Typing of subclasses and light chains of human monoclonal immunoglobulins by particle counting immunoassay (PACIA)

Journal oflmmunologicaIMethods, 69 (1984) 229-241

229

Elsevier JIM03051

Typing of Subclasses and Light Chains of Human Monoclonal Immunoglobulins by Particle Counting Immunoassay (PACIA) C.G.M. Magnusson 1, D.L. Delacroix, J.P. Vaerman and P.L. Masson Universitb Catholique de Louvain, International Institute of Cellular and Molecular Pathology, Unit of Experimental Medicine, 75 avenue Hippocrate, B- 1200 Brussels, Belgium

(Received 31 August 1983, accepted 28 December 1983)

The subclasses of monoclonal IgGs and IgAs were identified by particle-counting immunoassay. The principle of the test is the inhibition of the agglutinating activity of either specific antisera or monoclonal antibodies (for IgA only) on latex particles coated with a monoclonal IgG or IgA of known subclass. The feasibility of assay of polyclonal Ig subclasses was demonstrated. However, the anti-IgG2 antiserum cross-reacted with an allotype (nG4m(b)) of IgG4. The possibility of typing monoclonal Igs for light chains by the same technique was also demonstrated. Results are obtained in 30 min, and the method requires only small amounts of purified immunoglobulins (Igs) and antisera or monoclonal antibodies. Key words: lg subclasses - human - immunoassay - latex - agglutination

Introduction T h e r e is increasing interest in the assay of I g G (Schur et al., 1970; Siber et al., 1980; Oxelius et al., 1982; Y o u n t , 1982) a n d I g A ( D e l a c r o i x et al., 1983a, b) subclasses in clinical p a t h o l o g y . Because of the relatively small antigenic differences b e t w e e n subclasses, highly specific reagents are necessary, a r e q u i r e m e n t which can o n l y b e achieved with either p o l y c l o n a l antisera that have been m e t i c u l o u s l y a b s o r b e d or with m o n o c l o n a l antibodies. A s o u r l a b o r a t o r y is d e v e l o p i n g the P A C I A (particle c o u n t i n g i m m u n o a s s a y ) technology ( M a s s o n et al., 1981) we have tried to a d a p t it to the assay of I g G a n d I g A subclasses. Because we have so far b e e n u n a b l e to o b t a i n all the necessary antisera with strict monospecificities we c a n n o t c o n s i d e r that the assay is r e a d y for r o u t i n e assay of p o l y c l o n a l subclasses. However, the p r o c e d u r e for t y p i n g m o n o c l o n a l I g G s a n d IgAs d e s c r i b e d here is so p r a c t i c a l

1 Correspondence to: C.G.M. Magnusson, Unit of Experimental Medicine, UCL-ICP 7430, 75 avenue Hippocrate, B-1200 Brussels, Belgium. 0022-1759/84/$03.00 © 1984 Elsevier Science Publishers B.V.

230 and easy that it deserves to be reported especially when it is compared with other techniques such as immunoprecipitation (Mancini et al., 1965), susceptibility to various proteases (Takatsuki and Osserman, 1964; Poulik and Schuster, 1965; Grey et al., 1968; Plaut et al., 1974), and inhibition of haemagglutination (Herbert, 1978; Van Loghem, 1978). PACIA may also be used, as we shall see, for typing the light chains of monoclonal Igs. PACIA, which is used for the quantification of various antigens (Collet-Cassart et al., 1981a; Magnusson et al., 1981), haptens (Collet-Cassart et al., 1981b), antibodies (Magnusson and Masson, 1982; Magnusson et al., 1983) and immune complexes (Cambiaso et al., 1977), is based on the agglutination of polystyrene particles ('latex'). The extent of agglutination is measured by optical, counting of the residual non-agglutinated particles. For the typing of monoclonal Igs, latex, coated with a given Ig subclass, is agglutinated by a specific anti-subclass antiserum or monoclonal antibody. Monoclonal Igs are identified and assayed by their inhibitory activities.

