A user's guide to the indirect solid-phase radioimmunoassay for the detection of cytomegalovirus-specific IgM antibodies

Journal of Virological Methods. 8 (1984) 271-282

271

Elsevier JVM 00308

REVIEW

ARTICLE

A USER’S GUIDE TO THE INDIRECT SOLID-PHASE RADIOIMMUNOASSAY FOR THE DETECTION OF CYTOMEGALOVIRUSSPECIFIC IgM ANTIBODIES

P.D. GRIFFITHS’

and H.O. KANGRO*

’ Virus Laboratory. Royal Free Hospital School of Medicine. London NW3, and 2 Virology Deparrmenr, St. Bartholomew’s Hospital, London ECI. U.K. (Accepted

14 February

1984)

In this review full laboratory detection commercial calculation

details

of specific IgM antibodies sources

of reagents,

high background

binding

congenital

pregnant

women.

cytomegalovirus

program

such as interference

procedure, available

the rapid dilution

from the authors.

by rheumatoid

radioimmunoassay

factor,

and 92% sensitivity

IgM

for identifying

for the

advice is given on readily available

deterioration

are avoided primary

of sera under test and

Problemsencountered of the radiolabel

by the methods

has been shown to give results of 100% specificity

infection

specific

indirect

Practical

found with some sera. If these problems

text then the radioimmunoassay detecting

cytomegalovirus.

a simple iodination

of the results with a computer

the assay are also detailed

are given of a solid-phase

against

and

given in the

with 89% sensitivity

cytomegalovirus

with

infection

for in

radioimmunoassay

INTRODUCTION

The diagnosis of acute infections by the detection of specific 1gM antibodies is a standard procedure for rubella (Cradock-Watson et al., 1979), hepatitis A (Lemon et al., 1980) hepatitis B (Tedder and Wilson-Croome, 1980) Epstein-Barr (Henle et al., 1974), and parvoviruses (Anderson et al., 1982). Cytomegalovirus (CMV) can now be added to the list since an indirect radioimmunoassay developed in our laboratories (Kangro, 1980; Griffiths, 198 1) has been confirmed in other laboratories to be capable of providing a specific and sensitive means ofdetectingparticular types ofinfection with this virus (Griffiths et al., 1982a,b). Since the original description of the method, the technical details of the assay have been modified progressively to produce a system which, we believe, is practical enough to become a standard diagnostic method available to major virus laboratories. The purpose of this review is to detail these changes and to emphasise those practical

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@ 1984 Elsevier Science Publishers

B.V

212

points

which must be followed

four simplifications detail: a commercial of serum dilution; LABORATORY

in order to obtain

highly specific results. In addition,

of the assay have been introduced source of antiserum; a computer

program

and these are described

a simple iodination for calculating

procedure;

in

a rapid form

the results.

DETAILS

Principle of assay The principle of the assay is illustrated in Fig. 1. A soluble extract of virus infected cells (test antigen) is dried onto the wells of microtitration plates and fixed with formalin. Any remaining protein binding sites in the plastic are blocked and dilutions of test sera are added to duplicate wells. Any IgG and IgM antibodies specific for

Fig

1. Principles

Virus-specific specific

of the radioimmunoassay.

proteins;

IgM antibodies:

(not virus specific).

0,

proteins

(a) Binding

of host cell origin;

, radiolabelled

goat

to test antigen.

(b) Binding

, CMV-specific /L

antl-human

to control

, CMV-

IgG antibodies;

IgM antiserum:

antigen. n.

