Detection of monoclonal influenza antibodies synthesized in culture by hybridoma cells with a solid-phase indirect immunofluorometric assay

Journal of VirologicalMethods, @ Elsevier/North-Holland

DETECTION

275-283

1 (1980)

Biomedical

215

Press

OF MONOCLONAL

INFLUENZA

ANTIBODIES

SYNTHESIZED

CULTURE BY HYBRIDOMA CELLS WITH A SOLID-PHASE IMMUNOFLUOROMETRIC

DONALD

ASSAY

ALAN P. KENDAL’

J. PHILLIPS’,

and CHARLES

IN

INDIRECT

, ROBERT

G. WEBSTER3,

PAUL M. FEORINO’

B. REIMER’

1Immunology and ’ Virology Divisions, Bureau of Laboratories, Center for Disease Control, Public Health Service,

U.S. Department

of Health and Human Services, Atlanta, GA 30333;

of Virology, St. Jude Childrens Research Hospital, Memphis, (Accepted

4 June

A solid-phase antibodies immune bound

indirect

sera is covalently

amounts

Mouse

antimouse

Ig and measuring screening

25- to 150-fold

assay

to polyaminostyrene

antibodies,

antigens.

produced

antigens,

the flourescent

intensity

more

sensitive

than

beads, Alternatively,

indirectly

monoclonal

IgG from hyper-

to which influenza

viruses are then

the virus is covalently cells in culture,

quantitated

by hybridoma

inhibition

of mouse Purified

with a filter-fluorometer.

synthesized

hemagglutination

reactions

virus as a model.

by hybridoma

and then

for low levels of antibodies

with influenza

for measuring

with Influenza

solid-phase

of solid-phase

mits rapid

bodies reactive

linked

to make

to the beads.

constant

immunofluorometric has been developed,

immunologically

directly

and ‘Division

U.S.A.

1980)

with antigen rabbit

TN 38101,

coupled

are reacted

by adding

The assay system cells in culture.

tests in detecting

with

FITC-labeled per-

It is about

monoclonal

anti-

virion HA protein.

INTRODUCTION

The development select for continuously

of procedures

to fuse immune

growing hybrid

(hybridoma)

splenic cells with myeloma cells secreting monoclonal

cells and antibody

(Kohler and Milstein, 1976) is expected to have important applications in research, diagnosis and clinical medicine. For many antigens, however, the yield of hybrid cells producing antibody of desired specificity is very low in each fusion experiment, so that many cell clones must be established

and screened for specific antibody

sible that use of the fluorescence-activated

production.

It is pos-

cell sorter will decrease the labor required for

this work (Parks et al., 1979) but the expense of the instrument will limit its availability. Most investigators must therefore rely on simpler assays to screen hybrid cell cultures, and in many instances ligand binding assays have been selected because these tests have the sensitivity needed to detect low concentrations of antibody that may be secreted by hybridoma cells in culture. The radioimmunoassay (RIA) has been the most frequently used method for detecting these antibodies by a solid-phase system. There are,

216

however, problems associated with the performance cern about the physical hazards and psychological

of RIA because of the growing conaversion associated with using even

low levels of radioisotopes, and because the disposal of radioactive waste is increasingly difficult and expensive (Byrne, 1980). Enzyme immunoassay, which is also highly sensitive, may not be sufficiently

precise for many purposes due to the inherent

reproducibly measuring enzyme activity. The immunofluorometric assay (IFA) is demonstrably and particularly stable assay for quantitative measurement

problems of

a safe, sensitive, reproducible of ligand binding (Deelder and

Ploem, 1974; Burgett et al., 1977; Reimer et al., 1978; Soini and Hemmila, 1979; Sundeen and Krakauer, 1979). Because relatively inexpensive equipment and stable reagents are used, the assay also is quite economical. We have therefore developed and evaluated a solid-phase indirect immunofluorometric assay (IIFA) for the measurement of binding by monoclonal antibodies to antigen, with the influenza viruses as a model system because the characteristics of many of their antigens are well-known (Dowdle et al., 1979) and because reagents, including monoclonal antibodies, are available for testing the assay.