Material and Methods Buffers

(1) GBS is glycine-buffered saline containing per liter 5.6 g NaCI, 7.5 g glycine and 4 g NaN 3, and adjusted to pH 9.2 with 1 N NaOH. (2) GBS-BSA, which is used for the dilution of coated latex particles, standards, myeloma sera and antisera, contains 10 g/1 of bovine serum albumin (BSA, lot 16, Fraction V; Miles Laboratories, Elkhart, IN). (3) Additive is GBS containing 20 g/1 dextran T-500 (Pharmacia, Uppsala) to enhance agglutination (Hellsing, 1969; Magnusson and Masson, 1982; Magnusson et al., 1983), 20.8 g/1 EDTA (ethylenediamine-tetraacetic acid, tetrasodium salt) and 168 g / l NaC1, which is used to avoid non-specific protein-protein interactions and Clq-mediated agglutination. The antiserum is diluted in this additive (Table II) prior to use. When monoclonal antibodies were used a more efficient additive was prepared by increasing the dextran T-500 concentration to 36 g/1 and reducing the NaC1 concentration to 33.6 g/1. Patients' sera

Monoclonal Igs were detected by electrophoresis on agarose gel at pH 8.6 and identified as IgG or IgA by immunoelectrophoresis. Patients' sera and a pool of sera from 1000 blood donors (NHS) were kept at - 2 0 ° C until used. A n tisera

Sheep antisera against the 4 human IgG subclasses were from Nordic Immunological Laboratories, Tilburg (lots no. 4-481; 5-481; 6-481 and 7-481) and Seward Laboratories, London (lots no. 2140; 2438; 2339A; 2096A). Rabbit antisera from the Red Cross and Transfusion Services, Amsterdam, were either agglutinating (antiIgG1, KH 161-51-A1; anti-IgG2, KH 162-05-A3; anti-IgG3, KH 163-45-A1; and

231 anti-IgG4, KH 164-46-A1) or precipitating (anti-IgG1, KH 161-01-P2 and anti-IgG3, K H 163-41-P1). Sheep precipitating antisera against anti-IgG2 (SH 162-06-P1) and anti-IgG4 (SH 164-04-P2) were also from the Dutch Red Cross. Polyclonal antisera against IgA1 and IgA2 were raised in rabbits and absorbed as previously described (Delacroix et al., 1982). Monoclonal antibodies against IgA1 and IgA2 (Delacroix et al., in preparation) were prepared as described elsewhere (Van Snick and Coulie, 1982). Rabbit anti-human light chains were obtained from Dako, Copenhagen (anti-kappa chain, lot 049E and anti-lambda chain, lot 109B). Proteins Myeloma proteins were purified by a combination of gel filtration on Ultrogel AcA 22 or 34 (LKB, Bromma), Pevikon block electrophoresis (C870, Serva, Heidelberg), and affinity chromatography on protein A-Sepharose (Pharmacia, Uppsala). The purity was assessed by immunoelectrophoresis with class specific antisera. Purified preparations of IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2 were kept at - 2 0 ° C in physiological saline containing 0.4 g/1 of NaN 3. Protein content was estimated by absorbance at 280 nm tElcm ~ 1% = 14). Latex preparation Purified monoclonal IgGs (100/zg) were coupled with BSA (900 #g) to 100 #1 carboxylated latex particles (100 g/l, 0.8/zm Estapor K150, lot 501P; Rh6ne-Poulenc, Courbevoie) by carbodiimide as previously described (Magnusson and Masson, 1982). Monoclonal IgAs (100 #g) were coupled to 100/~1 latex in the same way. We used BSA as protein ballast to avoid spontaneous aggregation of the IgG-coated latex. Latex preparations were stable for 6 months at 4°C and more than 18 months at - 2 0 ° C . Before use latex was diluted 8 times (final concentration 0.625 g/l) in GBS-BSA and sonicated for 10 sec in a Branson Sonifier B12 (Danbury, CT 06810). PA CIA PACIA and its instrumentation have been described in detail elsewhere (Magnusson et al., 1981; Masson et al., 1981). For the assay the sample after suitable dilution is placed in the inner row of the sampler called DIAS. A 30 #1 aliquot is automatically mixed with 30 #1 of the additive supplemented or not with the specific antibodies and 30/zl of latex. After continuous vortexing for 25 min at 37°C the suspension is diluted and then aspirated into a modified cell-counter where the non-agglutinated particles are counted. This number is expressed on the recorder by peak height proportional to the concentration of antigen as it inhibits the agglutinating activity of the specific antibodies. Prediluted samples are automatically assayed at a rate of 50 analyses per h with a throughput time of 30 min. The automated instrument is now produced by Acade, Brussels. However, it should be stated that the assay may be done with much simpler and cheaper equipment (Cambiaso et al., 1977; Masson et al., 1981). Conditions for typing The dilutions of the antisera and samples as well as the number of tests that can be done with 1 ml of antiserum are presented in Table I. Because some samples may

