Immunoglobulin E assayed after pepsin digestion by an automated and highly sensitive particle counting immunoassay: Application to human cord blood

Immunoglobulin E assayed after pepsin digestion by an automated and highly sensitive particle counting immunoassay: Application to human cord blood C. G. M. Magnusson, B.Sc., and P. L. Masson, M.D. Brussels, Belgium

We describe in this article a fully automated, universal assay for serum IgE after pepsin digestion of the sample and subsequent assay of the released Fc'~fragment by particle counting immunoassay (PACIA). The sensitivity and the range of the assay were easily modulated by changing the concentration of dextran in the reaction medium. In the application to cord serum, the sensitivity reached 0.1 IU/ml in 30 min incubation time and with a troughput of 50 analyses per hour. Within-day and between-day coefficients of variation did not exceed 7.6% for IgE levels covering a wide range of the standard curve. Dependable accuracy was demonstrated by linearity tests, analytical recoveries (89% to 112%), and correlation with PRIST on 48 samples from children ages 1 to 14 yr (y = 0.97x + 5.92; r = 0.987). Minor discrepancies between the two methods were attributed to a slight serum effect in PRIST. PACIA applied to 348 cord serum samples demonstrated a statistically significant influence of sex and race on the cord IgE level. In European neonates boys had significantly (p = 0.019) higher geometrical mean cord lgE levels (n = 142; 0.46 lU/ml; range <0.10 to 30 IU/ml) than girls (n = 146; 0.33 lU/ml; range <0.10 to 8.0 IU/ml), which was attributed to a predominance of boys (X2 = 4.29; p < 0.05) having more often elevated cord lgE (<1.20 IU/ml) than girls. Neonates of African-Asian origin had significantly (p < 0.00005) higher cord lgE levels (n = 60; 1.05 IU/ml; range <0.I0 to 125.0 IU/ml) than European neonates (n = 288; 0.39 1U/ml; range <0.10 to 30.0 lU/ml). (J ALLERGYCLtN IMMUNOL 75:513-24, 1985.)

Several articles have demonstrated that a high IgE level in neonates TM and infants 59 is an early warning sign for subsequent development of allergy. 10. ~1However, at such a young age the IgE content of the blood is very lOW, 12 and because of technical difficulties few techniques are sensitive and accurate enough to provide reliable results. Because of the high frequency of allergy and the possibility of partial prevention,l~ ~3 one can envision screening all neonates or at least those at high risk." This type of work requires assays that are cheap, reliable at the level of 1.2 to 1.3 IU/ ml of IgE, m 14 and ideally fully automated. We will describe in this article the adaptation of the PACIA 15 to the determination of serum IgE with parFrom the Unit of Experimental Medicine, International Institute of Cellular and MolecularPathology, Universit6Catholiquede Louvain, Brussels, Belgium. Supported by Technicon Corporation, Tarrytown, N. Y. Received for publication Jan. 9, 1984. Accepted for publication Aug. 29, 1984. Reprint requests: Carl G. M. Magnusson, B.Sc., Department of Clinical Immunology,KarolinskaHospital, S-104 01 Stockholm, Sweden.

Abbreviations used

RIST: DAB: BAC: PBS: NRS: PACIA: GBS: EDTA:

Radioimmunosorbent test Double antibody Bromoacetylcellulose Phosphate-buffered saline Normal rabbit serum Particle counting immunoassay Glycine-buffered saline Ethylenediaminetetraacetic acid

ticular reference to cord serum and demonstrate that this technique fulfills the criteria mentioned above. The principle of PACIA is on the basis of agglutinating activity of the antigen on latex particles coated with antibodies. The extent of agglutination is measured by optical counting of the residual number of nonagglutinated particles. The technique and the instrument have been described in detail elsewhere, iS, ~6 In the field of allergy, PACIA has already been adapted to the assay of total IgE, 16IgE antibodies,17 and blocking antibodies. 18, 19 A technical novelty in this article

513

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Magnusson and Masson

is the use of a pepsin digestion before the assay 2~that eliminates possible interferences caused by nonspecific agglutinators 21, 2~ or agglutinating inhibitors and releases a protease-resistant fragment of IgE, namely the Fc" fragment, 17' 23 which is then assayed by PACIA. Interestingly, the introduction of the digestion step increased the intrinsic sensitivity of the assay about fourfold for IgE. The application of PACIA to the assay of IgE in 348 cord sera has not only allowed us to compare our results with those reported in the literature but also to detect a statistical influence of sex and race on the IgE level. The results of cord IgE as predictor of subsequent development of atopy in a group of these newborn infants as well as the relation between cord IgE and various gestational, serologic, and parental factors will be reported elsewhere. 14

