Journal of Immunological Methods, 24 (1978) 183--191

183

© Elsevier/North-Holland Biomedical Press

A MICRO IMMUNOASSAY M E T H O D F O R MEASURING IgG A NTI B ODI ES USING STAPHYLOCOCCAL PROTEIN-A

D.M. MORAN *, BARBARA E. DUPE and SUSAN GAUNTLETT Beecham Pharmaceuticals, Research Division, Brockham Park, Betchworth, Surrey, U.K.

(Received 11 April 1978, accepted 20 May 1978)

A micro radioimmunoassay procedure has been developed for assaying IgG antibodies to ragweed and grass allergen components. The method offers a rapid and reproducible procedure for estimating antibodies in human sera and is well adapted for monitoring immunological responses following hyposensitisation therapy. INTRODUCTION Staphylococcal protein-A has been used in a variety of systems t o d e t e c t and assay IgG class antibodies (Hjelm et al., 1972). Recently, Foucard and Johansson (1976) described such a m e t h o d for assaying IgG antibodies specific to T i m o t h y grass allergen in hum a n sera. We have now developed a high capacity microassay procedure, based on a similar procedure to t hat described by the above workers, but using Sepharose-linked protein-A in a modified Farr-type assay system. In this com m uni cat i on, we report the assay conditions e m p l o y e d for measuring IgG antibodies to ragweed antigen E, and rye group I allergens in h u m a n sera. MATERIALS AND METHODS Antigens

Ragweed antigen E (AGE) was obtained from NIH in 50% glycerol and used w i t h o u t f u r t h e r purification or extraction. Rye group I was obtained as a gift from Dr. D. Marsh (Johns Hopkins Medical School). Ragweed ex tract e m p l o y e d for raising antisera in animals was prepared by aqueous extraction of A m b r o s i a elatior pollen (ex Greer Labs.) by the m e t h o d similar to t h a t described previously for grass pollen extracts (Moran et al., 1976). A n tisera

The h u m a n sera e m p l o y e d in these studies were obtained from individuals * Communications should be addressed to Dr. D.M. Moran.

184

who had undergone i m m u n o t h e r a p y within the last 24 months with the respective allergen extract. Normal serum was collected from healthy volunteers. Ragweed specific animal sera was obtained from rats (outbred Wistars). The immunisation schedules employed are described in the text.

Reagents Staphylococcal protein-A--Sepharose conjugate was obtained from Pharmacia. The material was dispersed in PBS containing 0.5% w/v BSA (bovine serum albumin) and 0.02% w/v sodium azide and stored at 4°C at a concentration of 5.0% w/v. Radioactive iodine was obtained as Na'25I from the Radiochemical Centre, Amersham.

Radio-labelling The basic radio-labelling procedure employed was essentially that described by Greenwood et al. (1963), using chloramine T. The iodinated materials were separated from u n b o u n d 12sI on a Sephadex G-25 column and stored in a stock buffer composed of PBS/0.5% w/v BSA/0.02% w/v sodium azide at --20°C. The immuno-reactivity of [1:SilAgE was f o u n d to be 60-70%; t h a t of ~2SI-labelled rye group I was approximately 70% using human sera. Specific activity for AgE was assessed by isotopic dilution. Figure 1 shows typical results for [12SilAgE. The data are plotted in terms of reciprocal counts bound by a given volume of serum as a function of added cold antigen E, and the specific activity, si, derived from the expression: Ci = k m s i

(1)

where c i are the counts taken up under conditions of antigen excess when mi

6O i/ci~105 50 (1/cpm) 40

T Mo

©

0

10 20 30 40 50 Cold AgE added (ng)

Fig. 1. P l o t o f r e c i p r o c a l counts bound o f 12SI-labelled ragweed antigen E t o h u m a n sera o b t a i n e d f r o m 4 ragweed s e n s i t i v e individuals as a f u n c t i o n o f increasing a m o u n t s o f cold antigen E a d d e d (i.e., as a f u n c t i o n o f decreasing specific activity). The v o l u m e o f s e r u m e m p l o y e d was a p p r o p r i a t e t o ensure t h a t antigen excess c o n d i t i o n s were established in all measurements.