232 TABLE I C O N D I T I O N S U S E D IN PACIA F O R T Y P I N G M O N O C L O N A L Ig SUBCLASSES A N D T H E I R LIGHT CHAINS Igs

Antiserum a dilution

Sample dilution

No. of tests with 1 ml antiserum 1500 3 000 9 000 12 000

IgG1 IgG2 IgG3 IgG4

50 100 300 400

10000 1000 10 000 1000

IgA1 IgA2 IgA1 IgA2

200 40 1000 b 1000 b

2 000 2 000 200 200

Kappa Lambda

150 1500

6 000 1200 30000 b 30000 b

10000 10 000

4500 45 000

a Antiserum dilution giving 65-75% agglutination. The number of tests that can be done by radial immunodiffusion with 1 ml of antiserum ranges from 40 to 50. b Ascitic fluid containing 1 m g / m l of monoclonal antibody.

contain

rheumatoid

activities incubation

of

factor

the purified

we

tested

monoclonal

with dithiothreitol

the

(0.3 mg/ml)

with 0.012% H20 2 as described

effect

of

Ig subclasses.

reduction Reduction

on was

the

inhibitory

achieved

by

f o r 15 m i n a t 3 7 ° C f o l l o w e d b y o x i d a t i o n

by Magnusson

e t al. ( 1 9 8 3 ) .

T A B L E II T I T R A T I O N a OF A N T I - I g G SUBCLASS A N T I S E R A A G G L U T I N A T I N G M O N O C L O N A L IgGC O A T E D LATEX Latex

AntiIgG1

AntiIgG2

AntiIgG3

AntiIgG4

IgG1 IgG2 IgG3 IgG4 b

Nordic 200 < 10 < 10 < 10

20 20 < 10 50

20 10 250 50

20 50 20 100

IgG1 IgG2 IgG3 IgG4 b

Red Cross 150 <10 10 < 10

(precipitating) 25 < 10 1000 <10 < 10 300 800 < 10

50 <10 < 10 2000

AntiIgG1

AntiIgG2

AntiIgG3

AntiIgG4

Seward 15 < 10 < 10 < 10

40 10 < 10 20

< 10 < 10 300 15

< 10 < 10 < 10 500

Red Cross 15 <10 < 10 < 10

(agglutinating) < 10 < 10 150 <10 < 10 40 20 < 10

< 10 <10 < 10 100

a Titre giving 50% agglutination. b IgG4 used to coat latex was from patient D o m and contained the allotype nG4m(b).

233 TABLE III TITRATION a OF ANTI-IgA ANTISERA AGGLUTINATING MONOCLONAL IgA-COATED LATEX Latex IgA1 IgA2

Polyclonal

Monoclonal b

anti-lgA1

anti-IgA2

anti-lgA1

anti-IgA2

800 < 10

< 10 80

70 0

0 75

a Titre giving 50% agglutination. b Percentage of agglutination caused by 1 ~g/ml of monoclonal antibodies.

Results

Antiserum specificity T h e specificity of a n t i s e r a f r o m v a r i o u s c o m m e r c i a l sources was c h e c k e d b y a g g l u t i n a t i o n of latex c o a t e d w i t h v a r i o u s p u r i f i e d m y e l o m a p r o t e i n s ( T a b l e II). T h e p r e c i p i t a t i n g a n t i - I g G a n t i s e r a f r o m the D u t c h R e d Cross, b e c a u s e they were the s t r o n g e s t a g g l u t i n a t o r s a n d d i s p l a y e d the best specificity ( T a b l e II) were u s e d i n all f u r t h e r e x p e r i m e n t s . H o w e v e r , the a n t i - I g G 2 a n t i s e r u m at a 800-fold d i l u t i o n a g g l u t i n a t e d IgG4-1atex 50%, w h e r e a s it a g g l u t i n a t e d IgG2-1atex to the s a m e e x t e n t at a 1000-fold d i l u t i o n ( T a b l e II). C o n t a m i n a t i o n of the m o n o c l o n a l I g G 4 used to c o a t latex w i t h traces of p o l y c l o n a l I g G 2 p r e s u m a b l y does n o t e x p l a i n this cross-agg l u t i n a t i o n . O u r p o l y c l o n a l a n d m o n o c l o n a l a n t i - I g A a n t i b o d i e s were also f o u n d s u i t a b l e for the assay o n the basis of their specificity a n d a g g l u t i n a t i n g activity ( T a b l e III).

