Comparison of ultrasensitive methods for the measurement of IgE

Journal of Immunologwal Methods, 58 (1983) 365-373 Elsevier Biomedical Press

365

Comparison of Ultrasensitive Methods for the Measurement of IgE 1 A. B r a d l e y E l s e n b r e y 2, T h o m a s Krebs, S a n d r a D u n n e t t e a n d G e r a l d J. G l e l c h 3 Departments of Immunology and Medwme, Mayo Medwal School, Allergw Daseases Research Laboratory, and Mayo Chmc and Foundatwn, Rochester, M N 55905, U S A

(Recewed 13 August 1982, accepted 8 November 1982)

Because the study of human lgE synthesis and regulation reqmres exqmsRely sensluve and rapidly performed methods for assay of m vitro IgE producaon, we compared 4 methods for radlometnc immunoassay (RIA) of human IgE double antibody RIA (DARIA), ultrasensmve enzymatic RIA (USERIA), sensmve paper ra&olmmunosorbent test (SPRIST) and mterotlter sohd-phase RIA (MSPRIA) IgE protein was measured m serum samples and cord bloods The USERIA and the MSPRIA consLstently detected levels of IgE as low as 27-35 pg/ml The DARIA and SPRIST were less sensmve Comparison of the USERIA and the MSPRIA favored the latter because (1) it is a more rapidly performed assay, (2) tt involves fewer mampulatlons, and (3) it has less vanabdlty We conclude that the MSPRIA for IgE is superior to the other methods both as a research tool for measunng minute quanuttes of IgE protein (as tn tissue culture supernatants) and for measunng IgE m serum samples where concentrataons fall below 1 ng/ml (0 42 IU/ml, 1 IU ffi 2 4 ng) Key words ultrasensmve methods for IgE - - ultrasenstuve enzyme RIA - - double antzbody R1A - - mtcrotlter sohd-phase RIA - - senstttve paper radlotmmunosorbent test

Introduction

Until recently, the study of human IgE synthesis and regulation has been hampered, m part, by the lack of sufficiently sensmve methods for assay of in v~tro production (Flser and Buckley, 1979, Zuraw et al, 1981) The ablhty to rapidly and consistently measure femtomolar (50 x 10-15 M) quantmes of IgE protein in small samples ( < 200 #1) permits extensive experimentation with regulatory factors (Zuraw et al, 1981) and subsets of lymphocytes in both normal and atoplc m&vlduals (Flser and Buckley, 1979, Zuraw et al, 1981) i Supported m part by grants from the National Institute of Allergy and Infectious Diseases, AI 11483, and by the Mayo Foundaaon z Present address Wayne State Umvermy School of Medicine, Detroit, MI 48201, U S A 3 Reprmt requests to Gerald J Glelch, M D , Department of Immunology, Mayo Chine, Rochester, MN 55905, U S A 0022-1759/83/0000-0000/$03 00 © 1983 Elsevier Science Pubhshers B V, Amsterdam

366 We report a comparison of 4 methods of radlometrlc lmmunoassay for human IgE the double-antibody radlolmmunoassay (DARIA) (Glelch et al, 1971), mlcrotlter sohd-phase radlolmmunoassay (MSPRIA) (Zolllnger et al, 1976), a PRIST '~ assay (Pharmacla Diagnostics AB, Uppsala) modified to increase sensitivity (R E Rockhn, personal commumcatlon, H e m a d y et al, 1983), and an application of the ultrasensitlve enzymic radmlmmunoassay (USERIA) (Harris et al, 1979. Hsu et al, 1981) for measurement of IgE SensItlwty, reproducibility, sample size and total length of assay time are compared In this study, we measured IgE protein in serum samples and human cord bloods and concluded that the MSPRIA is superior to the other methods, both as a research tool, as in measuring minute quantmes of IgE produced in tissue culture supernatants, and for measurmg IgE in body fluids where concentrations are expected to be less than 1 0 n g / m l (0 42 I U / m l , 1 IU = 2 4 ng) (Bazaral and Hamburger, 1972, Gletch and Dunnette, 1977, Nerenberg and Prasad, 1981)