MATERIALS

AND METHODS

Monoclonal antibodies For the production of monoclonal antibodies to influenza A/New Jersey/76 (HswlNl), hybridomas were derived from splenocytes from immune adult Balb/c mice by fusion with mouse myeloma P3-X63-Ag8

(non-secretor)

cells similar to the method described by

Keamey et al. (1979). Hybridoma tissue culture fluids and ascitic fluids containing monoclonal antibodies to other influenza virus types, subtypes, or host antigen were similarly prepared but using the P3-X63-Ag8 (secretor) mouse myeloma cell line as previously reported (Webster et al., 1979). Viruses A/New Jersey/76 (HswlNl) was an expired whole-virus vaccine that had been commercially prepared in 1976 and subsequently stored at 4°C. Influenza strains A/Brazil/ 11/78 (HlNl), A/Texas/l/77 (H3N2) and B/Hong Kong/l/78 were propagated by inoculating the allantoic cavity of lOday-old embryonated chicken eggs with approfimately lo4 egg infectious doses. After 2 days at 34°C the allantoic fluids were harvested and stored in aliquots at -70°C for subsequent use in the preparation of solid-phase antigen. Virus preparations used to immunize rabbits were purified by differential and density gradient centrifugation procedures (Kendal et al., 1979).

211

Solid-phase reactants The procedures

for preparing

scribed in detail elsewhere. pared by nitration

solid-phase

reactants

Briefly, solid-phase

and reduction

of uniform

used in the IIFA have been de-

polyaminostyrene

diameter

(PAS) beads were pre-

(1.1 @I) polystyrene

(Dow Diag-

nostics, Indianapolis, IN) spheres (Reimer et al., 1978). Antigen or antibody were then covalently coupled to the solid-phase PAS through diazo-coupling reactions (Phillips et al., 1.980). For this study, hyperimmune sera to different influenza strains were prepared by inoculation of rabbits in the footpads with about 5 mg of purified virus mixed with Freund’s complete adjuvant, followed at about 3-4 weeks by an intravenous injection of purified virus. Sera were collected about 2 weeks later and had hemagglutination-inhibition (HI) titers of > 5000. The IgG antibody fraction of each serum was isolated from hyperimmune rabbit sera by DEAE Sephadex A.50 chromatography (Reirner et al., 1975) and was used for the preparation of solid-phase, diazo-coupled influenza antibody. A stock suspension of solid-phase antibody beads was adjusted (counting chamber) to 3.7 X 10s beads/ml in phosphate-buffered saline (PBS) containing 05% normal goat serum (NGS) and 0.1% sodium azide. The normal serum was treated with heat, trypsin and periodate to inactivate non-specific inhibitors (Dawdle et al., 1979). Assay procedure The IIFA was initiated by vortex mixing 0.1 ml of the stock solid-phase antibody with 0.1 ml of virus-infected allantoic fluid in 12 X 75 mm disposable borosilicate tubes. In preliminary tests, the virus dose corresponding to about 50% of the antigen-binding capacity of the solid-phase antibody was determined, and binding of this amount of virus was essentially complete after an incubation of 1 h at 37°C. Because antibody-coupled beads were in excess, washing

of these beads after the presentation

of virus antigen could be

omitted without affecting test results. Next, a 0.1 ml test sample of hybridoma culture fluid of ascitic fluid from mice inoculated with hybridoma cells was added and incubated 1 h at 37°C. Unbound mouse Ig was removed by resuspending the solid-phase complex in 21) ml of PBS, pH 8.0, containing 025% treated normal goat serum and centrifuging at 1950 g for 15 min at 20°C. The supernatant fluids were decanted and the wash procedure repeated once. To detect binding of mouse antibody, we added 0.05 ml of prediluted fluorescein isothiocyanate (FITC) conjugated goat antimouse Ig (Research Plus Laboratories, Denville, NJ) to the solid-phase pellet, mixed and incubated 1 hat 37’C. Conjugate predilution had been previously established as the dilution of conjugate needed to saturate antigen beads that were already saturated with mouse antibody. Accordingly, the conjugate was diluted l/20 in 10% heated, trypsin-periodate-treated normal rabbit serum, stored at 4’C for 48 h and centrifuged 100,000 g for 30 min to remove immune complexes. The supematant fluid was stored frozen in aliquots, thawed and diluted l/S in 0.5% NGS at the time of use. Excess conjugate was removed from the solid-phase com-

278

plex by three cycles of resuspension

and centrifugation

of the solid-phase

as described

above except with PBS, pH 8 .O, containing 0.15 l.(g Tween 2O/ml (PBS/Tw20). The final bead pellet was resuspended in 1 .O ml of PBS/Tw20, and the presence of mouse antibody was measured goat antimouse

indirectly

from the fluorescent

response

Ig by using a Gilson Spectra/G10 filter-fluorometer

of the FITC-conjugated (Gilson Medical Elec-

tronics, Middleton, WI) equipped with a digital readout and previously described (Phillips et al., 1980) narrow-band interference filters for blue excitation of FITC. In the case of A/New Jersey/76 (HSw lN1) antigen, solid-phase antigen was prepared by direct chemical coupling (diazotization) of purified virus to PAS beads (see Table l), but other procedures were unchanged.