TABLE IV INHIBITION a OF ANTI-IgG SUBCLASS ANTISERA b BY PURIFIED MONOCLONAL Igs Inhibitory c protein (100 p,g/ml)

Anti-IgG1

Anti-IgG2

Anti-IgG3

Anti-IgG4

IgG1 IgG2 IgG3 IgG4Kin

100 <2 < 0.5 < 0.5

< 0.2 100 < 0.5 <2

< 0.1 < 0.1 100 < 0.1

< 0.1 <1 < 0.5 100

a Expressed in /~g/ml of the reference IgG subclass corresponding to the agglutination system. The antisera were used at a dilution causing 65 to 75% agglutination, which was a suitable compromise between sensitivity and precision (Magnusson et al., 1983). b Dutch Red Cross precipitating antisera. c Two purified IgG1, IgG2, and IgG3, and 4 lgG4 were used. IgG4-1atex was prepared with IgG4Dom. IgG4Deb and IgG4Kin slightly inhibited whereas the other 2, IgG4Dom and IgG4Ros were almost as inhibitory as IgG2 (see text).

234

The specificity of Dutch Red Cross anti-IgG antisera and the cross-reaction of anti-IgG2 with IgG4 were confirmed by inhibition experiments (Table IV). No major cross-reactions were observed for IgG1, IgG2, and IgG3. Out of the 4 tested purified monoclonal IgG4, 2 - - IgG4Dom and IgG4Ros - - strongly inhibited also the anti-IgG2 antiserum. This inhibition reached about 90% but was not complete even after addition of 100 /~g/ml of IgG4Dom or IgG4Ros, suggesting that the anti-IgG2 antiserum contained a large proportion of cross-reacting antibodies which we could not absorb out without losing most of the agglutinating activity. As the cross-reacting antibodies were inhibited by only 2 of the monoclonal IgG4 we attribute the cross-reaction to the presence in these IgG4s of an allotypic determinant, nG4m(b), which is isotypic for IgG2. This explanation is plausible since the supplier states that this anti-IgG2 antiserum contains antibodies directed against such an isoallotypic determinant. The inhibition experiments with purified monoclonal IgA confirmed the monospecificities of our polyclonal and monoclonal anti-IgA antibodies (Table V). Standards

Standard curves with purified monoclonal Igs in GBS-BSA were similar in shape and sensitivity for the 4 IgG (Fig. 1) and the 2 IgA (not shown) subclasses. Owing to the crude estimates of protein concentrations by absorbance at 280 nm the results were expressed as percentages of the NHS pool. The inhibition by a suitable dilution of this NHS pool (Table I) is indicated in Fig. 1 (O) and corresponded to 10-25% inhibition, chosen to get as good discrimination as possible between normal sera and sera with an M-component. Serial dilutions of NHS were in general parallel with standard curves, except for IgG1, for which at dilutions lower than 1/1000 inhibitory activity was weaker than expected (data not shown). Anti-allotypic antibodies a n d / o r anti-IgG autoantibodies (rheumatoid factor) present at low dilutions of NHS could have been responsible for these slight differences from standard curves. Such interfering factors were not likely to affect seriously the assay as the samples were tested at 1/1000 or 1/10,000 dilutions. Furthermore, if samples contain high titres of rheumatoid factor this could be inactivated by treatment with dithiothreitol, which only slightly affected the inhibitory activities of monoclonal IgG1 and IgG3.