Materials and Methods DARIA The D A R I A for human IgE has been described in detad elsewhere (Gleich et al, 1971, Gleich and Dunnette, 1977, Dunnette and Gleich, 1981) Briefly, IgE standard, or sample, in a volume of 25-500 #1 is added to 10 x 75 disposable glass tubes and the volume is adjusted to 500 #1 with 0 1 M potassium phosphate buffer, p H 7 5, containing 1% bovine serum albumin and 0 1% N a N 3 (BSA buffer) Rabbit anti-human IgE(ND)-Fc (100 lal) diluted 1 50,000 in BSA buffer and 125I-labeled h u m a n IgE diluted to 1 n g / m l (100 #1) in BSA buffer are added, mixed, and incubated at 20°C for 18-96 h Burro anti-rabbit IgG (100 gl, added in an amount to ensure antibody excess for rabbit IgG) and normal rabbit serum (1 20, 100 #1) are added, rmxed, and then incubated for 2 h at room temperature The tubes are centrifuged at 3420 × g for 20-30 m m at 4°C, the supernatants decanted and the tubes drained for 10 mln to rmmmlze the fluid associated with the pellet Samples are counted for 5 - 1 0 m m each in a g a m m a scmtdlatlon counter The sensmvIty of the D A R I A (normally about 1 n g / m l ) can be increased to 0 3 - 0 5 n g / m l by decreasing the amount of antl-IgE by a factor of 10, decreasing the first stage volume to 0 5 ml and increasing the m c u b a t m n times to 3 days m the first stage of the assay, 1 day for antMgE plus test material, and to 2 days after a d d m o n of the radmlabeled IgE USERIA The assay was performed in a manner slrmlar to that described by Hsu et al (1981) [3H]AMP (New England Nuclear, Boston, MA) was prepared as described The optimal pH, sonic strength, and antibody concentration for coating polyvlnyl chloride U-bottom rmcrotlter plates (Dynatech Laboratories, Alexandria, VA) was deternuned by checkerboard titration m the following manner (Harris et al, 1979) mouse monoclonal anti-human IgE antibodies from 2 clones, 7 12 and 4 15, specific

367 for the Fc region of IgE (londly prowded by Dr A Saxon) were combined m a 1 1 mole ratio and dduted to 1 and 100 /xg/ml m phosphate and carbonate buffers ranging m pH and molanty from 10 5 to 5 8 and 60 mM to 7 5 mM, respectively A 100/~1 ahquot of each combination was added in duphcate to wells and the plates were incubated overmght at 4°C m a hurm&fied box The plates were washed 5 times with 175 #1 Dulbecco's phosphate-buffered sahne (PBS) containing 0 05% Tween 20 and 0 1% NaN 3 (PBS-Tween), and each well recewed 100/~1 (1 5 ng) of purified IgE(PS) which had previously been labeled with 125I by the iodine monochloride method (Bale et al, 1966, Dunnette and Glelch, 1981) After incubating at 37°C for 3 5 h, the wells were again washed 5 times with 175 ~1 of PBS-Tween and counted m a gamma scmtdlat~on counter Those condlUons which pernutted the greatest binding of ~25I-labeled IgE were selected for routine preparation of coated plates For the assay of samples containing IgE, plates were coated by the method outlined above using a combined monoclonal antibody concentration of 10/~g/ml m 7 5 mM carbonate buffer (pH 9 3) After recovering the well contents (the coating solution can be used up to 6 times without slgmficant loss of sensltwlty), the plates were washed 5 times with 175 /~1 of PBS-Tween A 100 /~1 volume of sample or standard, diluted m 0 1 M KH2POa-Na2HPO4, pH 7 5, 0 1% NaN 3, 1% Tween 20, 0 2% bowne serum alburmn (BSA-Tween) was added to each well and the plates were incubated overnight m a hurm&fied box at 4°C The wells were washed 5 times with 175 ~1 of PBS-Tween and 100/.tl of rabbit anu-human IgE(PS)-Fc (that had been absorbed with normal human serum) dduted 1 4000 m 10% normal mouse serum (NMS) plus 10% normal goat serum (NGS) was added to each well Following a 3 5 h incubation period at room temperature, the wells were again washed 5 times with 175 /~1 of PBS-Tween and 100/tl (5 ng) of alkahne phosphatase conjugated goat anti-rabbit IgG (Boehrmger Mannhelm Blochemlcals, Indianapolis, IN) &luted m PBS with 10% NGS and 10% NMS was added to each well After another 3 5 h incubation period at room temperature, the wells were aspirated and the excess conjugate was removed by five 175 ttl washes with PBS-Tween and 2 washes w~th 175 #1 of 100 mM dlethanolanune (pH 9 6) A 100/~1 volume of [3H]AMP (1 × 10 6 cpm) m 10 mM dlethanolarmne (pH 9) was added to each well and the plates were incubated for 18 h at 37°C A 50 ~1 ahquot from each well was applied to plastic disposable columns (Isolab, Akron, OH) containing 1 5 ml packed volume DEAE-Sephadex A-25 (Pharmacla), eqmhbrated with &stilled water, and eluted with 3 ml of a 1 20 ddutlon of PBS containing 0 1 mM adenosine (Sigma Chemical C o , St Louis, MO) m three 1 ml steps A 1 ml ahquot from each column eluate was combined w~th 4 ml scmtdlatlon counting flmd (NaUonal Dmgnostlcs, Somervdle, NJ) and counted in a beta counter (LS100, Beckman Instruments, Irvlne, CA) to 0 5% error Senstttve PRIST ®