RESULTS

To evaluate the assay for detecting monoclonal antibodies, nine samples of tissue culture fluid from hybridomas (identified by other methods as secreting antibodies to various influenza A or B antigens or egg-host antigens, as well as some specific antibodyfree fluids) were selected in Memphis and tested in Atlanta under coded conditions (Fig. la). Each fluid was assayed by using influenza A/Texas/l/77 (H3N2), A/Brazil/ll/78 (HlNl) and B/Hong Kong/l/78 solid-phase antigens that were prepared by adding infected allantoic fluids to PAS beads bearing covalently coupled IgG from the serum of rabbits hyperimmunized with the homologous virus. The influenza A strains selected contain related type-specific internal matrix (M) protein and ribonucleoprotein (NP) antigens but unrelated subtype-specific surface glycoproteins. All virus-specific antigens of influenza B are distinct from those of influenza A. Analysis of accumulated

data for 30 background

samples indicated that a fluorescence

measurement of twice background represented 15 standard deviations above the mean background determination and was routinely used as our discrimination point. Fluid No. 1 (Fig. 1A) reacted only with A/Texas/l/77 solid-phase antigen and was therefore identified as specific for one of the su?face glycoproteins characteristic of this virus, H3 or N2. Fliuds Nos. 2 and 3 reacted only with A/Brazil/l l/78 solid-phase antigen and therefore were identified as specific for either Hl or Nl surface glycoproteins. Because fluid No. 4 reacted with all three antigens, it was identified as specific for some avian host component common to all influenza viruses grown in embryonated chicken eggs (Harboe, 1973). Fluid No. 5 was specific for influenza B and fluid No. 6, because it was reactive with both influenza A antigens, was identified as type-A specific. The assay gave a false-negative result with fluid No. 7, known from other tests to be specific for influenza A matrix protein. This suggests that, in contrast to NP, relatively low amounts of free M protein are present in the allantoic fluid virus samples used to prepare solid-phase antigen; this is not surprising in view of the known association of M protein with cell and virion membranes which leaves little of the protein available to antibody.

279

u&-v4

2

2 “t-HA

3

6 &A

A-NP

H&T

7 A-M

SELECTED HYBRIDOMA FLUIDS

.E

0

14 Hswl-HA

30 HswZ-HA

76 HsW-HA

Antigens, immunologically

5 HOST

attached to solid-phase:

0 A/Erazi~/ll/78(HlN11 •AfT@xos/~/77(H3N2)

q B/Hong Antigen,covalently

attached to solId-phase:

mAtNew

Kongl’lj7B Jersey~76WswlNl)

Fig. 1. Solid-phase indirect ~muno~uoromet~c assay (IIFA) results with hyb~doma culture fluid. A) Selected fluids, independently and previously shown by other methods (e.g. hemagglutinin-inh.ibilion, n~r~~da~-in~bition and enzyme-immunoassay tests) to contain antibodies specific for type, subtype or host antigens of the influenza viruses and negative control ffuids, were tested under coded conditions by IIFA using undiluted, untreated fluids. B) Undiluted, untreated fluids from cultures, obtained by polyethyIene glycol fusion of spleen cells from mice, immunized with inactivated A/New Jersey/76 (GwlNl) vaccine with P3-X63-Ag8 myebma cells, were screened by IIFA for the presence of virus-specific antibody after actively growing hybridoma cells were detected (about 2-3 weeks post-fusion).