,

% IgM molecules

273

CMV bind to the solid phase and, after washing,

the IgM antibodies

by the addition

IgM antiserum

of a radiolabelled

anti-human

When the assay has been completed, bound

to the virus-specific

sources:

direct binding

portion

all wells contain

of the solid-phase.

of the radiolabel;

attachment

alone are detected

(Fig. la).

radioactivity

This originates

which has not from three main

of serum IgM to viral compo-

nents of the test antigen with subsequent binding of the radiolabel; attachment of IgM molecules to cellular components of the antigen followed by radiolabel binding. Reduction in non-specific binding due to the first two sources is achieved by incorporating blocking proteins into the reagents (see below) while the extent of the latter binding is assessed by testing each serum dilution, in duplicate, against a control antigen prepared from uninfected cell cultures (Fig. lb). In each assay, replicates of wells containing no human serum are set up to provide the background count for both test and control antigens. For each serum dilution the binding ratio against test antigen (BRt) is defined as the mean cpm for serum + mean background for test antigen. Figure 2 shows that at low serum dilutions, such as l/100, the binding sites on the plastic are saturated but that, as the serum is diluted progressively, a logarithmic decline in BRt is found until the cpm reach the background level. (BRt = 1.0.) The binding ratio against

counts per

control

antigen

(BRc) is similarly

defined

as the mean

15

minute IXlV21 10

Binding ratio or specific binding index

“

10

5

1-b 1

2

4

Serum

Fig. 2. Titration

8

16

dilution

ofa serum

(SBI).

test antigen

64

factor MO~21

containing

Upper panel: ., = cpm against ratio against

32

cytomegalovirus specific IgM anttbodies. 0, = cpm against control antigen.

test antigen;

(BRt); o, = binding ratio against control

cpm = Counts per minute. Lower panel: ., = Binding

antigen (BRc); n , = specific

binding index

274

cpm for serum + mean background for control antigen. Figure 2 shows that BRc has a different regression against serum dilution with a relatively high value initially which declines

rapidly.

binding

index (SBI) is calculated

To determine

Only SBIs > 2.0 are considered

whether

specific IgM antibody

for each serum dilution to indicate

the presence

is present,

the specific

where SBI = BRt -+ BRc. of specific IgM antibodies.

Figure 2 also shows the effect of serum dilution on SBI. This value increases as the serum is diluted, not due to an increase in BRt but to a decrease in BRc. It follows that, if sera are to be screened for the presence of specific IgM antibodies, they should be tested at moderate dilution e.g. l/400, l/1600, since the SBI is maximal at this point. If a serum gives a positive screen result it can subsequently be titrated using a series of doubling dilutions. In practice, 8 two-fold dilutions are used from l/100 to l/12,800 and the titre of the serum is defined as the point where the plot of BRt against serum dilution crosses the BRt = 2 axis. Cord sera are usually screened at dilutions of l/50 and l/100 since they generally contain lower antibody titres and also give much lower BRc values than do adult sera. As an alternative to titrating each serum to end-point, the content of specific antibody can be estimated in terms of the BRt found at one serum dilution. A dilution of l/400 is often used for this purpose since the BRt of most sera is declining logarithmically at this dilution (see Fig. 2). There is a good correlation between the BRt at l/400 and the titre of the same serum determined by dilution to end-point. If a group of sera are tested in one assay, their average content of specific antibody may be conveniently estimated from the geometric mean binding ratio (GMBR) found at serum dilutions of l/400 and the GMBR is thus analogous to GMT. The BRc has been clearly shown to be proportional to the total IgM content of the serum under test. This is true for adult and cord sera although only data for the latter samples will be presented. During an investigation into congenital CMV infection, 197 cord sera were tested under code (Griffiths et al., 1982b). In total, 68 sera were identified in an arbitrary manner as reacting excessively with control antigen since their binding ratios were > 4.0 at a serum dilution of l/100. After the code was broken, the results were examined to determine if such non-specific binding was especially common amongst any particular group of patients. In addition to the proportion of sera giving binding ratios > 4.0, the GMBR was calculated for each group of cord sera. The results in Table 1 show that cord sera from uninfected babies with raised levels of total IgM or those from congenitally infected babies (who also had raised levels of total IgM due to intrauterine antigenic stimulation) were most likely to bind excessively to control antigen. Whether assessed by the proportion of sera in each group giving binding ratios > 4.0 or by the geometric mean binding ratio, no significant differences were seen between cord sera with raised total IgM levels and those from congenitally infected babies. In contrast, each of these two groups of sera had significantly more non-specific binding than did any of the other three groups of sera shown in Table 1. False-positive reactions are produced readily in this assay by the combination of

I

>4.0/no.

cord sera:

babies

’ Determined

by l-test.