Phadebas IgE PRIST &scs were purchased from Pharmacla Diagnostics (Plscataway, N J) Reagents used were the Phadebas PRIST &scs, and PBS-Tween and

368

125I-labeled rabbtt anti-human IgE(PS)-Fc tracer prepared in this laboratory Dilutions were made in BSA-Tween, unless culture supernatants were tested, in whtch case dilutions were made in RPMI 1640 (GIBCO, Grand Island, NY) with 10% fetal calf serum (FCS) The procedure followed that described by Hemady et al (1983) Briefly, discs (Phadebas Phadezym-PRIST mterface k i t ) w e r e placed m the bottom of 12 mm × 55 mm round bottom polystyrene tubes (Pharmacla) and 300 /~l of sample or standard solution were added The tubes were incubated for 20-24 h at room temperature on a horizontal shaker (Lab-Line, Melrose Park, IL) at 150 rpm The tubes were asptrated and the discs washed 3 times with 2 5 ml PBS-Tween with 10 min soaking periods between washes with a final aspiration after residual moisture had pooled Radiolodlnated anti-human IgE (200/tl, 10 ng) was added to each tube, and the tubes incubated again for 20-24 h at room temperature The d i s c s were washed 3 t i m e s , as described above, and counted in a gamma scmtdlatlon counter

MSPRIA In the plastic plate radlometric assay for IgE (MSPRIA) flexible plastic U-bottom plates (Dynatech Laboratories, Alexandria, VA) were rinsed with distilled deiontzed water and coated with monoclonal mouse anti-human IgE as in the U S E R I A (100/~l of 10 /~g antibody per well, diluted in 7 5 mM HCO 3 buffer, pH 9 3), at 5°C overnight The coating antibody solution was removed and wells were washed 3 times with 175 pl PBS-Tween to remove free coatmg antibody Next, 175/~l of 5% newborn bovine serum in PBS (blocking solution) with 0 1% N a N 3 was added and incubated at 4°C In hunudlfled atmosphere for 3 h The blocking solution was removed by aspiration, and the plates washed 5 times with PBS-Tween Samples and standards were diluted in BSA-Tween, and 100 /~l of sample were added to each well Wells were incubated overnight at 5°C in a humidified box Wells were aspirated, washed 5 ttmes with 175/~l of PBS-Tween, and 100/~l of radlotodmated affinity chromatography purified anti-human IgE * (5 ng/well) were added to each well Wells were incubated for 4 h at room temperature, washed l0 times with 175 #l of PBS-Tween, cut out with scissors, and counted in a gamma scintillation counter

Stattstwal analysts Calibration curves for the various immunoassays were generated by least square regression curves of best fit with first or second order equattons using a Hewlett Packard 9845B computer and program no 09845-15011 for regression analysis The analysis of covanance was tested on a Hewlett-Packard 9810