TABLE V I N H I B I T I O N a OF ANTI-IgA SUBCLASS A N T I S E R A BY P U R I F I E D M O N O C L O N A L Igs Inhibitory b

Polyclonal

protein (100 la g / m l )

anti-lgA1

anti-IgA2

Monoclonal anti-IgA1

anti-lgA2

IgA1 IgA2

100 <1

<1 100

100 < 0.1

< 0.1 100

a Expressed in # g / m l of the reference IgA subclasses corresponding to the agglutination system. b Two proteins of each subclass were tested.

235 100

IgGl

I00

9O 80 70

"~ SO 4O 30 2O

20 I0

o,~

100

o12

'o~ ....

;

2

s

~

IgG3

,o o;, 10(]

90

9C

O0

8O

0.2

'

'o~ ....

;

],

'

'?

....

:~

'

' ~ ....

,b

IgG4

7O 6O

50

Q. 4O

4O ................................................ 30

3O

20

20

10 o~

o'.2 '

'o.s

';

2 IgG( pglml )

s

io

i 1o al

i 0.2

i

'oT":i

IgO [ ihlll ml )

Fig. 1. I n h i b i t i o n curves o b t a i n e d w i t h purified m y e l o m a p r o t e i n s of the 4 I g G subclasses for the a g g l u t i n a t i n g activity of anti-subclass antisera o n m o n o c l o n a l I g G - c o a t e d latex. The a n t i s e r u m d i l u t i o n s used are i n d i c a t e d in T a b l e I. The i n h i b i t i o n s caused b y the 1 / 1 0 0 0 or 1 / 1 0 , 0 0 0 d i l u t i o n s of the N H S p o o l (Table I) are i n d i c a t e d ( O ) .

IgG myelomas The 4 IgG subclasses were quantified in 72 sera with monoclonal IgG detected after agarose gel electrophoresis and immunoelectrophoresis. They were easily distinguished by PACIA from the 20 sera taken at random among samples sent to our laboratory for routine protein analysis. Among the monoclonal IgGs 50 IgG1, 13 IgG2, 4 IgG3 and 5 IgG4 were identified (Fig. 2). Their relative concentration as measured by PACIA agreed well with the intensity of the corresponding electrophoretic bands (Fig. 3) and, as usual in myelomas, the levels of the other polyclonal subclasses were clearly suppressed (Figs. 2 and 3). No ambiguous results were obtained except for 2 myeloma sera that strongly inhibited both anti-IgG2 and anti-IgG4 antisera. The possible simultaneous presence of 2 monoclonal Igs, 1 IgG2

236 1000

IgG1- assay

?

800 600 400

200

,l~lq l~ ~ I000 -

8O.

IgG 2 - a s s a y

B00

'

600

z

400

o

£

200

3000 IgG3- assay C

2000

8 " 1000

0

: 4OO 20O 0

8000

IgG/,- assay

6000 40OO

400" 200

~ l - - I ~ l 11 Control

IgG1

IgG2 IgG3 IgG4

sera

MYELOMA Fig. 2. Estimation of the IgG subclass content of different sera. The results are expressed in percentages of the NHS pool. At the recommended dilutions some samples gave no inhibition. They were not reassayed at lower dilution. The concentration was therefore considered below a certain limit that was indicated by dotted lines. The encircled numbers refer to the samples of the agarose gel electrophoresis (Fig. 3).

and the other IgG4, was not considered because the agarose gel electrophoresis of the sera clearly showed a single monoclonal component. The IgG2 nature of these monoclonal components was unlikely because, as we have seen earlier, the anti-IgG4 antiserum was not inhibited by IgG2. We therefore concluded that these 2 myeloma proteins belonged to the IgG4 subclass and reacted with the anti-IgG2 antiserum because they were of the nG4m(b) allotype. As expected, like the other IgG2 myeloma proteins tested (n = 7) they inhibited the agglutinating activity of the anti-IgG2 antiserum toward IgG4-1atex whereas the 3 other IgG4 myeloma sera failed to inhibit this system. Thus IgG2-positive myelomas should also be tested for

237

m

NHS

PATIENTS' SERA

G1

G2

G3

G4

MYELOMA

Fig. 3. Agarose-gelelectrophoresisat pH 8.6, showingpatients' sera and various myelomasera containing the 4 IgG subclasses. The numbers of the samples correspond to those given in Fig. 2.

IgG4, since polyclonal anti-IgG2 antisera often seem to cross-react with IgG4 (nGm4(b)) (Howard and Rivella, 1969).

