Quantitation of IgE antibodies by radioimmunoassay in the presence of high concentrations of non-IgE antibodies of the same specificity

Journal oflmmunologicalMethods, 85 (1985) 39-52 Elsevier

39

JIM03704

Quantitation of IgE Antibodies by Radioimmunoassay in the Presence of High Concentrations of Non-IgE Antibodies of the Same Specificity 1 Seiji H a b a a n d A l f r e d N i s o n o f f 2 Rosenstiel Research Center, Department of Biology, Brandeis University, Waltham, MA 02254, U.S.A. (Received 22 July 1985, accepted 2 August 1985)

Radioimmunoassays for mouse IgE antibodies, based on adherence to an antigen-coated surface, are precise and sensitive, but errors can be introduced by the presence of relatively high concentrations of non-lgE antibodies of the same specificity. Such errors are caused by competition for the limited number of antigenic determinants on the antigen-coated surface. In this paper we explore further the quantitative aspects of the 'competition effect'. An easily applied method is described, based on preferential precipitation of non-IgE antibodies by ammonium sulfate, that permits analysis of IgE antibodies in the presence of large amounts of non-IgE antibodies (that are principally IgG). For IgE anti-At, the maximum permissible ratio is extended from - 1500 : 1 to at least 40,000 : 1. We have also determined the effect of IgG antibodies or whole mouse serum on PCA reactions of mouse IgE antibodies, carried out in rats. Key words: I g E - quantitation -

anti-arsonate- radioimmunoassay

Introduction

Radioimmunoassays for IgE of a given specificity have an advantage, as compared to PCA or Prausnitz-Kiastner reactions, of greater accuracy and sensitivity (Wide et al., 1968; Pauwels et al., 1977; Karlsson et al., 1979). We recently reported the development of a radioimmunoassay for IgE antibody with anti-Ar specificity that is also adaptable to the measurement of the CRI A content of the antibody (Haba et al., 1984). The lower limit of sensitivity is 40-80 pg of anti-Ar IgE 1 Supported by Grants AI-17751 and AI-22068 from the National Institutes of Health. 2 To whom correspondence should be addressed. Abbreviations: Ar, p-azophenylarsonate; BSA, bovine serum albumin; CAF1, ( B A L B / c × A / J ) F ¢ CFA, complete Freund's adjuvant; CRI, idiotype that is cross-reactive within a strain; CRI A, major CRI associated with anti-At antibodies of A / J mice; KLH, keyhole limpet hemocyanin; PCA, passive cutaneous anaphylaxis; RAMFab, rabbit anti-mouse Fab; RAMIgE, rabbit anti-mouse IgE; 40%S, 40% of saturation. 0022-1759/85/$03.30 © 1985 Elsevier Science Publishers B.V. (Biomedical Division)

40 contained in a volume of 50 t~l of diluted serum sample. A limiting factor in this assay, which makes use of BSA-Ar-coated polyvinylchloride microtiter plates (Klinman et al., 1976), is the ratio of total anti-Ar (mainly IgG) to IgE anti-At in the sample. This is due to competition for the limited number of haptenic sites on a BSA-Ar-coated well. When, in our assay, more than 50-100 ng of anti-At is added to a well its capacity is exceeded and the percentage bound decreases as the total amount of anti-At used is further increased. If 100 pg of IgE is present, the ratio, 50 ng : 100 pg, equals 500 : 1. This limit can be increased somewhat, up to - 1500 : 1, by correcting the data for the competition effect, using standard solutions containing known amounts of IgG and IgE anti-Ar (Haba et al., 1984). Because, with some immunization protocols, ratios of total-to-IgE antibody markedly exceeding 1500:1 occur frequently, a method for increasing the useful range of ratios is desirable. The present paper describes a simple procedure that can be used to extend the ratio to at least 40,000 : 1 ; for example, it can detect 100 n g / m l IgE anti-At in the presence of 4 m g / m l total anti-Ar. The method is based on preferential precipitation of IgG from serum with ammonium sulfate, leaving a large fraction of the IgE in the supernatant. It is not necessary to dialyze the sample after precipitation. We have also investigated the effect of whole serum or IgG anti-Ar antibodies on PCA reactions induced by IgE anti-Ar antibodies.

Materials and Methods

Experimental animals A / J , B A L B / c and CAF 1 mice were obtained from the Jackson Laboratory, Bar Harbor, ME. They were 8-12 weeks old at the start of experiments. Male C D rats (retired breeders) were obtained from the Charles River Breeding Laboratories, Wilmington, MA.

