reactivity of monoclonal antibodies to potato virus a in various types of enzyme-linked...

Potato Research 37 (1994) 135 - 141

Reactivity of monoclonal antibodies to potato virus A in various types of enzyme-linked immunosorbent assay

M. F I L I G A R O V , ~ I , N. CEIS, O V S K A I , F. FRAN[EK2 and P. DI~DI(23

l lnstitute of Experimental Botany, Academy of Sciences of the Czech Republic, Na Karlovce la, CZ-160 00 Praha 6, Czech Republic 21nstitute of Molecular Genetics, Academy of Sciences of the Czech Republic. Vfdefask,'i 1083, CZ- 140 00 Praha 4. Czech Republic 31nstitute for Potato Research, CZ-580 03 Havlff:kf.w Brod, Czech Republic

Accepted for publication: 9 December 1993

Additional keywords: PVA, selection, assay reactivity, ELISA-types, Solaaum tttberosunt L.

Summary

A panel of mouse monoclonal antibodies (MAbs) was prepared against potato virus A (PVA) and their reactivity was tested in various types of ELISA. The type of ELISA as well as the methods of MAbs purification played an essential role. All monoclonal antibodies reacted with PVA antigen in IDAS-ELISA but MAbs used in DASI-ELISA and in DAS2-ELISA, in combination with polyclonal antibodies, showed different reactivity, The reactivity of MAbs in DAS-ELISA was dependent on the individual MAb used as a coating or as a conjugate. Two MAbs showed highest reactivity as coating antibodies but only one of them as a conjugate. The results demonstrate the importance of using different type of ELISA in selecting MAbs for routine diagnosis.

Introduction

The potyvi rus potato virus A ( P V A ) causes widespread economic damage in most po ta to -g rowing countr ies . A t present in Czech Republ ic a large number of po ta to samples are rou t ine ly checked by means of E L I S A for the presence of var ious viruses including PVA. Monoc lona l an t ibod ies ( M A b s ) have been shown to e l iminate many nonspecif ic reac t ions and great ly increase sensi t ivi ty (Koh le r & Milstein, 1975) as well as offer ing the possibi l i ty of p roduc ing a po ten t ia l ly unl imi ted supply of highly specific and uniform ant ibodies .

This pape r descr ibes the product ion and charac ter iza t ion of a panel of M A b s to PVA, the influence of purif icat ion methods of M A b s from ascitic fluid on their sensitivity, the dif ferences in their reaetivi t ies as dependen t on various types of E L I S A and the utility of PVA-speci f ic M A b s in diagnost ic assays by means of D A S - E L I S A .

Materials and methods

Viruses P V A isola ted from po ta to cv. Lichte Indust r ie ( isolate LI) was ob ta ined from Ins t i tu te of Po ta to Research and Breeding, Havlfe, kfiv Brod, r and was ma in ta ined in Nicotiana tabacum cv. Samsun. The leaves of infected plants were

Potato Research 3 7 (1994) 13 5

M. FILIGAROVA, N. (~E13,OVSKA, F. FRANI~K AND P. D~DIC

harvested three to six weeks after inoculation. The virus was purified by high speed centrifugation followed by equilibrium density gradient centrifugation in cesium chloride solutions, according to the method of (~e~ovskfi et al. (1991). Virus concentrations were estimated spectrophotometrically E2r I).01%,=2.8 (Stace-Smith & Tremaine, 1970). The purified virus was then used for rabbit anti-PVA antiserum production and for immunisation of mice.

Generation of hybridomas BALB/c mice were immunized intraperitoneally with four doses of purified PVA emulsified with the adjuvant AI-Span-Oil (Sevac, Prague) as follows: day 0, 6.5 Iag purified PVA; day 21, 11 pg; day 42, 20 pg: day 68, 110 Iag. The hybridomas were generated by the fusion of spleen cells from immune mice with the mouse myeloma cell line Sp2/0-Agl4 (Galfr6 & Milstein, 1981). The hybrid population in HAT medium was distributed into 800 wells of microtitre plates, qupernatants from growing cultures were tested for the presence of the PVA specific antibody by IDAS- ELISA (see below) using as antigen sap from virus-free and PVA - infective N. tabacum plants and swine anti-mouse immunoglobulins horseradish peroxidase (SEVAC, CSFR) as conjugate. Hybridomas from wells testing positive for PVA were cloned by limiting dilution and then propagated to obtain inocula for the production of ascites in BALB/c mice primed with mineral oil. Stable hybridoma lines were preserved in liquid nitrogen. Six hybridomas producing monoclonal antibodies of the IgG class were selected for further evaluation.