’ cpm with serum / cpm without

by x2 test.

serum.

s/25 (20)

others

a Determined

3/26 (12)

2/35 ( 6)

9/18 (50)

49/93 (53)

tested (%)

pregnancies

from seronegative

pregnancies

from seropositive

total IgM levels

with raised

infected

from congenitally

binding

ratio

No. of sera with

the total IgM content

of selected

between

Characteristics

Relationship

TABLE

IBM




>0.05

total

infected babies

<0.05



>o.os

raised

congenitally

antigen

with

to

to control

from

cord sera:

compared

P value” when

of cord sera and their binding

2.78

2.44

2.31

4.86

4.38

of 100

serum dilution

IgM

<0.05



>o.os

total

infected babies

<0.02



>0.05

with raised

from

to

congenitally

cord sera:

mean binding ratiob at

P valueC when compared

Geometric

216

specific IgG antibodies

and rheumatoid

sera giving apparently

positive reactions

after absorption

of RF before

factor (RF) of IgM class (see Fig. 3). All whole must be retested after serum fractionation

the result can be taken

specific IgM antibodies.

The former

procedure

removes

while the latter removes

the RF from the serum under

to indicate

the presence

or of

the specific IgG antibodies test.

Details of procedure Preliminary experiments In the early reports of this assay we used an antiserum which we prepared ourselves by affinity chromatography using highly purified human IgM as ligand. We have since found a commercial source of equally pure antiserum which gives excellent results (goat anti-human IgM, TAG0 4102, Tissue Culture Services Ltd.). This antiserum is iodinated using the iodogen method (Salacinski et al., 1979). A solution of iodogen (Pierce Chemical Co.) containing 80 ug/ml is prepared in dichloromethane and 100 ul is dispensed into each of a series of 1 ml polypropylene vials using a glass syringe. The solvent is allowed to evaporate at room temperature in a fume cupboard and the vials are then stored in the dark at -20°C. When required, an iodogen vial is removed from the freezer and allowed to reach room temperature. The antiserum (50 ~1) is added followed by 10 ul of [‘251]sodium iodide containing 1 mCi (Radiochemical Centre, Amersham). The reaction is allowed to proceed at room temperature for a time which is constant for each batch of vials; typically 15-30 min. The extent of radioiodide incorporation is proportional to this incubation time. If cord sera are to be tested a high (70-80%) incorporation of iodide is required; if adult sera are to be tested, a moderate (60%) incorporation will suffice. The iodination reaction is terminated by aspiration of the vial contents which are layered on top of a 10 cm 10 ml column of Sephadex G-50 (Pharmacia, superfine

Fig. 3. Binding of rheumatoid factor.

factor to the solid phase. Symbols as for Fig. 1

277

grade) equilibrated

with PBS containing

rum is eluted from the column 0.5 ml, are collected. pooled,

diluted

The fractions

containing

with an equal volume

This stock label solution

1 mg/ml

bovine serum albumin.

using this buffer and 30fractions,each of elution

The antise-

ofapproximately

the first peak of radioactivity buffer and stored

are

at 4°C.

can be used to test adult sera for approximately

3 wk after

which time a fresh radiolabel should be prepared. The deterioration in the stock radiolabel is partly explained by the increasing proportion of the radioiodide which elutes from the labelled antiserum. To illustrate this, the proportion of radioactivity which was not precipitable with 10% trichloracetic acid (i.e. was not protein-bound) was determined daily after iodination. The results (see Fig. 4) show that after lo-14 days approximately 10% of the radioactivity was no longer protein-bound. The performance of such a radiolabel can be improved by re-eluting it through a Sephadex G-50 column, but iodination with the iodogen method is so simple to perform that we find it easier to prepare a new radiolabel. Deterioration of the radiolabel is even more rapid if cord sera are to be tested; reliable results are then only obtained within 24 h of iodination of the antiserum. The reason for this is not clear but is illustrated by the results in Table 2. Five sera, four known to contain low or moderate levels of specific IgM antibodies and one negative control serum, were titrated on each of three consecutive days. Starting on the day that the radiolabel was prepared, microtitration plates containing the solid-phase antigens were manufactured daily and the radiolabel was first used within 24 h of iodination. Table 2 shows the results obtained at serum dilutions of l/200. On the first day, four sera had titres varying from 280 to 5970 whilst the fifth serum gave a clearly negative result. On the second day the titres of all four positive sera declined and, on the third