MATERIAL AND METHODS Preparation of antiserum and latex. Immunization of rabbits against the Fc" and Fc fragments of the monoclonal IgE (DES) prepared by pepsin and papain digestion, respectively, has been described. '6 F(ab')2 fragments of antiserum lgG were isolated and coupled to carboxylated latex particles (0.8 txm, Estapor, lot no. K150 501P, Rh6nePoulenc, Courbevoie, France) by carbodiimide." Standard curve. A reference serum from Behringwerke (lot no. 046506K) containing 10,400 IU/ml of IgE was diluted in a pool of NRS to produce eight concentrations ranging from 0.1 to 20 lU/ml (1 IU = 2.42 ng2'). Pepsin treatment. Cord sera (100 Ixl) or the standards in NRS (100 I.~l) were treated with 200 pJ of 0.2 N HC1 containing 5 mg/ml of pepsin (Sigma Chemical Co., St. Louis, Mo.) for 30 min at room temperature. The reaction was stopped by the addition of 60 la.1of2M Tris NaOH, pH 11.3. IgE values that were too high were reassayed after appropriate dilution in pepsin-digested NRS prepared as above. Sera. Serum from children and umbilical cord veins were randomly collected between November 1979 to June 1981 at the Cliniques Universitaires Saint-Luc, Brussels, and kept at - 2 0 ~ C until the serum was assayed in duplicate. PACIA. The automated sample handling and the instrument have been described in detail elsewhere.~5' 16 The instrumentation comprises four modules: a sampler ensuring agitation, incubation, and dilution; a peristaltic pump; an optical counter; and a recorder. The pepsin-treated samples were placed in the inner row of the sampler, and then all reagents were automatically added and incubated for 25 rain under vortex mixing. Since this work was completed, a new version of the instrument called IMPACT has been designed by Acade s.a., Brussels, Belgium. It executes the entire assay automatically including the pepsin digestion step. It takes 50 min on this instrument for one assay, and 60 assays are performed per hour. * *Magnusson CGM: Unpublished data.

J. ALLERGY CLIN. IMMUNOL. APRIL 1985

Additives. The sensitivity and the range of the method were modulated by introducing into the reaction mixture different additives consisting of GBS-EDTA (0.17M of glycine, 0.10M NaC1, and 50 mM of EDTA adjusted to pH 9.2 with 1 N NaOH) and various concentrations of NaC1 and dextran T-500 TM 25 (Pharmacia AB, Uppsala, Sweden). Ultracentrifugation. Isokinetic sucrose gradients (5% to 21% w/v) were prepared in nitrocellulose tubes (12 ml) according to the method of Johns and Stanworth. -'~ The samples were layered on top of the gradients and centrifuged in a swinging bucket rotor Ti-41 in a Beckman Spinco L265B ultracentrifuge (Beckman Instruments Inc., Spinco Div., Palo Alto, Calif). Fractions were collected from the top of the gradients with continuous monitoring of the optical density at 280 nm by means of an ISCO gradient fractionator, model 640 (Instrumentation Specialties Company, Lincoln, Neb.). PRIST. Total serum IgE for correlation and linearity studies was assayed according to the manufacturer's instructions with Phadebas PRIST kits purchased from Pharmacia AB, Uppsala, Sweden. Statistical analysis. As a result of the non-Gaussian distribution of IgE levels, the geometrical mean Ibr the groups was calculated after natural log conversion of the IgE values. Groups were compared by Student's t test and the nonparametric Mann-Whitney U test with correction for ties.

RESULTS Effect of pepsin treatment Pepsin digestion of IgE increased the sensitivity of the assay about four times, which is demonstrated by the calibration curves obtained with digested and nondigested IgE (Fig. 1). We prepared the standards by diluting in NRS the reference serum containing 10,400 IU/ml of IgE. Each standard was digested separately in a first experiment, whereas in a second experiment, an IgE concentration of 400 IU/ml in NRS was digested and then diluted in predigested NRS. The two curves were superposable. Therefore, the presence of NRS and the concentration of IgE did not affect the digestion, at least in the conditions of these experiments, i.e., 30 min of incubation at room temperature and 5 mg/ml of pepsin at pH 1.5. The third curve was obtained with nondigested IgE diluted in digested NRS and illustrates clearly a fourfold change in the sensitivity (Fig. 1). In a control experiment the IgE standard of 400 IU/ml was submitted to pH 1.5 in the absence of pepsin and then diluted in digested NRS. This treatment decreased the sensitivity, a fact that indicated that denaturation of low pH alone could not account for the increase in sensitivity observed after pepsin digestion. It should be noted that undigested NRS displayed a relatively strong agglutinating activity that disappeared after pepsin treatment and that was probably related to the presence in NRS of antiallotype anti-

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FIG. 1. Effect of pepsin digestion on the assay of IgE by PACIA. Calibration curve in NRS after pepsin digestion of each standard (o e). Calibration curve obtained by diluting one pepsin digested standard of 400 IU/ml in digested NRS (o o). Calibration curve of nondigested IgE diluted in digested NRS (=---~=). Calibration curve of nondigested IgE but exposed to pH 1.5 for 30 min and then diluted in digested NRS (D D). Calibration curve of nondigested IgE prepared with nondigested NRS (& &). All digestions were done at room temperature for 30 min at 5 mg/ml of pepsin.