Antigens and adjuvants Keyhole limpet hemocyanin ( K L H ) was purchased from Calbiochem (La Jolla, CA); bovine serum albumin, fraction V, from Sigma (St. Louis, MO); a m m o n i u m sulfate (special enzyme grade) from S c h w a r z / M a n n (Cambridge, MA); CFA from Difco (Detroit, MI). Alum was prepared by the method of Levine and Vaz (1970) from aluminum sulfate and sodium hydroxide (Fisher Scientific, Pittsburgh, PA). The alum was mixed with antigen just before use; 4 mg alum and 5/~g antigen in 0.5 ml of saline were inoculated i.p. When CFA was used for immunization of mice it was emulsified with an equal volume of a solution containing 100/~g of antigen; 0.2 ml of emulsion was inoculated i.p. Three IgE monoclonal antibodies were used in this work. SE20.2 (lgE~, anti-Ar, C R I b ) and SE1.3 (IgEK, anti-Ar, CRIA) have been described ( H a b a et al., 1985). TIB-142 (IgEK, anti-TNP) was derived from hybridoma cells obtained from the American Type Culture Collection. The cells had been donated by M. Wabl (Rudolph et al., 1981). Each of the monoclonal antibodies was prepared from ascitic fluid. Hybridomas SE20.2 and SE1.3, which are of A / J derivation, were grown in

41 CAF1 mice. The TIB-142 hybridoma, of B A L B / c origin, was grown in B A L B / c mice. Mice were treated with pristane 1 3 weeks before introducing the hybridoma cells i.p. SE20.2 and SE1.3 were affinity purified, using BGG-Ar conjugated to Sepharose4B, as previously described (Haba et al., 1985). To purify protein TIB-142, the ascitic fluid was treated with saturated ammonium sulfate on ice and a fraction soluble at 40% of saturation but insoluble at 60% was obtained. The antibody was then affinity purified on Sepharose-4B conjugated with DNP-BSA. The monoclonal antibody was eluted with 0.15 M dinitrophenol, pH 9.0, and dialyzed extensively. Residual hapten was removed by passage through a small column of the anion exchanger, Dowex-1, equilibrated with borate-buffered saline, pH 8.0. The hapten was undetectable spectrophotometrically (absorbance < 0.02 at 400 nm in alkaline solution) after the latter step; calculations showed that less than 4% of the binding sites of the purified antibodies were occupied by hapten. Note that the above procedure makes use of dinitrophenol rather than the trinitrophenol for which TIB-142 is specific. It proved very difficult to remove trinitrophenol from the antibody. Rabbit antibodies to mouse I g E and Fab

Rabbit anti-IgE antiserum (RAMIgE) was obtained after 3 or more subcutaneous inoculations at multiple sites of 200 /~g portions of protein TIB-142, first in CFA then IFA. The rabbit antiserum was adsorbed by passage through a column of Sepharose-4B conjugated to a crude globulin fraction of normal A / J serum; the latter was obtained by 2 precipitations of serum with ammonium sulfate at 50% saturation. The pass-through fraction of the rabbit antiserum was precipitated with ammonium sulfate (50% saturation), redissolved at one-fourth the original concentration and dialyzed. It was then further adsorbed with Sepharose-4B doubly conjugated to mouse IgM (globulin fraction of MOPC 104E ascites) and mouse IgA (globulin fraction of MOPC 315 ascites). These fractions also contained other B A L B / c mouse globulins. Both MOPC 104E and MOPC 315 possess X chains. The adsorbed rabbit anti-IgE was affinity purified on a column of Sepharose-4B conjugated to protein SE1.3 (IgE•). The antibody was eluted in the cold with 3 M NaSCN and dialyzed immediately against borate-buffered saline, pH 8.0. The dialyzed fraction was finally passed through a small column of a non-specific A / J globulin coupled to Sepharose 4B. When tested by radioimmunoassay, with BSA-Ar-coated wells, the 125I-labeled purified anti-IgE gave a strong signal when monoclonal IgE anti-Ar antibodies were tested; 100 pg of IgE anti-Ar yielded a number of counts about 3 times background. In contrast 2000 ng of monoclonal anti-Ar antibodies of other classes gave no more than 1.5 times background. The monoclonal antibodies tested were of the IgM, IgA, IgG1, IgGza, IgGzb and IgG 3 isotypes. Rabbit antibodies to non-specific mouse Fab fragments (RAMFab) were prepared by an immunization protocol very similar to that used for mouse IgE. The antibodies were affinity purified on a mouse Fab-Sepharose 4B column, using 0.1 M glycine-HC1, pH 2.3 for elution.

