunization protocols and purification of secretory IgA have been detailed elsewhere (Montgomery et al., 1974). The purification of an anti-mouse a-chain reagent was accomplished by passage of 4.0 ml of anti-mouse IgA, heavy chain specific antisera (Litton Bionetics) over an IgA-Sepharose column. The anti-mouse a-chain was eluted with I M acetic acid, dialyzed against phosphate buffered saline, pH 7.8, concentrated by vacuum dialysis and clarified by high speed centrifugation. The purified anti-a (25 pg) was labelled with 1 mCi of carrier free '2sI (Amersham) using the chloramine-T procedure (Hunter and Greenw o o d , 1962). The specific activity of the labelled preparation was 0.6 t~Ci/ #mole. ['31I]a_N-(4-hydroxyphenacetyl)-e-N-(2,4 dinitrophenyl)-L-lysine ([13'I]HDL, 10 mCi/pmole) was prepared as described by Wang and Montgomery (1973).

317 Several agaroses were tested for optimal resolving power and minimal electroendosmotic properties. The agaroses tested included Sea Kern ME (Marine Colloids), Sea Kern HGT-P (Marine Colloids) and Agarose EF (LKB Instruments). Acrylamide (X5521), N,N-methylene-bisacrylamide and TEMED were purchased from Eastman. Ampholine carrier ampholytes (pH 3.5--10) were purchased from LKB Instruments.

Flat plate isoelectric focusing Polyacrylamide gels (5%) were prepared from a mixture of the following reagents: 25.0 ml dist. H20, 5.7 ml 30% acrylamide/bisacrylamide (28.5 g acrylamide, 1.5 g bisacrylamide in 100 ml dist. H20), 21 /J1 TEMED and 1.7 ml ampholines (pH 3.5--10). The solution was de-gassed, 1.5 ml 1.6% ammonium persulfate added, and poured into a glass sandwich (2 mm gasket thickness, Ampholine Electrofocusing Kit, LKB Instruments), the inner surfaces of which were covered with Gel Bond Film (7 mil thickness, Marine Colloids). Gels were allowed to set for 4 h at r o o m temperature. Proteins (100--150 pg in 0.001 M phosphate buffer, pH 7.8) were applied at the cathodal end and focusing was carried o u t on the LKB Multiphor apparatus for 30 min at 50 V, 16 h at 200 V, maximum 10 mA at 10°C. Electrode solutions were 1 M NaOH at the cathode, 1 M phosphoric acid at the anode. Gels were stained in 0.1% Coomassie Brilliant Blue R250 (Eastman) in e t h a n o l : a c e t i c a c i d : d i s t . H20 ( 9 : 2 : 9 ) for 30 min and destained for 24 h in ethanol : acetic acid : dist. H20 (6 : 1 : 13) with periodic changing of the destaining solution. Agarose gels were prepared by the method of Rosen et al. (1979). Briefly, agarose (0.8%, w/v) and sorbitol (10%, w/v) were dissolved in dist. H20 by heating to 100°C with constant stirring. Ampholines (0.8 ml, pH 3.5--10) were added to 16.0 ml of agarose held at 70°C. After mixing, the solution was poured onto a plastic sheet (110 mm × 125 mm, LKB Instruments), allowed to solidify and transferred to a humid chamber where it was left overnight at 4°C. Proteins were applied at the cathodal end using filter paper strips. Isoelectric focusing was performed at 1WC using the LKB Multiphor apparatus for 30 min at 50 V, maximum 5 mA; 90 min at 100 V, m a x i m u m 10 mA and 120 min at 400 V, maximum 20 mA, sequentially. Gels were stained in 0.1% Coomassie Brilliant Blue and destained as described above, except that destaining was completed in 3 h. Composite gels (1.0% acrylamide-0.75% agarose) were prepared using a modification of Peacock and Dingman (1968). Acrylamide/bisacrylamide (0.16 ml, 10%; 9.5 g acrylamide, 0.5 g bisacrylamide in 100 ml dist. H~O), ampholine (0.8 ml, pH 3.5--10), TEMED (10 pl) and a m m o n i u m persulfate (0.47 ml, 1.6%) were added to agarose (14.6 ml, 0.8% containing 10% sorbitol) held at 70°C. The solution was mixed and poured onto a plastic sheet (110 mm × 125 ram), allowed to set at r o o m temperature for 10 min and transferred to a humid chamber at 4°C for at least 2 h or overnight to

318 allow for complete polymerization. Conditions for sample application, isoelectric focusing and staining of the gels were identical to those described for the agarose gels.