Purification of immunoglobulin and conjugation with enz ynm The ascitic fluid was clarified by centrifugation at 1000 g to remove cells. lmmunoglobulin (IgG) was precipitated from mouse ascitic fluid with 50% (v/v) saturated (NI-I4)2S0 4 solution adjusted to pH 7.4 by adding drops of I M NaOH or with caprylic acid (Steinbuch & Audran, 1969). The concentrations of IgG were determined by A2sll.lcm I~.1%=1.4. The ascitic fluids and purified monoclonal antibodies were stored frozen at -20 ~ or with 0.1% NaN 3, at 4 ~ Samples of lgG were conjugated to alkaline phosphatase (Boehringer 567 752) by a one-step glutaraldehyde method as described by Avrameas, 1969, using 2500 units enzyme/mg globulin.

A swine anti-mouse IgG (UMG, CSFR) was conjugated to alkaline phosphatase as described above.

Rabbit polyclonal antiserum was purified by caprylic acid precipitation and immunoglobulins were conjugated as MAb-IgG.

Determination of antibody subclass The IgG isotypes were determined by Ouchterlony gel diffusion using swine isotype- specific antisera.

ELISA The extract of PVA-infected or virus-free leaves from tobacco or from potato were used. The leaves were ground in PBS buffer pH 7.2 containing 0.05% Tween 20, 2%

136 Potato Research 37

REACTION OF MONOCLONAL ANTIBODIES TO PVA IN VARIOUS TYPES OF ELISA

polyvinyl-pyrrolidone (m.w. 24000), 0.2% ovalbumin and 1M urea (PBS-TPOU) in a ratio of I:10 (g/roll.

The PVA samples included twelve isolates of PVA of different European origin and seven PVA-infectcd potato cultivars.

Three types of ELISA test were used. Experimental conditions were optimized for each form of ELISA.

Indirect double antibody sandwich ELISA (IDAS-ELISA). The wells of microtitre plates (UMG, CSFR) were coated by incubation for 3 h at 37 ~ with rabbit anti-PVA polyclonal antibodies (PAbs) at I Iag/ml in 50 mM sodium carbonate buffer (coating buffer) at pH 9.6 (200 t-fl/well). After washing the wells four times with PBS containing 0.(/5% Tween 20 (PBS-T), the samples were added and incubated overnight at 4 ~ The wells were washed as above and isolated MAbs ( 1 ~Jg/ml) was incubated for 2 h at 37 ~ After washing a swine anti-mouse IgG-alkaline phosphatase conjugate (SWAM-AP) in a 1:1000 dilution was incubated for 4 h at 37 ~ and was then detected with p-nitrophenyl phosphate at I M diethanolamine buffer at pH 9.8.

Double antibody sandwich ELISA (DAS-ELISA). This type of ELISA was performed according to Clark & Adams (1977). The wells were coated either with PAbs (1 [ag/ml) or with individual MAbs (1 jJg/ml) before overnight incubation with plant sap. After washing, antigen bound by PAbs was detected using rabbit polyclonal IgG-alkaline phosphatase conjugate (PAbs-AP), antigen bound by MAb was detected using MAb-alkaline phosphatase conjugate (MAb-AP) and each conjugate was diluted 1:1000.

D A S r E L I S A . The wells of microtiter plates were coated with PAbs (1 jag/ml) and incubated 3 h at 37 ~ The samples diluted in PBS-TPOU were added. After overnight incubation at 4 ~ the MAbs-AP (diluted 1:200 and 1:1000) was added and incubated for 4 h at 37 ~

DAS,-ELISA. Wells were first coated with MAbs (I and 5 iag/ml), the samples incubated overnight at 4 ~ and PAbs-AP at a 1:1000 dilution was then incubated for 4 h at 37 ~

Indirect ELISA. For the plate-trapped antigen form of indirect ELISA (Mowat, 1985), three steps were considered which were separated by washing the wells as described previously. For step [ the wells contained purified virus (100 and 200 ng/ml) in a coating buffer, for step 2 this was replaced by MAbs (5 I.Ig/ml) and for step 3 the linked MAbs were detected using SWAM-AP in dilution 1:1000.