42 Affinity purified RAMFab and RAMIgE were iodinated with carrier-free ~25I (Amersham, Arlington Heights, IL) by the chloramine-T method (Hunter, 1970). The initial specific activities were approximately 2 and 40 /zCi//~g of protein, respectively.

Radioimmunoassays Radioimmunoassays were carried out in the wells of polyvinylchloride microtiter plates (Fisher Scientific Co., Pittsburgh, PA) (Klinman et al., 1976). In each case the wells were saturated with 3% horse serum after coating with the desired antigen or antibody. Unknown samples were incubated for 6 h and radiolabeled developing reagents overnight at room temperature. To assay for total anti-Ar, wells were coated with BSA-Ar (1 mg/ml). After applying the unknown, wells were developed with 75 ng 125I-labeled affinity purified RAMFab. Affinity purified A / J anti-Ar antibodies from hyperimmune sera were used as the standard. The assay for IgE anti-Ar was very similar, except for the use of ~25I-labeled, affinity purified RAMIgE (12 ng) as developing reagent. The monoclonal anti-Ar antibody, SE20.2 (IgE~), was used as the standard in this assay. To quantify total IgE, wells were coated with a mixture containing a 3 : 1 ratio of normal rabbit IgG to affinity purified RAMIgE, at a total concentration of 5 t~g/ml. The 2 reagents were diluted and mixtures prepared immediately before coating the wells, which were then saturated with 3% horse serum. After exposure to the unknown sample and washing, 125I-labeled affinity purified RAMIgE (12 ng) was used for development. Protein SE20.2 was again used as the standard. Other methods Proteins were diazotized, using p-arsanilic acid, as described elsewhere (Nisonoff, 1967). Saturated ammonium sulfate was prepared at room temperature; its pH was adjusted to 7.2 with concentrated ammonium hydroxide. Precipitations with ammonium sulfate were carried out in test tubes immersed in an ice bath. The final ammonium sulfate concentrations referred to in the text are therefore based on its solubility at room temperature. The total volume of each sample after addition of ammonium sulfate was 100 /~1; i.e., to obtain 40% saturation, 40 /~1 of saturated ammonium sulfate was added to 60/~1 of sample. The sample was stirred magnetically during the addition. PCA tests for IgE anti-Ar were carried out in rats by the method of Ovary (1964) (Haba et al., 1984); 0.1 ml was used for each spot. Blue spots >/10 mm in diameter were considered positive. Results

In an earlier paper we presented data on the inhibitory effect of non-IgE anti-Ar antibodies on the radioimmunoassay for IgE anti-Ar (Haba et al., 1984). In the present work we extended these studies by using a wide range of concentrations of IgE anti-Ar and total anti-At. Anti-Ar antibodies with a very low content of IgE were affinity purified (Dohi and Nisonoff, 1979) from pooled ascites (Tung et al., 1976) of A / J mice repetitively immunized with K L H - A r in CFA. With this protocol

43

A o_

c~

B

0 nn

~e

C 4 0

D L

0

o.

,

~ ,

,

l I00

,

TOTAL

,

i

\

~

\o i

ANTI-Ar

I 200

,

,

'\ i

//

I 400

//

I I000

(ng)

Fig. 1. Effect of increasing amounts of non-IgE anti-Ar on the radioimmunoassay for a fixed a m o u n t of IgE anti-Ar. Protein SE20.2 (IgEx, anti-Ar) was mixed with increasing amounts of affinity purified A / J anti-Ar antibodies that lacked detectable IgE. The amounts of SE20.2 used were: A, 1000 pg; B, 500 pg; C, 250 pg; D, 125 pg; E, no SE20.2 present.