Sucrose density gradient isoelectric focusing The sucrose microscale isoelectric focusing technique was employed as detailed previously (Montgomery and Skandera, 1978). Purified MOPC-315 IgA (25 pg in 0.001 M phosphate buffer, pH 7.8) was applied and focused for 18 h at 4 ° C. Gradients were harvested by collecting single drop fractions in 100 pl of 0.15 M NaC1. After the pH measurement of individual fractions, 50 pl aliquots were removed and analyzed for the presence of DNP binding activity using a solid phase radioimmunoassay. The radioimmunoassay employed DNP-BSA coated plates and an 12sI anti-mouse a-chain reagent and followed the identical protocol used for detecting focused fractions of rabbit IgA (Montgomery and Skandera, 1978). The radioimmunoassay binding data were corrected for machine background and the cpm/50 ill of test fraction was plotted against the individual pH values.

A u toradiograp hy Hapten binding by immunoglobulins focused on composite gels was assessed by the method of Williamson (1971). The gels were coated with a 1 pM solution of ['~II]HDL, developed at 37°C for 15 min and fixed with 18% (w/v) Na2SO4 in water. The gels were washed, dried and a contact autoradiograph made using Kodak SB5 X-ray film. RESULTS Initial experimentation assessed the resolving properties of several agaroses. In our hands, Agarose EF (LKB Instruments) yielded the best resolution for both IgA and IgM proteins whether used alone or in composite gels and therefore was employed throughout the present studies. In addition, IgA and IgM immunoglobulins would not enter any of the three gel systems if loaded at the anodal end. Increasing the focusing time to 48 h and reversal of the electrode solutions did not influence the results. For all of the gel systems it was necessary to load IgA and IgM immunoglobulins at the cathodal end.

Comparative resolution of IgA in 3 gel systems Fig. 1 shows comparative spectrotypes obtained after protein staining for 5 IgA protein preparations in three gel systems: acrylamide (5%), agarose (0.8%) and composite (1.0% acrylamide-0.75% agarose) gels. The IgA protein preparations tested included: purified IgA (Vu) myeloma, containing a

319

Agarose

Acrylamide

Composite m

¸:¸

3 4 5 pH

a

b

c

d

e

a

i:i! b

c

6

7 d

e

a

b

c

d

e

8

Fig. 1. Isoelectric focusing patterns of IgA in 5% acrylamide, 0.8% agarose and composite (1.0% acrylamide-0.75% agarose) gels. Samples include: purified human IgA myeloma (Vu) (track a), purified mouse IgA myeloma MOPC-315 (track b), purified mouse IgA myeloma TEPC-15 (track c), purified sIgA from rabbit PR62 milk (track d) and ammonium sulfate fraction of rabbit C262 milk (track e). Samples (100--150 ~g) were loaded at the cathodal end and gels contained pH 3.5--10 ampholines. Proteins were visualized by staining with 0.1% Coomassie Blue (see Materials and Methods).

mixture of monomers and polymers (track a); purified mouse MOPC-315 IgA {track b); purified mouse TEPC-15 IgA {track c); purified rabbit polymeric sIgA, PR62 (track d) and an ammonium sulfate cut of rabbit milk from C262 (track e). Overall the composite gel was found to focus IgA into more clearly defined banding patterns than either the acrylamide or agarose gels, although individual IgA proteins showed differing degrees of variation within each system. For example, while TEPC-15 IgA exhibited discernible banding patterns in each system, spectrotype differences did occur. TEPC-15 IgA displayed two distinct sets of bands (pI 4.6--5.0 and 5.1--5.8) in the acrylamide gel (track c), one major (pI 4.5--5.1) and one minor (pI 5.2--5.7) set of bands in the agarose gel (track c) and one set of bands (pI 4.7--5.5) in the composite gel (track c). MOPC-315 IgA exhibited pI ranges of 3.8--4.7 in acrylamide (track b), 4.0--4.8 in agarose (track b) and 4.2--4.7 in the composite gel (track b). For MOPC-315 the banding resolution was best on the composite gel. The most pronounced differences occurred for human IgA (track a) and rabbit sIgA preparations (tracks d and e). Streaking was evident for all 3 immunoglobulins in the acrylamide gel (tracks a, d and e). Streaking was absent in the agarose gel {tracks a, d and e) but distinct bands were not discernible. The composite gel yielded discernible banding patterns for all 3 IgA preparations (tracks a, d and e}. Table 1 summarizes the isoelectric focusing data for the immunoglobulin preparations shown in the composite gel (Fig. 1).