In all types of ELISA tests, p-nitrophenyl phosphate ( 1 mg/ml) was added and 30 rain later the A405 was measured with an ELISA reader (Dynatech Minireader Ill.

Potato Research 37 (1994) 137

M. FILIGAROVA, N. CEROVSKA, F. FRAN[K AND P. DEDIC

Results

Hybridomas secreting antibodies specific for PVA were used to produce MAbs in ascites fluids. Six stable hybridomas produced immunoglobulin of the IgG1 (MAb PVA-151, 187, 328 and 634), IgG2 a (MAb PVA-534) and IgG2 b (MAb PVA-290) subclasses were selected for further work. Preliminary tests showed that the same IgG concentration of MAbs purified from ascitic fluids by caprylic acid precipitation gave an A405 values as two-fold greater than given with the same IgG concentration of MAbs purified by (NH4)2SO4 in IDAS-ELISA. Further experiments were therefore done to ascertain whether the different reactivity of MAbs revealed by these earlier tests would also be found in DAS2-ELISA and DAS-ELISA. The data presented in Table 1 show that MAb PVA-151 precipitated by caprylic acid and used as a coat and a conjugate gave the highest positive A405 value. Similar results were obtained in IgG purification experiments repeated three times. The results with other MAbs and results in DASz-ELISA (data not shown) also indicated that the method of their purification influenced sensitivity of individual MAb.

Various types of ELISA with PVA leaf extracts documented the differences in reactivity of these MAbs. In preliminary experiments the specificities of MAbs to PVA determined conditions for testing their reactivity in different types of ELISA. For comparative studies, the optimum concentrations of MAb-IgG were found to be 1 pg/ml and 5 lag/ml and optimum dilutions of conjugates 1:200 and 1:1000. Higher concentrations gave higher A405 values and a few MAbs >2.45, and thus it was impossible to differentiate MAbs reactivity. Lower concentrations yielded a negative or negligible reaction from some MAbs. These two optimum concentrations and dilutions show differences between individual MAbs (Tables 2 and 3). The results in Table 2 demonstrate the highest sensitivity of MAbs PVA-634 and PVA-151 with IDAS-ELISA. From Table 3 it is also evident that AP-conjugates of MAbs PVA-151 and PVA-634 gave the best reaction with DASI-ELISA at the usual incubation conditions. By contrast, the best Mab for coating in DAS2-ELISA was PVA-534.

In further tests of DAS-ELISA homologous and heterologous MAb combinations were used on the same plate (Table 4). Because lower concentrations of IgG and higher dilution of conjugate are preferred for routine detection, both were used for comparative studies of reactivity of individual MAbs. The best combination of MAbs in this type of test came from using PVA-151 as a coating antibody and also in the function of conjugate. There are other possibilities for using PVA-328 and PVA-187 as a coats but only with PVA-151 as a conjugate.

Other tests were done using sap from different PVA-infected potato cultivars (data not shown). MAb PVA-151 reacted with all tested PVA samples in this type of ELISA and was chosen for the construction of a diagnostic kit. The results obtained in the plate-trapped antigen form of indirect ELISA revealed that either not all MAbs reacted with the same epitope of the virus or they have different affinities. This is the objective of our further study.

Because large numbers of experiments were done and the virus concentration in the saps varied between experiments, it was decided to present the results in the



Table 1. Absorbance values obta ined in D A S - E L I S A I exper iments to compare the reactivity of M A b PVA-151 purified by two different methods.

Mab (as a coat) isolated by caprylic acid (NH4)2SO 4

AP-conjugate of M A b isolated by: A4052 _+ S.D.

caprylic acid >2.45 1.76 _+ 0.04 (NH4)2SO 4 0.80 + 0.05 0.68 _+ 0.07

J Wells were coated with M A b at 1 lug/ml concent ra t ion and M A b - A P conjugate was used at a 1 : 1000 dilution. 2 A405 values _+ S.D. of three exper iments of two wells.