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bodies and anti-F(ab')2 autoantibodies (Fig. 1). Careful selection of NRS that has low agglutinating activity before digestion is therefore important. S e l e c t i o n of s t a n d a r d

diluent

In spite of the pepsin digestion that should minimize protein-protein interactions, we observed a significant effect of the protein content of the reaction medium on the agglutinating process. When decreasing concentrations of bovine serum albumin (5% to 1% w/v) were compared to NRS as diluent for the IgE standards, the agglutination gradually increased to a maximum of 15% in the middle of the curve (data not shown). It was therefore critical to have a suitable diluent mimicking as well as possible the protein content of the samples. A pool of NRS fulfilled the requirements as demonstrated by the linearity data and the analytical recoveries. Conditions

of d i g e s t i o n

Nine IgE standards in NRS were digested for 30 min at room temperature at pepsin concentrations of 0.5, 1.0, 5.0, and 10 mg/ml. Only minor differences of the standard curves obtained were observed (data not shown), and 0.5 mg/ml were probably sufficient to complete the digestion. However, as patients' sera may differ in susceptibilities to pepsin, we selected a concentration of 5.0 mg/ml. Since pepsin could affect the agglutinating reaction either by digesting IgE itself

FIG. 2. Ultracentrifugation of pepsin digests of lgE. One IgE standard (1000 IU/ml) was digested with pepsin (5 mg/ ml) at room temperature for various times (0 to 120 min). After tenfold dilution in buffered saline, the digests were run through isokinetic sucrose gradients, and Fc"-containing fragments were assayed in the fractions after another pepsin digestion as in the routine method. The positions of IgE, IgG, albumin, and pepsin are indicated for comparison.

or the bulk of proteins, we tried to distinguish the two modes of action by performing two experiments (data not shown). First, a high IgE concentration was digested for five different times (0 to 120 min) and diluted in predigested NRS (30 min). Second, NRS was digested for five different times (0 to 120 min) and used to dilute a high predigested IgE concentration (30 min). The results demonstrated that almost maximum agglutination was already obtained after 71/2 min digestion of IgE, whereas the agglutination continuously increased with the digestion time for NRS and approached a plateau after 30 to 60 min. These experiments demonstrated that the digestion of the bulk of proteins was more time-dependent and influenced the agglutinating reaction more than the digestion of IgE itself. Ultracentrifugation

of IgE digests

An IgE standard of 1000 IU/ml was digested with pepsin (5 mg/ml) at room temperature for various

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PRIST IgE (IU/rnl) FIG. 5. Correlation between PRIST and PACIA on the IgE results from 48 infants' sera (r = 0.99; y = 0.97.x + 5.95). Additive II was used for PACIA. In both methods each assay was carried out in duplicate, and all samples were assayed undiluted except for the six highest values that were assayed in PRIST after tenfold dilution. With these six 'samples omitted the coefficient of correlation was r = 0.98, and the linear regression was y -- 1.36.x + 0.83.

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incubation times. The digests were diluted 10 times in buffered saline and ultracentrifuged through an isokinetic sucrose gradient. The various fractions were then assayed by PACIA including the digestion step (Fig. 2). A single symmetrical peak of agglutinating activity was obtained after 120 min of digestion. This material had an estimated molecular weight of 35,000 that agrees well with the molecular weight of the Fc"~ fragment known to be heat and protease resistant.23, 27 After 30 min of incubation, the time chosen for our routine assay, the peak was slightly asymmetrical, but the major part was in the form of the Fc", fragment. After 7Vz min the intact IgE was barely detectable, and the digested material was resolved in three peaks with molecular weights of about 35,000 (3.2 S), 100,000 (4.9 S), and 150,000 (6.4 S), prob-

ably corresponding to the Fc",, the 5 S, pepsin,* and the F(ab')2, fragment, respectively.27 These data demonstrate the extreme resistance of the Fc", fragment toward pepsin digestion, and one should note that once released, the Fc" fragment is not degraded further even after 120 min of digestion time.