‘21

0

1

2

3

4

5

6

7

8

I I II 11 10 11 12 13 14 15

9

Deys after lodinatlon Fig. 4. Effect of storage on the proportion

of unbound

radioactivity

present in the stock radiolabel.

1

2.19

1.84

0.99

5.48

4.29

1.14

3

4

5

= binding

BR,

ratio

ratio

SBI = specific binding

= binding

Wj

control

against

index (BR,/BR,f.

antigen.

test antigen.

against

detected

1.15

2.w

at this serum dilution.


280

355

1410

2.40”

2.w

5970

Titer

2.22*

SBI

i.11

4.20

5.21

7.98

8.21

BR,

Day 2

five cord sera tested on three consecutive

I&l antibodies

3.55

8.52

2

specific

3.73

8.29

t

a Indicates

RR,

BR,

Day

Serum

number

results from

Radioimmunoassay

TABLE 2

1.03

2.14 1.M

I .96

2.19”

2.71”

2.94 2.38

2.6P

SBI

3.12

BR,

days

‘cl00

160

25s

1020

3950

Titer

I.59

4.4 1

5.39

6.82

7.26

BR,

Day

3

1.O?

2.39

2.84

3.R3

4.01

BRc

1xi2

1.84

1.90

I.78

1.81

SBI



130


2400

Titer

279

day, two of them failed to react whilst a third (serum negative

control

serum gave consistently

out the experiment. dilutions detect

Close inspection

(not shown), specific

low binding

results

both antigens

through-

of the results in Table 2, and those of all other

shows that the deterioration

IgM antibodies

3) had a very low titre. The

against

in the ability

from both a decrease

of the radiolabel

in binding

to

to the test

antigen and an increase in binding to control antigen. The latter effect explains why serum 1 failed to react at a dilution of 200 but did react at all further serum dilutions up to 2400. If RF is to be removed by absorption, the potency of the absorption reagents must be determined. Latex beads with human IgG attached can be prepared locally or a commercial source can be used (Rapitex, Hoechst Pharmaceuticals). The potency of the beads is best assessed by selecting a serum from a patient with rheumatoid arthritis with a strongly positive slide agglutination test for RF which also has a moderate to high CMV IgG antibody titre. The serum (10 ~1) is dispensed into each of a seriesoftest tubes and increasing volumes (lo-200 pl) of beads added to sequential tubes. The total volume in each tube is made up to 0.5 ml with PBS and the tubes are incubated at 37’C for 1 h with occasional shaking. The tubes are then centrifuged at 4,000 X g for 30 min and doubling dilutions of the supernatants are prepared and tested by RIA. The minimal volume of beads which completely abolishes the false-positive RIA reaction is determined and twice this volume is used in subsequent assays. Experiments have shown that this procedure does not remove any of the serum IgM so that there is no risk of absorption of RF producing false-negative RIA-IgM results. Suitable preparations of test and control antigens are available commercially (Hoechst Pharmaceuticals, ref. OREA 04105 and OREE 04105, respectively). For each batch, chessboard titrations are set up to determine the optimal dilution using a negative serum and two positive sera of similar antibody titres but which differ in their reactivity with control antigen since the SBI can be greatly affected by the BRc of a given positive serum. Thus, an antigen dilution must be chosen not simply to produce the highest titre in a serum with a high SBI but to produce the best discrimination between a negative serum and a positive serum with a high BRc. Typical chessboard experiments with the commercial materials described above would produce optima when diluted l/15 for adult sera or l/12 for cord sera. Routine performance of the assay The solid phase is prepared by reconstituting the freeze-dried test and control antigens with 1 ml of distilled water and diluting to the previously determined optimum with PBS-B. Using a 12-well multichannel pipette (Flow Laboratories) and a reservoir, portions (50 ~1) of the test and control antigens are added to U-shaped wells of separate polyvinyl chloride microtitration plates (Dynatech M24) and allowed to dry (2-3 h) aided by the flow of cold air from a convector fan. The antigens are fixed by the addition to each well of 100 ul of a 10% (v/v) solution of saturated formalin in PBS (pH 6.5). After incubation for 20 min at room temperature the formalin is shaken