185

cold antigen is present, m is the absolute mass of antigen bound, k is a constant and a is the activity of the labelled antigen added. When no cold antigen is added, eq. (1) becomes (2)

Co = kmSo

where So = a/mo and mo is the mass of labelled antigen added with activity a. Division of eq. (1) by (2), and substitution of si, for a/mo + mi affords Co

_

Ci

m0 + m i

(3)

m0

Consequently 1

1 -

Ci

moCo

mi +

-

(4)

-

moCo

and the intercept of 1/ci against m i when 1/C i 0 yields m0. Specific activity values for ['2SI]AgE typically ranged from 2,000 to 12,000 cpm/ng, l:SI-labelled rye group I specific activity levels were also of a similar order b u t binding results in this case wer~ quoted in terms of a standard serum, arbitrarily defined as containing 5,000 units/ml. It is noted that ragweed AgE, in our hands, did n o t require separation from the glycerol vehicle prior to radio-labelling as indicated recently by Gleich et al. (1977}. =

Radioimmunoassay procedure employed

The basic procedure used is described below. The effect of changes in the experimental variables employed is considered in the main text. Two-fold serial dilutions of test sera were prepared in PBS/0.5% BSA buffer. Aliquots of diluted serum (25 pl) were added to U-shaped wells of disposable polystyrene microtitre plates (ex Dynatech). Radioactive antigen (25 gl) diluted in the above buffer and in concentration sufficient to ensure antigen excess (see text}, was .then added to each well. For the majority of experiments, approximately 100,000 cpm were added to each well. The solution was then left for at least 3 h at ambient temperature. Protein-A/ Sepharose suspension (50 pl, 5.0 w/v in PBS/0.5% w/v BSA) was added to each well and the microtitre plate was shaken for a minimum of 60 min using a micro-shaker (Dynatech). The plate was then spun using a microtitre plate centrifuge b u c k e t (Wheeler and Hatcher, 1977) at 1,000 rev./min for 10 min. The supernatants were aspirated from each well and the suspension resuspended in 100 pl of PBS/0.5% w/v BSA. This process was repeated 5 times. The individual wells of the microtitre plate were separated using ordinary scissors. The wells, containing the residual Sepharose--Protein-A phase, were then placed in carrier tubes and counted in a Packard gamma counter.

186 RESULTS

Capacity o f protein-A--Sepharose conjugate to bind immunogIobulin Typical results for [~2SI]AgE uptake by human sera obtained from ragweed sensitive individuals, as a function of the a m o u n t of protein-A immunosorbent employed, are shown in Fig. 2. The data show that the capacity of the immunosorbent used for assay purpose (i.e., 2.5 mg/assay) was not significantly limiting for serum levels as high as 5 tH. However, in practice, it was found necessary to restrict the volume of test serum employed to a m a x i m u m of 2.5 pl in order to ensure consistent quantitative absorption of immunoglobulin.

The influence o f incubation times Incubation of labelled antigen with sera for various times, ranging from 1--24 h at ambient temperature showed that essentially total reaction was achieved after 2 h. A period of 3 h was used routinely for assay purposes. The effect of varying the incubation time of the protein-A immunosorbent with prior reacted antibody antigen solutions is shown in Table 1. The data illustrate that essentially invariant levels of binding were achieved following 60-min reaction times at ambient temperature. Examination of the residual supernatant solution confirmed that all specific antibody binding capacity was removed by this relatively short immunosorbent reaction time. A period of 1 h was used routinely for all assay measurements.

Serum 18

6000=)

~O,- 51.ti

O 25t at E

200

0

,

'"O i.25pi

o % 6000] ,