Results and Discussion

Standard curves for the radlometrlc assays were generated with purified IgE protein (PS) (Dunnette and Glelch, 1981), World Health Organization (WHO) * We also tested t25I-labeled lmmunosorbent purified anti-human IgE (PRIST reagent) from Pharmacta Diagnostics, using 10 ng/well it was 5-12-fold less sensUlve than the antl-lgE prepared m our laboratory

369

standard serum (no 68-341), American standard serum (Evans, 1981), and a secondary standard prepared m our laboratory by poohng serum from 7 normal m&wduals and cahbrated as contmmng 177 ng IgE/ml against the WHO standard Representatwe standard curves are presented m Fig 1 Although each assay method enabled measurement of IgE concentrations to less than 500 pg/ml, the qualmes of each method are so &stmct that they wdl be &scussed m&wdually before compansons will be made General charactenstlcs of each assay method are presented m Table I DARIA

The DARIA ts our laboratory standard as the assay has given consistently reproductble results over the last 12 years However, the assay is 1Lrmted by the long lncubaUon t~mes needed to achieve tugh sensltwxty and by an mabdlty m our laboratory to consistently measure levels below 300 pg/ m l On the other hand, Sampson and Buckley (1981) have measured as httle ag 100 pg/ml using a 6 day incubation of the first stage anubody and further dilution of the first stage antibody

lO

2

B 1 0

m

0

m

1

o ._1

2

o 0 0 _1

3 I

4

t0

3 Log I~g/O 3 ml

2

5 I I 2 3 Log pg/ml

I 4

1O0~ =

16 10

o Io~ 0 ..I

0 .J

10

10 16

.i

i

[

2 3 Log p g / m l

20

I

I

I

I

1

2

3

4

Log pg/ml Fig 1 Representatwe standard curves for each of the 4 assay methods A standard curve for the sensitive DARIA using punfied IgE(PS) Loglt Is calculated loge y / ( l - y ) B/BO is cpm bound/cpm bound with no mhJbmng protein B USERIA standard curve using the U S standard serum (900 IU/ml) as the IgE standard (1 I U = 24 ng) (Bazaral and Hamburger, 1972, Yungonger and Gielch, 1973) Log ~ TC ts calculated logj0 [(cpm released- background)/(cpm substrate a d d e d - background)× 100] Background is measured as nonspeclfic hydrolysis of substrate in parallel samples without enzyme C SPRIST using the calibrated secondary standard (177 ng/ml) pool of sera from 7 nonatoplc mdlwduals Log % TC ]s calculated loglo [(cpm b o u n d - c p m in antigen blanks)/(cpm tracer added)x 100] D MSPRIA using plates coated with mouse monoclonal anti-human IgE and the U S standard serum as the IgE standard Log ~ TC Is calculated as in the SPRIST

370 TABLE I COMPARISONS OF SENSITIVE R A D I O I M M U N O A S S A Y S FOR IgE

Range ( p g / m l ) Limit of sensitivity a CV (%) Sample slze/rephcate (t~l) Length of assay (days) Equipment needed

DARIA

USERIA

SPRIST

MSPRIA

300-13 500 NAb 8 3~ 300 5 Centrifuge gamma counter

12-4 200 27 83 6 100 3-4 Beta counter

84-16 749 125 10-20 d 300 3 Horizontal shaker gamma counter

9-41 873 35 18 8 100 2 Gamma counter

Limit of sensltiwty defined as background + 2 S D ( p g / m l ) b Not applicable by cratenon used m footnote a (vide supra) From the standard assay, lnsuff~oent number of sensmve assays run d Hemady et al, 1983 a