of immunization, IgE anti-Ar was undetectable by radioimmunoassay or by the PCA test for IgE anti-Ar. That immunization with CFA yields low but detectable titers of IgE antibodies was reported by H a m a o k a et al. (1974). We observed very low titers of IgE anti-Ar after 1 or 2 injections and it became undetectable after subsequent inoculations. The effect of the presence of this non-IgE anti-Ar on assays for IgE anti-Ar was then measured. The IgE anti-Ar used was the monoclonal antibody SE20.2 (IgE•). Fig. 1 shows the effect of the presence of increasing amounts of total anti-Ar on the assay for IgE anti-Ar, present at a concentration of 125, 250, 500, or 1000 pg in a 50 t~l sample. It is evident that the amount of labeled anti-IgE bound to a well decreases with increasing levels of non-IgE anti-Ar and that the decrease becomes substantial as the total amount of anti-Ar applied to a well exceeds - 100 ng. Fig. 2 shows similar data obtained by adding an anti-Ar antiserum low in IgE (obtained by using CFA + K L H - A r for immunization) to a serum sample that was relatively rich in IgE anti-Ar antibodies (obtained by immunization with alum as adjuvant); i.e., the source of IgE antibody was an immune serum rather than a monoclonal antibody. The ratio of total anti-Ar to IgE anti-Ar in the IgE-rich serum was - 100 : 1. The numbers plotted on the abscissa include the non-IgE anti-Ar present in both components of the mixture. Also included in Fig. 2, for comparison, are data obtained with the monoclonal IgE antibody, SE20.2 (500 pg). In each case, the amount of labeled rabbit anti-IgE bound to a well decreased significantly when the total anti-Ar content exceeded 50-100 ng per well, and the shapes of the inhibition curves are similar with protein SE20.2 or serum as the source of IgE anti-Ar.

44

I,o ×

16

c] z D ©

12

W H

8

A

E3

o

o.

co

~~

~

TOTAL

ANTI-At

(ng)

Fig. 2. Effect of increasing amounts of non-IgE anti-Ar on the radioimmunoassay for a fixed amount of IgE anti-At. The sources of IgE and non-IgE antibodies were immune sera, as described in the text, except for curve E, for which protein SE20.2 was used as the lgE component. The amounts of lgE anti-At used were: A. 1000 pg; B, 500 pg; C, 250 pg; D, no IgE: E, 500 pg SE20.2.

Effect of ammonium sulfate on radioimmunoassays for total anti-Ar, IgE anti-Ar and total lgE Subsequent sections will deal with the e n r i c h m e n t of IgE a n t i - A r by preferential p r e c i p i t a t i o n of I g G a n t i - A r with a m m o n i u m sulfate. Since dialysis of n u m e r o u s s a m p l e s after such t r e a t m e n t w o u l d be very time c o n s u m i n g we a s c e r t a i n e d the effect of the presence of a m m o n i u m sulfate on r a d i o i m m u n o a s s a y s . Total a n t i - A t (12 ng), IgE a n t i - A t (512 pg) or total IgE (1.8 rig) were assayed in the presence a n d a b s e n c e of a m m o n i u m sulfate. In each case a m m o n i u m sulfate at 1%S, 2%S or 3%S h a d no d e t e c t a b l e effect on the assay. (The s y m b o l %S refers to p e r c e n t a g e of saturation.) The a p p a r e n t yields in the 3 assays at 5%S were 89%, 83% and 90% respectively ( m e a n values of d u p l i c a t e experiments). In the e x p e r i m e n t s d e s c r i b e d b e l o w final c o n c e n t r a t i o n s of a m m o n i u m sulfate up to 42.5%S were used. To avoid corrections for the effect of a m m o n i u m sulfate on assays it was therefore necessary to dilute samples at least 14-fold before testing. If we set a lower limit for l g E a n t i - A r of 80 pg, the m i n i m u m c o n c e n t r a t i o n in e x p e r i m e n t a l samples must be 14 x 80 x 1.74 = 1950 pg in 50/~1, or a b o u t 40 n g / m l of IgE a n t i - A t . The value 1.74 corrects for the dilution that occurs u p o n a d d i t i o n of a m m o n i u m sulfate (42.5%S).

Enrichment by ammonium sulfate precipitation of IgE anti-Ar in sera containing non-IgG and IgE anti-Ar antibodies A p p r o p r i a t e a n t i - A r antisera, c o n t a i n i n g or lacking IgE anti-Ar, were mixed so as to yield final c o n c e n t r a t i o n s of 3.2 m g / m l total a n t i - A r a n d 1.0 f f g / m l IgE a n t i - A t (ratio, 3200:1). T h e value for total a n t i - A r was o b t a i n e d by r a d i o i m m u n o a s s a y , using ~25I-labeled R A M F a b as d e v e l o p i n g reagent. W h e n assayed for IgE a n t i - A r at a 1 : 100 dilution the c o n c e n t r a t i o n calculated for the original m i x t u r e was 67 n g / m l

None

35 37.5 40 42.5

35 37.5 40 42.5

35 37.5 40 42.5

35 37.5 40 42.5

Undiluted

Undiluted

1:1

1:2

1:3

85, 83, 65, 49,

91, 98, 80, 50,

106, 102, 89, 58,

116, 84, 88, 50,

81 71 61 41

92 88 71 45

103 90 78 52

115 103 78 47

22 14 4 2

20 6 3 1

13 4 1 0

41, 39 19,15 5, 5 2, 2

29, 18, 5, 2,

20, 9, 3, 3,

22, 3, 1, 0,

100 (3170 ~ g / m l )