Calibration curve A plot of peak heights corresponding to the number of nonagglutinated particles versus log IgE concentration formed a sigmoidal curve extending from 0.1 to 20 IU/ml (Fig. 3). According to the method of Kaiser, 28 we set the limit of detection at three times the noise level, i.e., a 2% decrease in the number of particles. ~7 This corresponded to about 0.05 IU/ml. Since the digestion and pH neutralization steps resuited in a 3,6 dilution of the sample, the theoretic sensitivity was about 0.01 IU/ml or 1 pg/30 txl of digested sample and assay tube.

Nonspecific agglutination Previously, 2~ we found that some human sera agglutinated latex coated with F(ab')2 fragments from nonimmunized rabbits. These agglutinators were *Magnusson CGM: Unpublished data.

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TABLE I. Analytical recovery of IgE by PACIA IgE recovered in NRS* Added concentration (lU/ml)

1.0 1.0 2.0 5.0:[:

lU/ml mean --. SD (recovery %)

1.12 1.03 1.78 4.85

-+ 0.10 _+ 0.09 -+ 0.09 -+ 0.36

IgE recovered in cord serat No. of samples

(112) (103) (89) (97)

Range of concentrations before addition of IgE (lU/ml)

12 12 10 16

0.10 0.10 0.10 0.10

to to to to

IgE recovered (lU/ml) (mean -+ SD)

0.48 0.50 0.33 0.87

1.10 1.02 1.92 5.03

Recovery (%)

-----0.08 _+ 0.10 --+-0.21 _+ 0.41

110 102 96 100.6

*Each NRS sample was treated with pepsin in duplicate and thereafter assayed in duplicate, i.e., a quadruple assay for each concentration. tEach cord serum was treated once with pepsin and thereafter assayed in duplicate. SAddivive II as used.

TABLE II. Influence of sample dilution on the assay of IgE (IU/ml) by PRIST and PACIA PRIST*

PAGIAt

Sample dilutions No. of sample

1/1

3/4

2/4

1/4

1/8

1/10

1/1

1/10

5753 5232 5813 5814 5467 5080 5827 4505 4780

32.5 15.8 13.4 . . . . . .

30.9 17.7 --

31.2 19.3 15.3 . . . . . .

36.4 23.2 15.8

--17.6

44.0 26.0 18.5 188 142 116 127 159 173

38.4 24.4 20.0 150 126 128 134 180 162

---162 136 128 140 172 166

. . . . . .

. . . . . .

. . . . . .

*Assayed in duplicate. ~'Assayed in duplicate with additive II.

probably IgM or IgG anti-F(ab')2 autoantibodies cross-reacting with rabbit F(ab')2. 2'~ 22 In this study we tested 20 sera with strong agglutinating activities and 10 sera devoid o f agglutinators on latex coated with F(ab')2 fragments o f nonimmunized rabbits with additive I. After pepsin digestion of both groups, no difference in the agglutinating activities of the positive and negative sera could be detected. The mean peak height was 100.8 _+ 0.50 with a coefficient of variation o f 0.5%. Additives

By changing the composition o f the additive, it was possible to adapt the sensitivity o f the assay. Additive I consisted o f GBS-EDTA, 0.6M NaC1, and 36 mg/ ml o f dextran T-500 and provided a suitable sensitivity for the assay of IgE in cord sera (Fig. 3). Additive II, which contained GBS-EDTA, 0.6M NaC1, and 20

mg/ml o f dextran T-500, elicited a slightly lower sensitivity and is convenient for infants (range 2 to 200 IU/ml). Additive III, which contained GBS-EDTA and 10 mg/ml of dextran-T500, elicited a range of 10 to 1000 IU/ml and was practical for adult samples since too high predilutions were avoided. Accuracy

One volume of four IgE standards in NRS was added to nine volumes of various samples of cord serum and assayed by PACIA in duplicate after pepsin digestion. The analytical recoveries ranged from 89% to 112% (Table I). The linearity o f the assay was demonstrated on three cord sera containing 10.1, 19.4, and 20.6 IU/ml o f IgE after five serial twofold dilutions in NRS (Fig. 4). The coefficients of variation on the values corrected by the dilutions were 5.6%, 8.8%, and 7.1%, respectively.