USERIA Although the USERIA was the most sensitive assay (as judged by its limit of sensitivity (Table I)), the assay to assay variation and the tediousness of preparing a large number of small columns winch must be eluted, with subsequent transfer of an ahquot to scintillation cocktail for counting, hmlt this assay to situations in which a positive/negative type of answer is required and only a relatively small number of samples need to be assayed SPRIST The slmphoty of this assay and the availability of the necessary reagents from a commercial source (Pharmacia) can make this assay readily available to a large number of laboratories The cost of reagents and the large sample sizes (300/d) are the pnnclpal hmitatlons The Pharmacla Phadebas Phadezym-PRIST interface kit (Pharmacla no 61190) and the RAST tracer (125I-labeled anti-human IgE) are available separately from Pharmacla MSPRIA The performance of this assay is critically dependent upon optimal coating of the plate with antibody and having a highly specific tracer antibody ()25I-labeled anti-human IgE) We have used 2 different sources of antibody for coating the plastic plates with success, the monoclonal antibodies from Dr Saxon, and an equine anti-human IgE from a commercial source (Kallestad Laboratories, Austin, TX, catalogue no 126) The checkerboard tltratlons of antibody concentration, pH and ionic strength must be performed for each coating antibody used Optimal conditions vaned for the antlsera tested in the plate assays so, for example, the equine anti-human IgE had optimal coating from pH 9 6 - 9 8 and the mouse monoclonal optimum was approxamately p H 9 3 Once plates are coated and

371

blocked, they can be drmned and subsequently stored at - 70°C for at least 4 weeks without loss of activity (data not shown) The tracer antibody (antl-IgE) must be specific Although the specificity of the affinity chromatography purified rabbit anti-human IgE(PS)-Fc used m these assays is well-documented (Glelch et al, 1980, Glelch et al, 1981), we tested the antiserum for spec~ficxty in the MSPRIA As seen in Fig 2, the assay showed cohnearlty for purified IgE(PS), the US standard IgE (Evans, 1981) and a secondary IgE standard prepared in our laboratory from a pool of sera from 7 mdlwduals For example, when we used the curve for the purified IgE(PS) to calculate the IgE concentration in the US standard, we found 822 I U / m l (assunung 1 IU = 2 4 ng) The U S IgE standard has a published value of 900 I U / m l (900 IU/vlal when diluted with dlstdled water as per NIAID instructions) (Evans, 1981) In a different assay (results not shown) we used the WHO standard (10,000 I U / m l or 24 # g / m l ) to prepare a standard curee and we measured 965 8 I U / m l (2 32 # g / m l ) for the U S standard In addition, when 116 samples were analyzed for IgE in the DARIA and the MSPRIA by 2 different individuals, the correlation was highly significant (r = + 0 93, P < 0 001) Further speoflclty tests using purified lmmunoglobuhns and heat mactlvauon of sera (Table II) showed the lack of cross-reaction with lmmunoglobuhns of each of the other classes Furthermore, we found that the specificity of the assay was for the heat-labile portion of the molecule (Dorrlngton and Bennlch, 1973) similar to the speofic~ty of the anti-IgE used in the radloallergosorbent test interference method for measurement of IgG blocking anubodles (Glelch et al, 1981)

12 II 8 4

Z 0 I-

0

•

4 0 _1

8 12 16

2o

1

I

I

I

I

I

2

3

4

5

6

7

Log dilution F~g 2 Specificity of MSPRIA by cohneanty of serum standards with punfied IgE protein We tested pooled sera from 7 nonatoplc mdlxaduals that had been cahbrated against the W H O standard IgE serum (secondary standard, A), the U S standard IgE serum (O) and purified IgE(PS) protein ( I ) m the same M S P R I A to exanune speclfioty by cohneanty of the curves By analys~s of covanance the lines were not statistically different (F = 0 8726, df = 4 36, P > 0 25) The ordinate gdves the logl0 5[ T C [loglo (cpm b o u n d - c p m m a n u g e n b l a n k s ) / ( c p m tracer a d d e d ) x 100] The abscissa gives the log of the d d u t m n of the standard sera and protein The mltml concentratmns were arbitrarily offset for clarity m the figure because the curves were coincident m the actual plot of log % TC vs log concentratmn

372 TABLE II SPECIFICITY OF ANTI-IgE IN THE MSPRIA

IgG a IgA ~ IgM c IgDC Normal human serum Heat inactivated d

Concentrations tested ( ~ g / m l )

IgE ( p g / m l )

01 01, 01, 01,

ND b ND 87 at l l 6 / ~ g / m l ND

10, 10, 1 1, 10,

105 102 116 100

23419 711 (3 0%) e

Cord serum Heat reactivated

140 82 (58 6%)