280

from the plate and each well is washed with 200 ul of 0.2% (w/v) gelatin PBS. This solution (v/v)

solution

solution

in

is likewise removed by shaking and replaced by 100 ul/well of a 5%

of normal

plates are then incubated

rabbit

serum (Flow

overnight

Laboratories

at 4°C in a humid

29-411-49)

in PBS. The

environment.

The dilutions of the sera under test are prepared in plates with 96 wells each of 1 ml capacity (Flow Laboratories 76-356-05). If each serum is to be tested at l/100, l/400, l/1600, and 116400 dilutions, then 1 ml of assay diluent (PBS containing 5% (v/v) normal rabbit serum and 1% (w/v) dextran TlO(Pharmacia 17-0250-O1))isdispensed into one well followed by 300 ul into each of the adjacent 3 wells. The serum under test (10 ~1) is added to the first well and 100 ul of this l/100 dilution is then transferred serially through the next three wells. Once all the sera have been diluted, the plates containing the solid phase are shaken to remove the rabbit serum and allowed to drain face-down for a few minutes. The first two rows, each of 8 wells, in the first test and first control plates are loaded with 50 ul/well of assay diluent alone to serve as background controls. From this point, 50 ul portions ofeach serum dilution are added in duplicate to test and in duplicate to control wells. The preparation of serum dilutions in the large volume 96-well plates greatly facilitates this process since the layout of diluted sera corresponds directly with that of the microplates containing the solid phase. Once this process is complete the microplates are incubated at 37°C for 3 h in a humid environment. The radiolabel dilution is prepared in label diluent (minimal essential medium containing 10% (v/v) normal rabbit serum and 0.5% (w/v) lactalbumin hydrolysate). The required volume of label diluent is made up and successive amounts of the stock label solution are added until the label diluent contains 30,000 cpm in 50 ul for adult sera or 60,000 cpm in 50 ul for cord sera. The microplates are then washed three times in 0.2% gelatin solution in PBS and 50 ul of the diluted radiolabel is added to each well. Incubation is continued for 2 h at 37°C after which each plate is washed 10 times in cold tap water. The plates may be stored dry at 4°C for several days until it is convenient to count the radioactivity

bound

to the solid phase.

Each well is cut from the microplates,

counted in a gamma scintillation spectrophotometer and the results recorded as cpm. The counting is greatly facilitated if a 16-well instrument is available (Nuclear Enterprises NE1600). Our instruments are now connected on-line to microcomputers so that the calculations described earlier and depicted in Fig. 2 are performed automatically. Copies of the program suitable for a Hewlett-Packard 125 are available from PDG and those for a Hewlett-Packard 85 from HOK. The programs calculate and print-out BRt, BRc and SBI for each serum dilution. They also calculate the percentage error between each pair of replicates (defined as 100 X difference between duplicates ; mean of duplicates) so that obvious laboratory errors can be readily identified. The percentage errors obtained when 100 CMV-IgM negative sera were diluted l/400 and tested in one assay were as follows: mean 7.9, standard deviation 6.4, upper 95% confidence limit 20.7. Thus, any sample with greater than 20% error

281

between

duplicates

should

be regarded

with suspicion.