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T A B L E III. Precision of PACIA in the assay of IgE in the cord serum Within run (n = 25)

Between run (n = 20)*

Concentrations IU/ml - SD

Coefficient of variation (%)

0.395 + 0.028

7.1

2.14 --- 0.123

5.9

10.34 - 0.786

7.6

Coefficient of variation (%)

Concentrations IU/ml _+ SD

0.399 1,00 2.01 4.04 9.86

_+ 0.021 _+ 0.054 + 0.107 + 0.176 + 0.596

5.3 5.3 5.3 4.4 6.0

*Each cord serum was treated once with pepsin and thereafter assayed in duplicate. T A B L E IV. Levels of IgE (IU/ml) in the cord serum of nonselected newborn infants

as measured by PACIA Nationality

Sex

Median

Geometric mean

+ 1 SD

Range

Mann-Whitney U test

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M

146

0.36

0.46

1.70

<0.10 to 30.0

European

F

142

0.27

0.33

1.06

European

M plus F

288

' 0.30

0.39

1.36

<0.10 to 8.0 <0.10 to 30.0

African-Asian

M plus F

60

0.85

1.05

5.38

African-Asian

M

26

1.10

1.27

6.59

African-Asian

F

34

0.61

0.92

4.66

Student's t test

p = 0.034

p = 0.019

p < 0.00005

p < 0.00005

<0.10 to 125.0 0.10 to 125.0 <0.10 to 14.0

T A B L E V. Cord IgE levels (IU/ml) of the African-Asian subgroups as measured by PACIA

Nationality

n

Median

Geometric mean

+ 1 SD

Central Africa* North Africat Japan-Formosa Lads-Vietnam Total

33 12 7 8 60

1.68 0.65 0.28 1.50 0.85

1.58 0.62 0.30 1.47 1.05

8.25 1.93 0.42 12.4 5.38

Range

0.10 to <0.10 to 0.22 to 0.10 to <0.10-

125.0 3.10 0.55 50.0 125.0

*Zaire, Cameroun, Upper Volta, Nigeria. tMorocco, Tunisia (includedhere are Turkey and Syria). Correlation

The correlation of results between PACIA (additive lI) and PRIST on 48 sera from children ages 1 to 14 yr was r = 0.987 with a linear regression of y = 0.97x + 5.95 (Fig. 5). PAC1A revealed higher values over the entire range except for the six samples with the highest IgE concentrations. It should be noted that these six samples were diluted l 0 times in PRIST

but were assayed undiluted by PACIA. We suspect that PRIST was responsible for the discrepancies, since PRIST results were found nonlinear when PRIST was tested on various samples including three samples from the correlation (Table II). The values tended to be abnormally low when the samples were undiluted, probably because of a serum effect that did not occur when the samples were diluted 10 times as

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for the six highest values of the correlation. After omitting these six values the correlation was r = 0.976 with the linear regression y = 1.36.x + 0.83.

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The IgE assay was repeated 25 times during the same day on a pool of cord sera supplemented with IgE to reach three concentrations and thereby to cover the whole range of the standard curve. The coefficient of variation ranged from 5.9% to 7.6% (Table III). The assays, now in duplicate, were also repeated daily for 20 days on the same pool of cord sera supplemented with IgE to reach five concentrations. In this assay the coefficient of variation ranged from 4.4% to 6.0% (Table III). The better coefficients of variation for interassays are probably explained by the fact that they were assayed in duplicate, whereas in the intraassays the samples were assayed once.

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IgE c o n c e n t r a t i o n s in cord sera

In 318 nonselected cord sera the IgE concentration ranged from less than 0.10 to 125 IU/ml (Fig. 6, Tables IV and V). Since several high values were found in neonates of foreign origin, the newborn infants were grouped as European and African-Asian neonates on the basis of their mother's nationality (n = 288 and n = 30, respectively). Another 30 samples were later added to the second group. The neonates with African-Asian origin had a higher geometrical mean cord IgE level than the European neonates, 1.05 and 0.39 IU/ml, respectively (Table IV). This difference was highly significant whether the mean was calculated by Student's t test (p < 0.00005) or Mann-Whitney U test (p < 0.00005). The African-Asian group was further divided into subgroups, since wide variations were observed within this group (Table V). The highest geometrical mean cord IgE levels were found in the central African (1.58 IU/ml) and the Laos-Vietnam (1.47 IU/ml) subgroups. The north African subgroup had an intermediate level (0.62 IU/ml), whereas the Japan-Formosa subgroup had a cord IgE level (0.30 IU/ml) comparable to that of the European group (0.39 IU/ ml). In view of these few results, it appears that higher cord IgE levels are found in newborn infants coming from tropical areas. In the European group significantly higher geometrical mean cord IgE levels were found in boys than in girls (Table IV) whether the mean was calculated by the Student's t test (p = 0.019) or the MannWhitney U test (p = 0.034). This difference between sexes can be explained by the fact that boys (n = 37; 25.3%) had significantly more often (p < 0.05; • = 4.29) cord IgE levels higher than 1.20 IU/ml ]4

AFRICAN ASIANS

EUROPEANS

n= 146

n=142

n =288

n =60

FIG. 6. IgE levels in the cord sera of the European and African-Asian groups. The horizontal lines represent the geometrical means and the broken line represents the lower limit of reliability of the assay from which the coefficient of variation exceeds 20%. Values below 0.10 IU per ml were extrapolated on the standard curve.