%

%

100 (8.1 # g / m l )

Total anti-Ar

120, 320, 530. 460,

150, 320, 680, 440,

370, 790, 880, 540,

200 350 580 340

200 510 740 480

300 880 830 450

(16) (33) (55) (40)

(18) (42) (71) (46)

(34) (84) (86) (49)

480, 730 (61) 990.1080(104) 800, 740 (77) 350, 390 (37)

70, 64 (7)

n g / m l (mean %)

IgE anti-At uncorrected h

Recovery in supernatant after treatment with ( N H 4 ) 2 S O 4

Total IgE

d d

d d

540, 610 470, 340

730, 780 440, 480

d 1000, 910 910. 850 560, 450

d 1010,1110 800, 740 350, 390

ng/ml

54, 61 47, 34

73, 78 44. 48

100, 91 91, 85 56, 45

101,111 80, 74 35, 39

%

IgE anti-Ar corr. for competition ~

83,100 96, 83

91,110 88,107

98,101 102,109 96, 87

120, 108 91, 94 70, 83

%

Corr. for competition and loss of lgE ~

Samples were prepared by mixing IgE-deficient and lgE-rich immune sera, as described under Results. The final concentrations of non-lgE anti-At and IgE anti-Ar were 3170 and 1.0 /Lg/ml, respectively; total IgE was 8.1 btg/ml. Precipitations with a m m o n i u m sulfate were carried out in duplicate. b Values adjusted for dilutions that occurred during the procedure but not corrected for the competition effect or loss of total IgE. Samples were diluted 100:1 for the radioimmunoassay. c The procedure for applying corrections for competition in the radioimmunoassay for IgE anti-Ar is described in the text. d Corrections are large and therefore unreliable; i.e., the ratio of non-lgE anti-Ar to lgE anti-At remained high after the precipitation with a m m o n i u m sulfate. IgE was determined by the isotype-specific radioimmunoassay.

A m m o n i u m sulfate % saturation

Sample dilution

E N R I C H M E N T OF lgE BY PRECIPITATION WITH A M M O N I U M S U L F A T E ~

TABLE I

46 instead of 1 # g / m l , the low value being due to the competition effect (Table I). A m m o n i u m sulfate was added in the cold, to final concentrations of 35%-42.5% of saturation (35%S-42.5%S), to portions of the serum mixture, undiluted or diluted 1 : 1, 1 : 2, or 1 : 3 with isotonic borate-buffered saline. Experiments were carried out in duplicate. After standing for 2 h, mixtures were centrifuged and radioimmunoassays were carried out on each supernatant, diluted to concentrations appropriate for the assay (minimum dilution, 1 : 100). Three radioimmunoassays were done on each sample: total anti-At; IgE anti-Ar; and total IgE. The amounts of IgE anti-Ar assayed after dilution were between 350 and 450 pg. The data in Table I are expressed as percentage of the amounts originally present in the mixture of antiserum (3.2 m g / m l total anti-Ar; 1.0 /~g/ml IgE anti-Ar; 8.1 /~g/ml total IgE) corrected for the dilution that occurred as a consequence of the precipitation procedure. It is evident, first, that the optimal concentrations of ammonium sulfate are 37.5%S or 40%S. A lower concentration (35%S) left substantial amounts of non-IgE anti-Ar in the supernatant ( - 15% to - 40% of the original amount present; column 4). When a m m o n i u m sulfate was added to a higher concentration (42.5%S), as much as half of the IgE was precipitated (column 3). At the higher dilutions of the serum, 1 : 2 or 1:3, the amount of non-IgE remaining in the supernatant, when 37.5%S a m m o n i u m sulfate was used, was unacceptably high. Optimal recoveries were obtained with: (a) undiluted serum + 37.5%S ammonium sulfate; or (b), serum diluted 1 : 1 + 37.5%S or 40%S ammonium sulfate. Small corrections for competition by non-IgE anti-Ar (columns 6 and 7) are considered acceptable since these corrections are readily applied with the aid of a standard curve. Corrections for loss of IgE (column 8) require the additional assay for total IgE. We would therefore focus on the recoveries in column 7 as being most indicative of the efficiency of the method. On the basis of these data the concentrations of IgE anti-Ar obtained were 85-111% of the theoretical values under conditions (a) and (b). The relevant values are underlined in Table I. It is obvious however, from the data in column 8, that corrections for loss of IgE (as measured by the isotype-specific assay) would extend the range of dilutions and of ammonium sulfate concentrations that can be used. We next applied the procedure to mixtures containing increasing ratios of total a n t i - A r : I g E anti-Ar (Table II). As in the previous experiments an immune serum relatively rich in IgE anti-Ar was used as the source of IgE antibody, and experiments were carried out in duplicate. To provide the needed high concentrations of non-IgE anti-Ar, we used affinity purified anti-Ar antibodies, obtained from the ascitic fluids of mice immunized with K L H - A r in CFA. Evidence for the absence of IgE anti-Ar antibody in the latter preparation was the failure to detect it by radioimmunoassay or PCA and, in addition, the failure to detect a significant concentration of total IgE by radioimmunoassay ( < 10 n g / m l in a solution containing 6.5 m g / m l of anti-Ar antibody). Before the assay, each mixture was diluted so that the final volume was twice the volume of IgE-containing serum used. A m m o n i u m sulfate was then added to a final concentration of 40%S. The second column of Table II lists the ratios of total anti-Ar: IgE anti-Ar tested (2800:1 to 39,300:1). The last 2 columns of Table II