Analysis by PACIA of electrophoretic distribution of lgG subclasses IgG subclasses are known to differ in electrophoretic mobility (Howard and Rivella, 1969). We confirm (Fig. 3) that monoclonal IgG1 has a variable distribution, that monoclonal IgG2 is predominantly in the slow region, that monoclonal IgG3 has essentially a cathodal mobility, and that monoclonal IgG4 is found exclusively in the fast/3-zone. The same distribution of polyclonal subclasses was observed after agarose gel electrophoresis of the NHS pool (Fig. 4). For this experiment we punched out small cylinders of gel (4 mm diameter), recovered the proteins by incubating the gel pieces in 200/~1 physiological saline (9 g/l) overnight and assayed the various subclasses in each electrophoretic.,fraction.

IgA myelomas Eighteen sera with monoclonal IgAs were diluted 2000 times in GBS-BSA (Table I) and typed with the polyclonal anti-IgA1 and anti-lgA2 antisera. Nine IgA1 and 9 IgA2 myelomas were identified without ambiguous results (Fig. 5). The inhibitory activities of these monoclonal IgAs markedly exceeded that of 10 patients' sera devoid of paraproteins. No significant difference in results was obtained when monoclonal antibodies were used in place of polyclonal antibodies (data not shown). Owing to their weaker affinity the more efficient additive was used, and the sera were assayed at a 200-fold dilution.

Light chains Control sera (n = 20) and myeloma sera containing a monoclonal IgG (n = 20) were diluted in GBS-BSA (Table I) and tested for inhibitory activities towards

238

E tm

:a. O

Fig. 4. Electrophoretic distribution of IgG subclasses in N H S as detected by PACIA after elution from the agarose gel.

1800

20000

IgAl-assay

1600

16000

14oo

12000

I 1200

BOO0

1000

4000

.~ 800

800

g 600

600

u 400

400

200

200

o

IgA2 -assay

i

Z

g

Control sera

0

IgA1 IgA2 MYEL.OMA

Control sera

IgAI

IgA2

MYELOMA

Fig. 5. Estimation of the IgA subclass content in various sera using polyclonal antisera. The results are expressed in percentages of the N H S pool. For dotted lines see explanations in Fig. 2.

239 I000C

100C z .S .9

g a.

lO

Contro[ sera

Myeloma sera

Fig. 6. Typing by PACIA of light chains in 10 patients' sera without monoclonal component and 20 myeloma sera. The results are expressed in percentage of the K/~ ratio in the NHS pool. When • or chains were undetectable at a 10,000-fold dilution (zx) they were not reassayed at lower dilutions but the value of the detection limit was used for the calculation of the ratio.

anti-light chain antibodies agglutinating latex coated with IgA1 (x) or IgA2 (~.) myeloma proteins. The standard curve consisted of serial dilutions of the NHS pool and the results were expressed as percentages of the kappa/lambda ratio of the myeloma sera to the kappa/lambda ratio of NHS. The type of Ig light chain in 20 tested myeloma sera was easily distinguished compared with the 10 control sera. Eleven Igs were of the kappa-type and 9 of the lambda-type (Fig. 6).

Discussion

We have shown that PACIA is suitable for typing IgG and IgA subclasses and light chains of myeloma proteins. As with inhibition of passive haemagglutination (Van Loghem, 1978) the use of purified monoclonal Igs to coat the particles decreases the risk of cross-reactions. Although coating particles with IgG could make them agglutinable by the C l q factor of complement or rheumatoid factor, as we have seen, the working conditions are such and the dilution of the samples is so high (1/1000 or more) that non-specific agglutination rarely occurs. Such agglutinators could anyway be easily inactivated by reduction with dithiothreitol. When the concentration of the monoclonal component does not clearly exceed the upper normal limit of the corresponding subclass, results have to be interpreted by reference to the electrophoretic pattern of the agarose gel and to the level of the