than girls (n = 22; 15.5%). No significant difference between sexes in the central African subgroup or in the African-Asian group as a whole was observed

(p > 0.10). Ultracentrifugation

Ultracentrifugation analysis of two cord sera containing 20.6 and 19.4 IU/ml, respectively, illustrated the specificity of the assay, the possibility of its use as a research tool and its high sensitivity. The cord sera were fractionated after fivefold dilution in bufferd saline on an isokinetic sucrose gradient, and lgE was assayed in the fractions after pepsin digestion. Cord serum lgE was recovered in the 7.8 S fraction (Fig. 7) that was expected for adult IgE. 26' 29 DISCUSSION Technical aspects

The wide variations in the reported levels of IgE i n cord serum illustrate the difficulties encountered by the various methods proposed for the assay of low concentrations of IgE (Table VI). Techniques on the basis of competition with a radiolabeled tracer such

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sucrose gradient. The distribution of IgE was studied by PACIA after pepsin digestion of the fractions (e-----e and c o). The positions of albumin, IgG, and IgE are indicated for comparison.

as RIST and BAC are known to be susceptible to various serum factors, k' 30-34Among these factors, for example, anti-IgE autoantibodies 35 and heterologous antibodies that react with the insolubilized anti-IgE immunoglobulins 36 will inhibit the binding of the tracer to the insolubilized antibodies and be responsible for abnormally high results. The DAB method,34.37 in which free and bound tracer is separated by precipitation with an anti-immunoglobulin antiserum is more reliable but laborious and very timeconsuming (96 hr) when very low levels of IgE are to be assayed. PRIST, 38 a sandwich technique on the basis of binding of IgE to insolubilized anti-IgE antibodies followed by detection with a radiolabeled antibody, is generally considered as the method of choice because this commercially available technique is simple, sensitive, and less prone to interfering factors, 3] We have demonstrated that PACIA can measure very low levels of IgE with precision and accuracy. For levels of 0.4 to 10 IU/ml, within-day and betweenday coefficients of variations did not exceed 7.6%. The importance of precision and accuracy is obvious when one considers that about 1.2 to 1.3 IU/ml in cord serum is the proposed clinical cutoff point for the prediction of future development of allergy. ~'' 14 Accuracy of PACIA was demonstrated by the linearity test and the analytical recoveries. In the correlation with PRIST (r = 0.99), the discrepancies observed could be attributed to serum effect on PRIST because

our linearity study of this test confirmed what was already suspected by others, 3t' 34 that PRIST gave too low values on undiluted serum. Interfering factors in PACIA are eliminated by peptic digestion. However, satisfactory accuracy was obtained, despite the digestion step, only if the standards and, when it was necessary, the samples were diluted in well chosen NRS, which mimics the protein composition of the serum samples. Earlier, z6 when we applied PACIA to the assay of IgE in adults, the sensitivity, 5 to 10 IU/ml, was limited by the necessity of a tenfold dilution of the sera to avoid nonspecific agglutination caused essentially by antibodies reacting with the rabbit F(ab')2 fragments coating latex. 2'' 22In the assay of other proteins, when pepsin cannot be used, these antibodies are inactivated by the addition of aggregated F(ab')2 fragments of nonimmunized rabbits. ,8.2. However, we prefer peptic digestion that does not only destroy antiF(ab')2 antibodies but also other possible interfering factors such as anti-IgE autoantibodies 3s 39,40 and putative allergen-IgE antibody complexes that could interfere in other assays. Because of the elimination of interferences by pepsin, PACIA can be applied to undiluted serum samples and various secretions, 4~ and agents known to enhance antigen-antibody reactions such as dextran ~8' 25 can be used to substantially increase the sensitivity with minimal risk of increasing nonspecific reactions. However, it is clear that the high sensitivity reached by PACIA, about 0.05 IU/ml in 30 rain of incubation time, is not only related to the addition of dextran and the weak dilution of samples but also to the digestion itself, which increased the sensitivity about four times. After pepsin treatment, which was demonstrated by ultracentrifugation, the agglutinator is clearly the Fc", fragment. This fragment probably displays a higher agglutinating activity than whole IgE by of'feting new antigenic determinants. However, the reduction in size could in itself account for the higher sensitivity. A small molecule diffuses more rapidly; hence its binding to antibody will be faster, and since the main antigenic determinants, recognized by our anti-IgE antiserum, are located in the Fc"~region, the risk of steric hindrance will also be decreased when the agglutinator is the Fc'~ fragment. In addition the stability of the agglutinates is probably reinforced by the mutual interactions between the protein layers coating the particles. This secondary bonding can be established only if the antigen bridge is short enough to keep the particles in close contact. An additional advantage of pepsin digestion is the use of a heat- and protease-resistant fragment as an antigenic target for