280

As described below, detection of hybridoma antibodies in the IIFA is possible ifM protein is made available on the solid-phase antigen. Negative results were obtained for two negative control fluids (Nos. 8 and 9) included in the coded samples (Fig. 1A). In those instances where highly purified antigen may be obtained in sufficient for covalent

attachment

directly

to the PAS beads, the IIFA test for detecting

quantity specific

antibody may be made simpler than when antigen is attached by immunologic methods to the solid-phase. This approach was evaluated by using influenza viruses which can be readily purified in the research laboratory or are sometimes available in bulk quantities for research use from vaccine manufacturers (Bucher et al., 1976). Therefore, a solidphase A/New Jersey/76 antigen was prepared by covalently linking to PAS beads expired A/New Jersey/76 whole-virus vaccine. Preliminary results with the IIFA demonstrated that the solid-phase A/New Jersey/76 antigen was capable of detecting antibodies in sera of mice vaccinated with the homologous antigen, and accordingly this solid-phase antigen was used to screen undiluted, untreated culture fluids obtained from hybridomas prepared with spleens from such mice and P3-X63-Ag8 myeloma cells. The IIFA detected hybridomas producing antibody reactive with the A/New Jersey/76 antigen (Fig. 1B). In an approach similar to that described for the test with immunologically bound solid-phase antigen (cf. Fig. lA), all fluids that showed a positive reaction by IIFA with A/New Jersey/76 antigen (and some negative fluids) were also treated by IIFA with influenza A (H3N2) antigen, to detect type-specific antibody, and with influenza B antigen, to detect antrbody reactive with non-specific host components common to all egg-grown influenza TABLE 1 Effect of Triton NlOl treatment on the indirect immunofluorometric assay (IIFA) titers of monoclonal antibodies to surface (HA) and internal (RNP and M) proteins of influenza A virus Monoclonal antibody titer? (IIFA)

A/NJ solid-phase antigenb

HA specific hybridoma fluid (No. 30, C-4) NP specific a&tic fluid WSN S/O M specific ascitic fluid (No. 174/l)

1,280

1,280

0

100

102,400

1,024

12,800

> 256

Untreated


Triton-trcatedc

Fold increase

a Serial two-fold dilutions of untreated fluids were tested, and the titers shown are the reciprocals of the highest dilutions producing a fluorescent intensity two-fold greater than the mean background determination. b 10 mg of purified, formalin-inactivated whole virus vaccine was covalently couple to 25 X 10”’ polyaminostyrene beads in a 25 ml reaction volume(Reimer et al., 1978;Phillips et al., 1980). c After covalent coupling to polyaminostyrene beads, A/New Jersey/76 virus was treated with 1.0% Triton N 10 1 in phosphate-buffered saline, pH 0.8, at room temperature for 5 min. Residual Triton N 10 1 was removed by 4 cycles of centrifugation and washing.

281 viruses (including the A/New Jersey/76 vaccine). In this way, several fluids (Fig. lB, Nos. 14,30,;6) were shown to be specific for A/New Jersey/76 and thus reactive with a subtype-specific surface glycoprotein, whereas several other fluids (Fig. lB, NOS. 5,41) cross.-eacted with influenza A and B viruses and therefore were judged to be non-specific for ?-al antigens.

A small number

of fluids (e g. Fig. lB, No. 25) exhibited

low but statistic-

ally significant levels of activity with both type A strains, suggesting the presence of typespecific antibody. Failure to detect high levels of type-specific antibodies, however, when screening hybridoma culture fluids with the solid-phase A/New Jersey/76 antigen, suggested that the solid-phase A/New Jersey/76 PAS beads might contain only intact virions so that the internal type-specific NP and M protein were not exposed. This contrasts with results of an enzyme-immunoassay using purified virus that detected type-specific antibodies (Hammond et al., 1980). Various methods were examined to prepare an antigen with exposed NP and M antigens, including treatment of antigen with non-ionic detergent before or after the virus was covalently attached to the PAS beads. These tests showed that treatment of virus after covalent attachment to PAS beads produced an antigen with greatly increased ability to detect monoclonal antibodies specific for NP and M antigens, without reducing the sensitivity for detecting antibody specific for the viral hemagglutinin (Table 1). Thus, detergent-treated antigen was useful for initial screening of hybridoma fluids to determine the presence of antibody reactive with type, subtype

TABLE 2

Comparison of the hemagglutination-inhibition test (HIT) and indirect immunofluorescent assay (IIFA) for the detection of mouse monoclonal antibodies to A/New Jersey/76 (HswlNl) hemagglutinin Monoclonal antibodiesa --. -Tissue culture fluids No. 30 No. 38 Mouse ascitic fluids No. 30 No. 38 a