320 TABLE 1 Isoelectric focusing properties of IgA and IgM immunoglobulins Immunoglobulin

Specificity a

IEF method b

Detection method c

pI range d

Mouse IgA MOPC-315

DNP

Composite Composite Composite Sucrose Composite

Stain [131I]HDL Stain RIA Stain

4.2--4.7 4.2--4.7 4.3--5.0 4.3--5.0 4.7--5.5

Composite

Stain

4.7--5.6

Composite Composite Composite Sucrose

Stain [131I]HDL Stain RIA

4.2--4.6 4.2--4.5 4.0--4.8 4.2--4.6 g

Agarose Composite

Stain Stain

5.8--6.3 5.6--6.4

TEPC-15

PC

Human IgA Vu Rabbit slgA PR62

DNP(Pn)

C262

DNP(Pn)

Mouse IgM MOPC-104E

Dextran

e e f f

a Specificity for myeloma or antibody preparations. DNP, dinitrophenyl; PC, phosphorylcholine; (Pn), carrier used for antibody induction, type III pneumococcus. b Composite gels; sucrose microscale density gradient isoelectric focusing; agarose gels. c Stain, protein stain ; [ 1311]HDL hapten overlay method ; RIA, radioimmunoassay. d Range for focused components in a given spectrotype. e Purified MOPC-315 IgA obtained from Litton Bionetics. f MOPC-315 IgA purified from ascites fluid of mice carrying the plasmacytoma. g Montgomery et al., 1979.

Hapten binding by IgA in the composite gel T h e i n t e g r i t y of I g A a f t e r isoelectric f o c u s i n g in c o m p o s i t e gels was e s t a b l i s h e d using the r a d i o a c t i v e h a p t e n o v e r l a y t e c h n i q u e . In this case t h e s p e c t r o t y p e s of t h e f o c u s e d D N P binding p r o t e i n s were visualized b y c o n t a c t a u t o r a d i o g r a p h s a f t e r t h e b i n d i n g o f [13~I]HDL. Fig. 2 s h o w s t h e s p e c t r o t y p e s o f M O P C - 3 1 5 IgA ( t r a c k a); a n t i - D N P a n t i b o d i e s p r e s e n t in p u r i f i e d sIgA f r o m P R 6 2 ( t r a c k b), a r a b b i t i m m u n i z e d w i t h DNP-Pn; a n d p u r i f i e d sIgA f r o m B M 9 3 ( t r a c k c), an u n i m m u n i z e d c o n t r o l r a b b i t . T h e D N P - b i n d i n g b a n d i n g p a t t e r n e x h i b i t e d b y M O P C - 3 1 5 I g A ( t r a c k a) corres p o n d e d e x a c t l y to t h e p r o t e i n b a n d i n g p a t t e r n s h o w n in t h e stained gel (Fig. 2, t r a c k a a n d Fig. 1, c o m p o s i t e gel, t r a c k b). In e a c h case t h e pI ranges w e r e identical: 4 . 2 - - 4 . 7 (see T a b l e 1). In t h e case o f r a b b i t P R 6 2 , the sIgA D N P - b i n d i n g s p e c t r o t y p e (Fig. 2, t r a c k b) c o n t a i n e d f e w e r b a n d s w i t h a m o r e r e s t r i c t e d p I range, 4 . 2 - - 4 . 5 , c o m p a r e d to t h e p a t t e r n o b t a i n e d w i t h p r o t e i n staining (Fig. 1, c o m p o s i t e gel t r a c k d, p I range 4 . 2 - - 4 . 6 ; see also