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Highly sensitive particle counting immunoassay

NUMBER 4

521

TABLE VI. Literature data for the level of IgE (IU/ml) in cord serum Year

Author(s)

N

Mean

1968 1971

Johansson 53 Stevenson et al. 54

RIST DAB

37 11

15.0 "'t 16.2"t

BAC DAB PRIST RIST RIST PRIST

33 150 26 26 26 24

2.1 ~ -0.39 0.64 3.79 0.22 ~'t

< 1 . 0 to 7.5* 0.08 to 16.0 ---<0.1 to 1.5

PRIST

30

1. I t

0.1 to 2.8

RIST

30

1.7t

0.1 to 17.0

Atopic mothers

RIST

33

2.45t

1978 1978 1979 1979 1980 1980 1982 1982 1983 1983 1983

Kjellman and Johansson' Kjellman and Johansson ~ Gamo and Maruyama 3~ Dannaeus et al. ~9 Dannaeus et al. $9 Ganju et al. 57 Thomas et al. 6~ Michel et al. 4 Basuyau et al. 6' Gordon et al. 62 Ringel et al. 46 Bousquet et al. 63 Bousquet et al. 63 Weil et al. $6

Atopic and nonatopic mothers Mexican-American white neonates (USA) Nonselected neonates (USA) Nonselected neonates White neonates White neonates White neonates Nonatopic neonates of nonatopic parents Atopic mothers

1971 1975 1976 1976 1976 1976

Bazaral et al. 33 Merrett et al. ~5 Johansson et al. 3~ Johansson et al. ~' Johansson et al. 3' Kjellman et al. '2

1976

PRIST PRIST PRIST PRIST PRIST PRIST PRIST ENZYGNOST PRIST PRIST PRIST

17 36 21 93 136 19 160 120 95 234 57

0.11i 0.24? 1.48t -0.32 0.29:~ 1.31t 0.2? 1.0 0.3 5.25*

<0.1 <0.1 <0.5 0 <0.5 0

1983 1984 1984 1984 1984 1984

Businco et al. 6" Magnusson z4 Magnusson '4 Magnusson# Magnusson# Magnusson#

PRIST PACIA PACIA PACIA PACIA PACIA

101 288 60 106 81 92

0.53~: 0.39t 1.05t 0.281 0.57t 0.20 ~*

0.5 <0.10 <0.10 <0.10 <0.10 <0.10

1976 1977

Method

Range 6.6 to 40.3* 7.3 to 48.2*

--

Nonatopic parents

to 0.15 to 0.7 to 12.0 to 1 to 5.5 to 0.75 -0 to 4.8 0 to 6.5 0 to 5.5 0.4 to 80.0* to to to to to to

Remarks

10 30.0 125.0 30.0 20.0 2.7

Nonatopic mothers Atopic mothers Black neonates (USA) Black neonates (USA) White neonates White neonates Nonatopic neonates White neonates Atopic parents Nonatopic parents Indian neonates of mothers with parasitosis Atopic parents or siblings European neonates African-Asian neonates Nonatopic parents Atopic parents Nonatopic neonates of nonatopic parents

*Values originally reported in nanograms per milliliters, but here they have been converted into international units per milliliters with 1 IU = 2.42 ng. 24 tGeometrical mean. SArithmetic mean. w newborn infants not presenting allergy symptoms within the first 18 months of life. I[Median. #Magnusson CGM: Manuscript in preparation. the assay o f IgE. Such a f r a g m e n t has a greater chance to be r e c o v e r e d intact in secretions 41 rich in proteases or in s e r u m samples not w e l l c o n s e r v e d . It has b e e n reported that antisera directed against the D1 determinants that pertain to the Fc"~ region 2~ are less suitable for r a d i o i m m u n o a s s a y s 42 than the antisera directed against the D 2 determinants,43' 44 which are located in the protease and heat-labile C terminal part o f the Fc~ fragment. 23 B e c a u s e o f this and a French article 45 on the inability o f P R I S T to detect f r a g m e n t e d

IgE in intestinal fluids, we treated IgE with pepsin and found that one can assay Fc"~ (D1) fragments by PRIST, and the binding c u r v e o f a serially diluted sample and o f the standards p r o v i d e d with the kit had even a slope 1.3 times steeper after pepsin digestion (data not shown). This change, however, was less evident than for P A C I A in w h i c h the pepsin treatment increased the sensitivity four times. In P R I S T the putative n e w antigenic determinants or the smaller size o f the antigen by accelerating its diffusion and re-