HITb

IIFAC

Fold increase

64 4

10 .ooo 100

156 25

51,200 102,400

3,200,000 3,200,OOO

62 31

Tissue culture fluids shown here were from cultures of cloned hybridoma cells. Fluids from unproducing hemagglutinin-specific antibodies generally have titers ranging from 100 to 1000 in the IIFA and 4 or less in the HIT. Mouse ascitic fluids were from pristane-treated mice which had been inoculated with cloned hybridoma cells. HI tests utilizing variants of Hswl influenza show that monoclonal antibodies Nos. 30 and 38 react with different epitopes on the hemagglutinin molecule. b Reciprocal of the highest dilution of inhibitor-treated tissue culture fluid or ascitic fluid causing complete inhibition of 4 hemagglutinin units. c Reciprocal of the highest dilution of untreated tissue culture fluid or ascitic fluid producing a fluorescent intensity of 2X the mean background fluorescence using normal tissue culture fluid or normal ascitic fluid for background determination. cloned hybridomas

282

(or host) antigen.

Several hybridomas

detected by the IIFA as secreting A/New Jersey/76

subtype-specific antibody were cloned and inoculated into pristane-treated mice. Titers of a&tic fluids produced in this way ranged from 25 to 150-fold higher by IIFA (highest dilution

producing

an IIFA response 2X mean background)

than by HI test (Table 2).

DISCUSSION

Our studies of the application of a solid-phase indirect ~munoffuorometric assay with influenza virus as a model system have shown that the assay is useful for detecting monoclonal antibody in a number of circumstances. When purified antigen is not readily available in large quantities, a solid-phase antigen may nevertheless be prepared by immunologic binding to the PAS beads. Solid-phase antibody preparation may be lyophilized and stored for several years without loss of potency (Reimer et al., 1978). Because the stability and specificity of the solid-phase Ig is the primary factor which affects the consistency of performance and discrimination of the test, freeze-dried reagents can assure long-term reproducibi~ty of the assay. Alternatively, when highly purified antigen is readily available, the test may be simplified somewhat by covalently attaching antigen io the PAS beads. Chemical modifications of a complex viral antigen may then be possible to alter the antigenic specificity of the test. When initially screening hybridoma cells for the production of influenza specific antibody, 150-200 samples/day were routinely tested by one person. A solid-phase antigen prepared with T&on-treated virus homologous to that with which mice were initially immunized was used. Preliminary determination of the specificity of antibody-containing fluids was a~comp~shed by the use of solid-phase antigen prepared with strains sharing type-specific or subtype-specific antigens. It has been reported, however, that even among the type-specific NP antigen of influenza viruses some antigenic differences exist that can be detected with cross-absorbed heterologous animal sera (Schild et al., 1979) or with monoclonal antibodies (Van Wyke et al., 1980). Therefore, it could be possible to misidentify a monoclonal antibody as being reactive with a subtype-specific surface glycoprotein

antigen

if, for example,

it reacted with an HlNl

but not with an H3N2

influenza A strain, when in fact the antibody was specific for an epitope on the NP of the HlNl strain. Once the specific antibody-synthes~~g hybridomas have been cloned and larger volumes of fluid and/or high titer antibodies produced in mouse ascites, then confirmatory

tests of the antibody

specificity

may be undertaken

using appropriate

methods that generally have less sensitivity than the IIFA. Specific advantages of the IIFA for use with influenza virus include (i) high sensitivity and precision for reliable detection of low levels of hybridoma antibody; (ii) the ability to test sera or other biological fluids directly, without pretreatment to inactivate nonspecific inhibitors; and (iii) the ability to detect binding of antibodies that may poorly inhibit biological activity and otherwise might go undetected (~publ~hed results). The availability of this IIFA should greatly facilitate the production and safe use of monoclonal antibodies. Preliminary results already indicate that the assay can be used for the detection of monoclonal antibodies to adenovirus and hepes virus.

283

ACKNOWLEDGEMENTS

Dr. Flo Roumillat

and Ms. Gale Galland provided expert assistance in these studies.

Robert

C. Webster received support

stitute

of Allergy and Infectious

by research grant AI08831

Diseases. Myeloma

from the National

cells were kindly

In-

provided by Dr.

J.F. Keamy, University of Alabama. Use of trade names is for identification only and does not constitute endorsement by the Public Health Service or by the U.S. Department of Health and Human Services. REFERENCES

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