Low IgE serum s Heat mactwated

85 N D ( < 10%) r

a DEAE fractlonatlon of Cohn fraction II of pooled sera b N D not detectable, below standard curve c Myeloma protein from starch block electrophoresls followed by Sephadex G-200 chromatography d Four hours at 56°C e Remaining react~vlty after inactivation f Assay detected to 9 p g / m l g From a patient whose serum had been studxed previously (Glelch et al, 1971), this serum had arbltrardy been assigned a value of 1 n g / m l

Conclusions Four sensitive radlometnc methods for measuring IgE have been compared double antibody radlolmmunoassay (DARIA), ultrasensltwe enzymatic ra&olmmunoassay (USERIA), sensitive PRIST (SPRIST), and multlsample plate ra&olmmunoassay (MSPRIA) All assays were able to measure < 300 pg IgE/ml, though the most sensitive assay, USERIA, was poorly reproducible The most wxdely accepted method, DARIA, was not as sensitive, and although tughly reproduoble, the long incubations and large sample volumes make the assay less useful for analysis of Ussue culture supernatants and other experimental samples The SPRIST should gain wide acceptance because the reagents are readdy available commercially, but the large sample volumes hrmt the usefulness of the assay The MSPRIA has been the most satisfactory of the sensmve radlometnc assays for human IgE m that ~t xs simple, has a relatively short assay Ume allowing for a shorter turn-around period between beginning an experiment and analysis of the data, and uses a small sample which ehmlnates the need for macro-cultures or pooled samples We conclude that the MSPRIA ~s the most satisfactory method to analyze rmnute quantmes of IgE ( < 0 5 I U / m l )

373

Acknowledgements We thank Dr Andrew Saxon for has generous gift of the monoclonal antibodies to IgE, Dr Ross Rockhn for supplying us with the details of the SPRIST assay, Dr John W Yungmger for has critique of tins manuscript, Mrs Lmda Calhster for secretanal assistance, and Mrs Cheryl Adolphson for edltonal assistance

References Bale, W F , R W Helmkamp, T P Davis, M J Izzo, R L Goodland, M A Contreras and I L Spar, 1966, Proc So(= Exp Blol Med 122, 407 Bazaral, M and R N Hamburger, 1972, J Allergy Chn Immunol 49, 189 Domngton, K J and H Benmch, 1973, J Biol Chem 248, 8378 Dunnette, S L and G J Glelch, 1981, m Methods m Enzymology, Vol 73, eds J J Langone and H Van Vunakas (Academic Press, New York) p 634 Evans, R , 1981, J Allergy Chn Immunol 68, 79 Flser, P M and R H Bucldey, 1979, J Immunol 123, 1788 Glelch, G J and S L Dunnette, 1977, J Allergy Chn Immunol 59, 337 Glelch, G J, A K Averbeck and H A Swedlund, 1971, J Lab Chn Med 77, 690 Glelch, G J , C R Adolphson and J W Yungmger, 1980, J Allergy Chn Immunol 65, 20 Glelch, G J , E M Zimmermann, M C Glelch and J W Yungmger, 1981, J Immunol 126, 575 Hams, C C, R H Yolken, H Krokan and I -C Hsu, 1979, Proc Natl Acad Scl U S A 76, 5336 Hemady, Z , F Blomberg, S Gelhs and R E Rockhn, 1983, J Allergy Chn Immunol m press Hsu, I -C, R H Yolken and C C Hams, 1981, m Methods m Enzymology, Vol 73, eds J J Langone and H Van Vunalos (Acadenuc Press, New York) p 383 Nerenberg, S T and R Prasad, 1981, m Methods m Enzymology, Vol 73, eds J J Langone and H Van Vunahs (Academic Press, New York) p 666 Sampson, H A and R H Buckley. 1981, J Immunol 127, 829 Yungmger, J W and G J Glelch, 1973, J Allergy Chn Immunol 51, 174 Zolhnger, W D , J M Dalrymple and M S Artenstem, 1976, J Immunol 117, 1788 Zuraw, B L, M Nonaka, C O'Hmr and D H Katz, 1981, J Immunol 127, 1169