ng/ml

1 200 630 340 180 340 180

IgE

t~g/ml

12.1 9.7 6.5 6.6 6.2 6.6

2800 : 1 5130 : 1 9120 : 1 19600:1 20800:1 39300:1

total anti-Ar: lgE anti-Ar

Ratio

86,85 80,77 86,80 83,75 82,88 81,76

% 4,5 5,5 6,6 7+5 4,3 3,4

%

Total anti-Ar

1060,1040 (88) 490, 480 (77) 300, 270 (84) 160, 150(86) 230, 250 (71) 150, 130 (78)

n g / m l (mean %)

IgE anti-Ar uncorrected

1110+1120 530, 510 330, 290 190, 160 280, 280 170, 150

ng/ml

lgE anti-Ar corr. for competition b

Recovery in supernatant after treatment with ( N H 4 ) 2 S O 4 Total IgE

93 83 91 97 82 89

mean %

108 105 110 123 96 113

mean %

Corr. for competition and loss of lgE ~

40%S. h See footnote c, Table I. See footnote e, Table I.

These experiments differ from those of Table I in that affinity purified antibodies, rather than serum, were used as the source of non-lgE anti-Ar. An antiserum was used as the source of IgE anti-Ar. In the final mixture, before fractionation, this antiserum was diluted 1 : 1. A m m o n i u m sulfate was ad d ed to

IgE

anti-Ar

Total

E N R I C H M E N T OF IgE BY T R E A T M E N T WI TH A M M O N I U M S U L F A T E "

TABLE II

100 (9) 80 (8) 120 (3) 210 (4) 60 (6) 70 (13) 210 (6) 220 (8) 270 (13)

~ g / m l (%) 850 710 1030 1090 610 350 490 320 340

ng/ml 118 : 1 113:1 117:1 193:1 98 : 1 200:1 429:1 688 : 1 794:1

10.7 19.8 12.6 15.0 3.3 12.5 11.3 12.2 8.0

/~g/ml

Total IgE

86 79 77 75 89 70 90 79 68

%

Recovery of total IgE

ng

PCA threshold ~

160 0.6 160 0.6 160 0.8 80 1.8 40 1.7 10 5.0 10 5.4 < l0 Not tested

PCA titer u

e To calculate thresholds the loss of total IgE during fractionation as well as the competition effect was taken into consideration.

In these experiments, in contrast to Tables I and 11, individual antisera, rather than artificial mixtures, were used. The IgE anti-Ar concentration was therefore not known before the analysis. For precipitations, samples were diluted 1 : 1 and ammonium sulfate was added to 40%S. b Values not corrected for the competition effect (see text). " Values corrected for the competition effect. u PCA titers of the original sera (not treated with amm oni um sulfate).

12.4 25.1 16.4 20.0 3.7 17.8 12.5 15.4 11.7

~g/ml

ng/ml

460 210 60 90 250 190 30 20 110

1060 1010 4110 5140 950 540 3480 2710 2090

Ratio (A) : (B)

/tg/ml

(B) IgE anti-Ar ~

(A) Total anti-Ar

Apparent lgE anti-Ar h

Total anti-Ar

Total IgE

Recovery in supernatant after fractionation

Before fractionation

A M M O N I U M SULFATE F R A C T I O N A T I O N OF A N T I S E R A C O N T A I N I N G H I G H RATIOS OF N O N - I g E A N T I - A r : lgE ANT1-Ar J