321 MOPC-315

4 ::=k

o 2 o

5

,,

pH

I

6 J

7'

a

b

c

3.0

4.0 5.0 pH

i

6.0

Fig. 2. Isoelectric spectrotypes of purified mouse myeloma IgA MOPC-315 (track a), purified sIgA from rabbit PR62 milk (track b) and purified sIgA from control rabbit BM93 milk (track c) run in composite (1.0% acrylamide-0.75% agarose) gels containing pH 3.5--10 ampholines. Samples (100--150 pg) were loaded at the cathodal end. The tracks represent DNP binding proteins visualized by contact autoradiographs following radioactive hapten overlay with [ 1311]HDL. Fig. 3. Isoelectric spectrotype of purified mouse myeloma IgA MOPC-315 using the microscale sucrose focusing technique. The ampholine range was obtained by mixing equal volumes of pH 3.5--5.0 and 4--6. Eluted drops were analyzed for pH and the presence of DNP-binding proteins. DNP-binding activity was assessed by solid phase radioimmunoassay employing an 12sI anti-mouse (~-chain reagent. A 25 /lg protein load was employed.

Table 1). T h e sIgA o b t a i n e d f r o m BM93, an u n i m m u n i z e d r a b b i t , did n o t display a n y DNP-binding p r o t e i n s (Fig. 2, t r a c k c).

Comparison o f the composite gel and sucrose density focusing system Figure 3 shows the s p e c t r o t y p e o f MOPC-315 IgA using the microscale sucrose d e n s i t y gradient focusing s y s t e m . This s p e c t r o t y p e was o b t a i n e d using a solid phase r a d i o i m m u n o a s s a y t o d e t e c t DNP-binding IgA proteins. T h e pI range o b t a i n e d with this t e c h n i q u e (Fig. 3, 4 . 3 - - 5 . 0 ) d i f f e r e d slightly f r o m t h a t o b t a i n e d in the c o m p o s i t e gel using e i t h e r the h a p t e n overlay (Fig. 2, t r a c k a, 4 . 2 - - 4 . 7 ) or p r o t e i n staining (Fig. 1, c o m p o s i t e gel, t r a c k b, 4 . 2 - - 4 . 7 ) . T h e s p e c t r o t y p e s s h o w n in Fig. 1 and 2 were o b t a i n e d using p u r i f i e d M O P C - 3 1 5 IgA ( L i t t o n Bionetics), while the microscale s p e c t r o t y p e s h o w n in Fig. 3 was o b t a i n e d using MOPC-315 IgA purified f r o m ascites fluid in o u r l a b o r a t o r y . When the latter p r e p a r a t i o n was f o c u s e d on t h e c o m p o s i t e gel, the pI range ( 4 . 3 - - 5 . 0 ) o f the f o c u s e d c o m p o n e n t s corre-

322

pH 7 a

b

Fig. 4. Isoelectric spectrotypes of purified mouse myeloma IgM MOPC-104E obtained on 0.8% agarose (track a) and composite (1.0% acrylamide-0.75% agarose) gel (track b). Samples (150 pg) were loaded at the cathodal end and gels contained pH 3.5--10 ampholines. Proteins were visualized by staining with 0.1% Coomassie Brilliant Blue.

sponded exactly to that obtained with the microscale sucrose system (see Table 1). In addition, it should be noted that the composite gel appears to be generally better suited to resolve individual components focusing in narrow pH ranges and to obtain reproducible spectrotypes. In 10 separate focusing runs the variation in the pI range of MOPC-315 IgA in the composite gel was less than 0.2 pH units with no change in the banding pattern.