240

other polyclonal subclasses which are usually suppressed. If doubt persists with respect to oligoclonality, the assay of subclasses a n d / o r light chains in punched out pieces of the electrophoretic gel will provide more clear-cut results. This procedure was applied to serum number 5 (Fig. 3) for which the ratio for myeloma serum over NHS for IgG1 was only 130%. In this way we confirmed that this patient had an IgGl-component migrating exactly as the band on the stained agarose gel (data not shown). The possibility of using this version of PACIA to assay polyclonal Ig subclasses is now being explored but we need first to find an anti-IgG2 antiserum that does not cross-react with IgG4. Monoclonal antibodies will presumably be useful in this respect and we have seen that, at least, for IgA subclasses, they were perfectly suitable despite their slightly weaker affinity. By comparing them with subclassspecific polyclonal antisera their specificity was demonstrated. Our results in typing monoclonal Igs with PACIA stress again the advantages of this technique, which does not require radioisotopes and uses stable reagents. Ig latex is stable for at least 18 months at - 2 0 ° C , is suited to automation, consumes tiny amounts of antibodies - - 1 ml of anti-IgG4 antiserum is sufficient for 12,000 tests - - and is adaptable to use in many types of assays, i.e. for antigens, haptens, antibodies, and immune complexes.

Acknowledgement We wish to thank Mrs. M. Magnusson for excellent technical assistance.

References Cambiaso, C.L., A.E. Leek, F. De Steenwinkel, J. Billen and P.L. Masson, 1977, J. Immunol. Methods 18, 33. Collet-Cassart, D., C.G.M. Magnusson, J.G. Ratcliffe, C.L. Cambiaso and P.L. Masson, 1981a, Clin. Chem. 27, 64. Collet-Cassart, D., C.G.M. Magnusson, C.L. Cambiaso, M. Lesne and P.L. Masson, 1981b, Clin. Chem. 27, 1205. Delacroix, D.L., C. Dive, J.C. Rambaud and J.P. Vaerman, 1982, Immunology 47, 383. Delacroix, D.L., K.B. Elkon, A.P. Geubel, H.F. Hodgson, C. Dive and J.P. Vaerman, 1983a, J. Clin. Invest. 71, 358. Delacroix, D.L., E. Liroux and J.P. Vaerman, 1983b, J. Clin. Immunol. 3, 51. Grey, H.M., C.A. Abel, W.J. Yount and H.G. Kunkel, 1968, J. Exp. Med. 128, 1223. Hellsing, K., 1969, Biochem. J. 114, 151. Herbert, W.J., 1978, in: Handbook of Experimental Immunology, Vol. 1, ed. D.M. Weir (Blackwell Scientific Publications, London) ch. 20. Howard, A. and G. Virella, 1969, Prot. Biol. Fluids 17, 449. Magnusson, C.G.M. and P.L. Masson, 1982, J. Allergy Clin. Immunol. 70, 326. Magnusson, C.G.M., D. Collet-Cassart, T.G. Merrett and P.L. Masson, 1981, Clin. Allergy 11,453. Magnusson, C.G.M., R. Djurup, B. Weeke and P.L. Masson, 1983, Int. Arch. Allergy Appl. lmmunol. 71, 144. Mancini, G., A.O. Carbonara and J.F. Heremans, 1965, Immunochemistry 2, 235.

241 Masson, P.L., C.L. Cambiaso, D. CoUet-Cassart, C.G.M. Magnusson, C.B. Richards and C.J.M. Sindic, 1981, Methods Enzymol. 74 (Part C), 106. Oxelius, V.-A., A.I. Berkel and L.,~. Hanson, 1982, New Engl. J. Med. 306, 515. Plaut, A.G., R. Wistar and J.D. Capra, 1974, J. Clin. Invest. 54, 1295. Poulik, M.D. and J. Shuster, 1965, Nature (London) 207, 1092. Schur, P.H., H. Borel, E.W. Gelfand, C.A. Alper and F.S. Rosen, 1970, New Engl. J. Med. 283, 631. Siber, G.R., P.H. Schur, A.L. Aisenberg, S.A. Weitzman and G. Schiffman, 1980, New Engl. J. Med. 303, 178. Takatsuki, K. and E.F. Osserman, 1964, Science 145, 498. Van Loghem, E., 1978, in: Handbook of Experimental Immunology, Vol. 1, ed. D.M. Weir (Blackwell Scientific Publications, London)ch. 11. Van Snick, J. and P. Coulie, 1982, J. Exp. Med. 155, 219. Yount, W.J., 1982, New Engl. J. Med. 306, 541.