TABLE Ill

49 indicate the recoveries of IgE anti-Ar corrected for the competition effect (next to last column) or for both the competition effect and loss of IgE (last column). Loss of IgE was quantified by the isotype-specific assay. For all of the mixtures tested, recoveries of IgE anti-Ar, corrected for competition, ranged from 82% to 97%. Values greater than 100%, obtained after correction for recovery of total IgE, are attributable to experimental error in the assays. We also applied the method to 9 serum samples, with ratios of total anti-At : IgE anti-Ar ranging from about 1000:1 to 7000:1 (Table III); 5 of these samples had ratios greater than 3000 : 1. (The actual ratios were not known until the ammonium sulfate precipitation method had been applied.) Samples were diluted 1 : 1 with borate-buffered saline, then precipitated with ammonium sulfate (40%S). In each case the precipitation reduced the ratio of total a n t i - A r : I g E anti-Ar to less than 800:1; in all but 3 instances the ratio was less than 300:1. Recoveries of lgE (determined by the isotype-specific assay) varied between 68% and 90% with a mean of 79%. PCA reactions were also carried out in rats with 8 of the above 9 samples, 7 of which had measurable PCA titers. On the assumption that our final values for lgE anti-Ar content are correct, the PCA thresholds, expressed as amount of IgE anti-Ar, were 0.6 to 5.4 ng in 0.1 ml. Using the monoclonal anti-Ar antibody SE20.2 (IgEx), the threshold was 1.5 ng. The relatively high values (5 and 5.4 ng) for the last 2 serum samples were attributable, at least in part, to the fact that their PCA titers were only 10:1, and this high final serum concentration interferes with the PCA reaction, reducing the apparent titer by a factor of about 2 to 4 (see below). The PCA titers of the other 5 antisera were 40 or more. Effect of serum or antibodies on the PCA reaction We determined the effects of normal A / J serum or non-IgE anti-Ar on the PCA endpoint, using protein SE20.2 as the source of IgE anti-Ar antibody. As a control, SE20.2 was diluted in 0.1% normal A / J serum, 99.9% saline. The threshold for the control corresponded to 1-1.5 ng. For SE20.2 dissolved in 10% mouse serum the threshold was 4 ng of IgE; with 2.5% mouse serum the value was 2 ng of IgE. Sixteen ~tg of IgG anti-Ar, in 0.1% normal mouse serum, had no significant effect on the PCA threshold. The ratio of IgG anti-Ar to IgE anti-Ar in the latter mixture was - 16,000 to 1 .

Discussion

The availability of monoclonal antibodies has permitted the development of radioimmunoassays for human (Wide et al., 1968), rat (Pauwels et al., 1977; Karlsson et al., 1979) and mouse (Liu et al., 1980) IgE antibodies of a given specificity. We recently described such an assay, sensitive to about 40-80 pg, for IgE anti-Ar antibodies, and for the CRI A content of such antibodies, derived from A / J mice ( H a b a et al., 1984). A limitation of the assay derives from the limited capacity of BSA-Ar coated wells of a microtiter plate ( - 50-100 ng of anti-Ar antibodies). As mentioned in the Introduction, errors due to the competition effect become signifi-