Isoelectric focusing of mouse myeloma IgM It was of interest to determine the behavior of another polymeric immunoglobulin in the composite gel isoelectric focusing system. Fig. 4 shows the spectrotypes obtained when MOPC-104E IgM was focused using the agarose (track a) or composite gel (track b). Table 1 summarizes the IgM isoelectric focusing data shown in Fig. 4. The IgM spectrotype obtained in the agarose gel shows two poorly resolved bands (pI 5.8--6.3); whereas, the composite gel shows clearly discernible bands (pI 5.6--6.4). DISCUSSION The physical nature of the composite gel permits isoelectric focusing of higher molecular Weight immunoglobulins such as IgA and IgM without the molecular sieving effects inherent in gels containing a higher percentage of acrylamide. The presence of 0.75% agarose reinforces the 1.0% acrylamide for ease of handling (Peacock and Dingman, 1968). Molecular sieving (as indicated by streaking) occurred in the acrylamide gel with rabbit sIgA preparations (10.8S; Montgomery et al., 1974) and the human IgA m y e l o m a containing a mixture of monomers and polymers (6.8 and 9--10S; Dorrington and R o c k e y , 1968). Differences in the focusing patterns of two murine IgA myelomas were also noted with MOPC-315 showing improvement of resolution in the composite gel. While the agarose gel showed no evidence of sieving, the resolution of individual bands was not as distinct as with the composite gel (Fig. 1). A similar improvement in resolution occurred when

323 the spectrotypes of murine MOPC-104E IgM run on agarose and composite gels were compared (Fig. 4). It was of interest to note that the IgA and IgM immunoglobulins would n o t enter any of the three gel systems if loaded at the anodal end. It appears possible that this observation may be due to properties associated with IgA and IgM immunoglobulins since rabbit IgG pI ranges were identical when loaded from either end. In comparing the composite gel spectrotypes visualized by protein staining (Fig. 1) and the hapten overlay technique (Fig. 2) for two DNP-binding immunoglobulins, the MOPC-315 IgA spectrotypes were superimposable and the PR62 sIgA antibody spectrotype (Fig. 2) contained one band less (summarized, Table 1). The absence of a band in the PR62 antibody spectrotype (hapten overlay) reflects the fact that the PR62 sIgA preparation was not purified on the basis of its specificity for DNP. The ability of the two IgA immunoglobulin preparations to bind hapten (Fig. 2) indicates that the integrity of the DNP binding site was maintained. Furthermore, it has been shown that dissociated heavy and light chains give spectrotypes markedly different from the parent molecule (Hoffman et al., 1972) indicating that the focusing seen in the composite gel was not due to chain dissociation. Additional substantiation that free focusing occurs in the composite gel system comes from comparing spectrotypes with those obtained in the microscale sucrose focusing system. Although slightly different pI ranges were noted when different MOPC-315 IgA preparations were focused on the composite gel (see Table 1), identical pI ranges were obtained when identical MOPC-315 IgA preparations were focused in the composite and microscale sucrose systems (Table 1). In the case of rabbit C262, a partially purified sIgA preparation, the focusing range for the anodal bands was 4.0--4.8 (see Table 1). Although this preparation contained IgG, confirmation that the anodal bands represented sIgA was obtained by diafiltration enrichment in which an Amicon XM300 retentate displayed an intensified anodal spectrotype. The pI range of the anodal bands (visualized by protein staining) corresponded to the IgA antibody spectrotype of rabbit C262 obtained previously using the microscale sucrose isoelectric focusing system (4.2--4.6, Montgomery et al., 1979). The purified TEPC-15 IgA spectrotype obtained with the composite gel (4.7--5.5, see Table 1 ) d i f f e r e d from that reported earlier using TEPC-15 ascites fluid in the microscale sucrose system (5.8-6.4, Gearhart et al., 1975). This disparity in pI range may have occurred because of differences in the use of purified TEPC-15 IgA vs. unfractionated ascites fluid. For the limited number of IgA proteins assessed in the current studies it was apparent that IgA displayed an acidic pI range (4.0--5.6, taken from the pI extremes presented in Table 1). The species and origin of the IgA (secretions vs. serum) did n o t significantly influence the pI range. These acidic pI ranges are in agreement for IgA values reported by other inves-