522

Magnusson and Masson

ducing steric hindrance could account for the steeper slope of the binding curve. It would be interesting to know if pepsin digestion could not reduce the serum effects observed, especially in RIST and BAC? ~ Of course for accurate conversion of results into international units per milliliters, the IgE standards have to be digested like the samples, and ideally one should use the Fc"~ fragment as radiolabeled tracer. Pepsin digestion of the sample before assay by PACIA is not limited to IgE, since it also works for other protease-resistant antigens, e.g., thyroid stimulating hormone, 2~ carcinoembryonic antigen,* and bacterial antigens, t

Clinical aspects The IgE content of cord blood that was mentioned previously is subject to wide variations (Table VI) caused mainly by technical difficulties. Also the differences in the populations studied can cause variations since a number of factors, mainly the incidence of atopy and race but also family history can influence the IgE level, a' la RIST is suspected to elicit falsely high results, a' 3J. 34 and until now, no other technique has convincingly confirmed the low levels of IgE found in cord blood with the PRIST technique except for a recent article in which an enzyme immunoassay was used. 46 Our data on the assay of IgE by PACIA in the cord serum of 288 European nonselected newborn infants agree with most PRIST results (Tables IV and VI). With PRIST, nonselected newborn infants from Sweden had a mean value of 0.39 IU/ml, 3~ a value identical to what we found in this study with PACIA. In a French study with PRIST4 in which the limit of detection was set at 0.5 IU/ml, 32% of the newborn infants had detectable IgE. We found 37% of the newborn infants with detectable IgE with this same level set as limit of detection for PACIA. Although the highest IgE levels in the Swedish and French studies reached 2.8 and 5.5 IU/ml, respectively, we found with PACIA newborn infants with levels up to 20 and 30 IU/ml in the European group and even higher in the African-Asian group (Fig. 6). Recently, in an extensive Swedish study 2 on IgE levels in cord serum, values up to 20 to 30 IU/ml were also found. However, in our study with PACIA, 59 European newborn infants (20.5%) had cord IgE levels higher than 1.20 IU/ml that is considered by us as the cutoff point for the prediction of allergy. 14 This percentage is clearly higher than that reported in the Swedish study in which only 5.3% had cord IgE levels *Mareschal GC: Unpublished data. tJouret M, Magnusson CGM: Unpublished data.

J. ALLERGY CLIN. IMMUNOL. APR/L 1985

1>1.30 IU/ml. 2 Perhaps these results could be explained by differences in either the methodology used or the population studied. Two major observations emerge from our study on cord sera. First, the IgE level in the European neonates was significantly higher in boys than in girls (Table IV). Although a difference was never reported for neonates, a difference in the IgE level between sexes has been reported on a population ages 6 to 75 yr or m o r e , 47 Tobacco smoking by males 47 was suggested as the partial cause of this difference since smoking is known to increase the IgE level. 48' 49 Since the influence of tobacco smoking can be excluded in the present study, our results strongly support the role of genetic factors to explain the differences between sexes. It is generally admitted that boys are about twice as often allergic than girls, 5~ 5~a fact remarkably well paralleled by the findings of others 2 and ours that boys compared to girls had significantly more often cord IgE levels above the cutoff point chosen for the prediction of allergy. ~o. J4 The fact that we did not observe any significant difference in cord IgE between sexes of the central African subgroup could be explained either by the limited number of cases available or by the fact that gender plays a weaker role than race and geographic factors. Second, cord IgE was significantly higher in the African-Asian neonates than in the European neonates (Table IV), but since African and Asian mothers could have very high lgE levels caused by parasitosis, traces of maternal blood could significantly increase the IgE level in cord blood, e.g., leakage during labor? 2 If contamination can be excluded and if the reported classic IgE does not cross the placenta, ~"27. ~3.53.54 either congenital or genetic factors, 55 perhaps associated with parasitosis, 5~ have to be envisioned. This is consistent with a recent article in which elevated cord IgE levels (geometric mean 5.25 IU/ml) of Indian neonates born to mothers with parasitic helminth infection were attributed to sensitization in utero by parasitic antigens? 6 However, these authors did not exclude a possible genetic basis for the high cord IgE levels. In favor of an eventual genetic explanation is the higher cord IgE levels found in black American neonates (geometric mean 1.48 IU/ml)? 7 We may conclude that in addition to the obvious advantages of not requiring radioisotopes, of being fully automated, and of requiring cheap and stable reagents, PACIA combined with pepsin digestion is one of the most nearly accurate, sensitive, and fast methods for the assay of IgE and is particularly suitable for the screening of neonatal IgE in view of allergy prediction. 14

VOLUME75 NUMBER4

Highly sensitive particle counting immunoassay

We wish to thank Mrs. M. Magnusson for excellent technical assistance and Mr. M. Delory for competent editorial work.

20.

REFERENCES

21.

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