50 cant when the ratio of non-IgE anti-Ar to lgE anti-Ar exceeds - 500 : 1 (using a minimal amount of IgE anti-Ar antibody in the test). The useful range of ratios can be extended to - 1500 : 1 by correcting for this competition effect, using standards prepared with a fixed amount of IgE anti-Ar and increasing amounts of IgG anti-Ar. The effect of competititon is illustrated by the data in Figs. 1 and 2. In these 2 figures the sources of IgE anti-Ar were, respectively, a monoclonal antibody, SE20.2, or an immune serum prepared with K L H - A r and alum as adjuvant. Antibodies lacking significant amounts of IgE were obtained by immunization with K L H - A r in complete Freund's adjuvant. When 150 ng of total anti-Ar is present in the sample applied to a well the reduction in apparent concentration of IgE anti-Ar is about 25%. If 100 pg of IgE anti-Ar (a value near the lower limit) is tested the corresponding ratio is 1500:1. Because ratios of total anti-Ar: IgE anti-Ar greatly exceeding 1500:1 are often encountered in immune sera a simple method for extending the useful range of the assay was desirable. We took advantage of the fact that ammonium sulfate, at an appropriate concentration, can precipitate a very large proportion of non-IgE anti-At (principally lgG) in a 1-2 h period while leaving > 80% of the IgE antibody in the supernatant. Because dialysis is time consuming it was expeditious to carry out assays without removing the ammonium sulfate. It was found that ammonium sulfate did not interfere with our radioimmunoassays when its concentration was 3% of saturation or less. To achieve this level, dilution of the sample after treatment with ammonium sulfate was necessary. As discussed under Results, this sets a lower limit for the IgE anti-At concentration in the original serum sample of about 40 n g / m l . (By comparison, the lower limit of IgE concentration in the PCA reaction for IgE anti-Ar is - 150 n g / m l , if one uses 0.l ml samples and dilutes the serum at least 10 : 1, as is customary.) Based on the data in Tables I and II, it was concluded that optimal results are obtained with undiluted antiserum or antiserum diluted 1 : 1, and final concentrations of ammonium sulfate of 37.5%S or 40%S. With lower concentrations of ammonium sulfate unacceptable amounts of non-IgE anti-Ar remain in the supernatant, and higher concentrations precipitate significant percentages of the IgE antibodies. Two corrections were made to the data on IgE anti-Ar recovered after precipitation: one. for the competition effect (Haba et al., 1984), makes use of data obtained with standards, i.e., with a fixed, known concentration of IgE anti-Ar and increasing amounts of non-IgE anti-Ar. Such corrections are practical up to values for total anti-Ar, per well in the assay, of - 150 ng, at which point the correction factor is approximately 1.3. The correction was negligible when less than 50 ng (total anti-Ar per well) was tested. A second correction can be made, based on the recovery of total IgE, as quantified by the isotype-specific assay. (Recoveries of total IgE are shown in the third column of Table I and the fourth column of Table II.) If one wishes to omit the isotype-specific assay for total IgE, the data corrected only for competition are of primary interest; these data are shown in the next to the last columns of Tables I and II. It is evident that recoveries of IgE anti-Ar greater than 75% are readily obtained under optimal conditions. These conditions can be extended considerably if the additional correction for total IgE recovery is made.

5]

As shown in Table II, precipitation with ammonium sulfate permits quantitation of IgE in samples with ratios of total anti-Ar : IgE anti-Ar up to - 40,000 : 1. This ratio begins to approach the useful maximum value. Since our method has a lower limit of - 4 0 n g / m l of IgE anti-Ar (owing in part to the need to dilute the ammonium sulfate before carrying out a radioimmunoassay) the ratio, 40,000:1, corresponds to a non-IgE anti-Ar concentration of 1.6 mg/ml. It is unusual to encounter serum concentrations very much higher than this value. The data discussed so far were obtained with mixtures containing known amounts of non-IgE anti-Ar and IgE anti-Ar; the source of IgE anti-Ar was either a monoclonal antibody or serum with a relatively high content of IgE anti-At. We also applied the technique to 9 unknown samples having high ratios of non-IgE:IgE anti-Ar antibodies. Here, it was not possible to compare the results with ' theoretical' values. It was found, however, that the fractionation with ammonium sulfate reduced the ratios of non-IgE anti-Ar:IgE anti-Ar to levels compatible with our radioimmunoassay. In addition, PCA thresholds agreed reasonably well with those predicted on the basis of radioimmunoassays; in other words, PCA titers were roughly proportional to values obtained for IgE anti-Ar concentrations. It is of interest that IgE anti-Ar can be quantified, after fractionation with ammonium sulfate, in certain samples in which it is virtually undetectable by radioimmunoassay prior to fractionation because of the competition effect. This was true, for example, of samples (Table II) in which the ratio of total anti-Ar : IgE anti-Ar was 20,000 : 1 or more. The IgE was, however, readily detected and quantified after the fractionation. In screening unknown sera for IgE anti-Ar, it can therefore be useful to apply the method to samples that are apparently devoid of lgE. In this context it should be mentioned that IgE anti-At could not be detected, even after ammonium sulfate fractionation, in the sera of mice repeatedly immunized with KLH-Ar in CFA, although small amounts were present in early bleedings. A question of theoretical interest is whether the fractionation succeeds only because the IgE is present at very low concentration or whether there is differential solubility of IgG and IgE in ammonium sulfate. That the latter is a significant factor was suggested by the results of adding ammonium sulfate (40%S) to an IgE anti-Ar monoclonal antibody (SE20.2) at a concentration of 2 mg/ml. More than 95% of the antibody remained in the supernatant. Under the same conditions a large proportion of a polyclonal mouse IgG preparation was precipitated. Our observation that apparent PCA titers are reduced (by a factor of 2 to 4) in the presence of 10% mouse serum, suggests that artificially low values may be obtained by this method with samples having low titers of IgE antibody. Serum diluted 40-fold was not inhibitory.

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