324 tigators ( 3 . 4 - - 6 . 4 ; G e a r h a r t et al., 1 9 7 5 ; M o n t g o m e r y a n d S k a n d e r a , 1 9 7 8 ; M o n t g o m e r y et al., m a n u s c r i p t s u b m i t t e d ) using t h e m i c r o s c a l e sucrose s y s t e m . O n t h e o t h e r h a n d , t h e s p e c t r o t y p e s o f IgM i m m u n o g l o b u l i n s f r o m various species ( m o u s e , h o r s e a n d h u m a n ) d o n o t a p p e a r t o be c o n f i n e d to a p a r t i c u l a r pI range (overall r a n g e 4 . 3 - - 8 . 8 ; Press a n d K l i n m a n , 1 9 7 3 ; K i m a n d K a r u s h , 1 9 7 4 ; G e a r h a r t et al., 1 9 7 5 ; R o s e n et al., 1 9 7 9 ) . T h u s the c o m p o s i t e gel s y s t e m p e r m i t s rapid, r e p r o d u c i b l e and high r e s o l u t i o n f o c u s i n g o f i n t a c t p o l y m e r i c I g A a n d IgM i m m u n o g l o b u l i n s . T h e t e c h n i q u e is suitable f o r s t u d y i n g t h e h e t e r o g e n e i t y o f small q u a n t i t i e s o f p o l y m e r i c i m m u n o g l o b u l i n s using b o t h p r o t e i n staining a n d r a d i o a c t i v e h a p t e n o v e r l a y . In a d d i t i o n , the d e v e l o p m e n t of l y m p h o c y t e h y b r i d o m a t e c h n o l o g y ( K o h l e r a n d Milstein, 1 9 7 5 ; K o h l e r et al., 1 9 7 6 ) h a d d e l i n e a t e d a n e e d to assess t h e a n t i b o d y h e t e r o g e n e i t y of large n u m b e r s o f c u l t u r e fluid s u p e r n a t a n t s f o r p o l y m e r i c as well as m o n o m e r i c i m m u n o g l o b u l i n s . T h e p r e s e n t m e t h o d will e n a b l e r a p i d analysis o f t h e p o l y m e r i c p r o d u c t s o f such h y b r i d cell lines. ACKNOWLEDGEMENT This w o r k was s u p p o r t e d b y U S P H S G r a n t D E - 0 2 6 2 3 . REFERENCES Awdeh, Z.L., A.R. Williamson and B.A. Askonas, 1969, Nature 219, 66. Bouman, H., W.D. Lusebrink, B. Havsteen and H. Stein, 1976, FEBS Lett. 64, 201. Dorrington, K.J. and J.H. Rockey, 1968, J. Biol. Chem. 243, 6511. Gearhart, P.J., N.H. Sigal and N.R. Klinman, 1975, Proc. Natl. Acad. Sci. U.S.A. 72, 1707. Hoffman, D.R., A.L. Grossberg and D. Pressman, 1972, J. Immunol. 108, 18. Hunter, W.M. and F.G. Greenwood, 1962, Nature 194,495. Johansson, B.G. and S. Hjerten, 1974, Anal. Biochem. 59, 200. Kim, Y.D. and F. Karush, 1974, Immunochemistry 11,147. Kohler, G. and C. Milstein, 1975, Nature 256,495. Kohler, G., S.C. Howe and C. Milstein, 1976, Eur. J. Immunol. 6, 292. Laas, T., 1972, J. Chromatogr. 66,347. Montgomery, P.C. and C.A. Skandera, 1978, J. Immunol. 121, 111. Montgomery, P.C., B.R. Rosner and J. Cohn, 1974, Immunol. Comm. 3,143. Montgomery, P.C., J.H. Rockey, R.L. Kahn and C.A. Skandera, 1975, J. Immunol. 115,904. Montgomery, P.C., C. Cohen, C.A. Skandera and K.M. Connelly, 1979, in: Immunology of Breast Milk, eds. P.L. Ogra and D. Dayton (Raven Press, New York) p. 115. Peacock, A.C. and C.W. Dingman, 1968, Biochemistry 7,668. Press, J.L. and N.R. Klinman, 1973, Immunochemistry 10,621. Rosen, A., K. Ek and P. Aman, 1979, J. Immunol. Methods 28, 1. Saravis, C.A. and N. Zamcheck, 1979, J. Immunol. Methods 29, 91. Wang, T.-I. and P.C. Montgomery, 1973, Immunochemistry 10,481. WiUiamson, A.R., 1971, Eur. J. Immunol. 1, 390.