The figure of 20% may seem

too large to be acceptable, but it corresponds to fairly small differences radioactivity (e.g. nl = 360 cpm, n2 = 440 cpm). Whether

the results are calculated

by computer

or human,

in bound

they must all be inspec-

ted critically. For example, when sera are screened at two dilutions (typically l/400, 111600, for adults) an occasional high titre serum is found whose SBI is < 2.0 at both of these dilutions. Such sera can be identified by their high BRt and BRc and the fact that SBI at 111600 is greater than SBI at l/400 (see Fig. 2). These sera should be retested at higher dilutions to reveal the presence of specific IgM antibodies. DISCUSSION

In this review full laboratory details are given to allow the detection of CMV-specifit IgM antibodies by indirect solid-phase radioimmunoassay. We believe that this system has now been sufficiently refined and evaluated for it to represent a standard method suitable for most virus diagnostic laboratories. When used to test sera from patients with intact immunity, the assay detects acute primary CMV infection with a specificity of 100% and a sensitivity of 92% (Griffiths, 1983). Thus, the assay is of value in investigating patients who have symptoms of, for example, infectious mononucleosis, acute hepatitis or for investigating women who might have CMV infection during pregnancy. In all of these groups of patients the possibility of false-positive reactions being produced by RF must always be evaluated by serum fractionation, to remove specific IgG antibodies, or by serum absorption, to remove RF. It is not acceptable to rely upon a negative latex agglutination result for RF for this purpose since the detection limit of this technique is far above theamount of RF which may interfere with the RIA. If scrupulous measures are not taken to remove RF then roughly equal numbers of true and false positive reactions will be found in sera from unselected pregnant women (Griffiths, 1981). In patients who are immunocompromised as a result of disease or therapy, the assay is specific for the virus but cannot reliably differentiate between primary and recurrent infections (Kangro et al., 1982; Pass et al., 1983). An occasional patient is found in whom severe disseminated primary CMV infection occurs without the production of specific IgM antibodies (Pass et al., 1983). In contrast, approximately one third of renal allograft recipients with recurrent CMV infections produce specific IgM antibodies (Kangro et al., 1982; Pass et al., 1983). The reason why such patients reawaken clones of lymphocytes to produce this class of immunoglobulin is not clear. It is tempting to speculate that specific IgM production may be related to the degree of virus dissemination in such patients but, as yet, it has not been possible to demonstrate this (Rasmussen et al., 1982; Pass et al., 1983). In order to detect the small amount of specific IgM antibody in cord sera from congenitally infected babies the assay is being used at the limit of its sensitivity. Cord sera with high titres are easily recognised but those with low titres present difficulties

282

due to the relatively high BRc at low serum dilutions. Under ideal conditions the assay has been shown to have a specificity of 100% and a sensitivity of 89% (Griffiths et al., 1982b). However,

as detailed

earlier, these ideal conditions

within 24 hours of the antiserum is a clearly reproducible

being iodinated.

phenomenon.

require that sera are tested

The reason for this is uncertain

but it

As a result, we suggest that the RIA will only be

of use to those laboratories which can collect cord sera and test them in batches. Although not shown here, results of low sensitivity have been obtained when used to test sera from infants with congenital or perinatal infections. We believe the reason for this is not that such infants lack specific IgM antibodies but that the rapid increase in serum IgM concentration which takes place after delivery makes it more difficult to detect specific IgM antibodies due to an increase in BRc. The reliable detection of CMV-specific IgM antibodies in such patients must await the development of other methods which may be less susceptible to the effects of non virus-specific IgM. Finally, in our laboratories the speed with which results are produced has been improved by processing of the raw radioactivity counts using microcomputers. This obviously eliminates tedious computation but the speed of availability of the results has little advantage when sera are being tested for specific IgM antibodies. However, we believe that this RIA offers many advantages to Blood Transfusion Centres who may wish to screen blood for CMV-IgG antibodies. The analogous assay for this class of specific immunoglobulin currently has two 60 min incubation stages so that results could be available soon after the samples reached the laboratory; with further optimisation the CMV status of the donor could be available within the time taken to cross-match his blood. REFERENCES

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