Table 2. Reactivity of monoclonal ant ibodies in IDAS-ELISA.

MAbs

Concen t ra t ion of monoclonal ant ibodies l/ag/ml 5 pg/ml

151 ++1 +++ 0 187 + + 0 290 + +++ 0 328 + +++ 0 534 + +++ 0 634 +++ +++ 0

control sap

I +++, A4o5=1.20-2.45: ++, A4o5=0.7-1.2: +, A4o5=0.1-0.7: 0, A4o5= <0,1 Control tests: Virus free sap A4o5=O (<0.04); D A S - E L I S A (polyclonal ant ibodies) A4o5=++.

Table 3. Reactivity of monoclonal ant ibodies in D A S I - E L I S A and DAS. -ELISA.

D A S v E L I S A Dilution of M A b s - A P

DAS2-ELISA Concent ra t ion of MAbs

1:1000 1:200 1 pg/ml 5 IJg/ml

MAbs 151 +++1 +++ + + 187 + + + + 290 + + 0 + 328 + + + ++ 534 + + ++ ++ 634 +++ +++ + +

l The range of A405 values is as in Table 2. Contro l tests: virus free sap A405=0 (<0.04): D A S - E L I S A (polyclonal ant ibodies) A405=++.


M. FILIGAROVA, N. CEI~OVSKfi,, F. FRAN~K AND P. D~DIC

Table 4. Reactivity of monoclonal antibodies in DAS-ELISA.

MAbs-AP conjugates 151 187 290 328 534 634

Mabs (coat) 151 +++1 + 0 + + 0 187 +++ + 0 + ++ 0 290 ++ + 0 + + 0 328 +++ + 0 + + + 534 ++ + 0 0 + ++ 634 0 0 0 0 0 (1

1 The range of A4o 5 values is as in Table 2. Control tests: virus-free sap A405=0 (<0.01)" DAS-ELISA (polyclonal antibodies) A405=++.

range of A405 readings (Tables 2 to 4). In general, the pat tern of reactivity of each M A b was similar in three to five replicates of each E L I S A type.

D i s c u s s i o n

Five MAbs to PV A were produced and character ized (Boonekamp et al., 1990). Three reacted with purified and non-purified PVA in indirect E L I S A and IDAS- E L I S A and might be useful for routine diagnosis. However , the usefulness and reactivity of these MAbs in D A S ~ - E L I S A and D A S - E L I S A was not documented .

Six MAbs were selected using I D A S - E L I S A with non-purified P V A leaf extracts and were compared in various types of ELISA. The reactivity of these individual MAbs was very different depending on the type of E L I S A used. M A b PVA-634, which showed highest affinity in I D A S - E L I S A , might be useful in D A S v E L I S A as conjugate but reacted weakly in D A S x - E L I S A when used as a coat. Additionally, this M A b did not react when used as a coat in D A S - E L I S A in combinat ion with all individual MAbs-AP. Tile type of E L I S A procedures in combinat ion with polyclonal antibodies or monoclonal ones alone played an essential role in selecting M A b for routine diagnosis.

Our MAbs differed in their ability to trap virus from infective sap when used in DAS~-ELISA in combinat ion with PAbs-AP. However , tlle same MAbs showed a different reactivity when used as conjugates in D A S I - E L I S A with PAbs as a coat (see Table 3). These results differ from those of polyclonal antibodies and MAbs to potato virus X (S6ber et al., 1988). When MAbs PVA-534 and PVA-328 were used as the first ant ibody and PAbs as the labelled second antibody, the assay was more sensitive than when the opposite order of antibodies was used. A similar pattern of reactivity of MAbs to soybean mosaic virus was observed by Hill et al. (1984). Binding of virus- specific rabbit polyclonal antisera to viral polypeptides could interfere with subsequent binding of MAbs to the same virus. Results of Bahrani et al. (1988), Van Regenmor te l (1984), Martin & Stace-Smith (1984) and Sherwood et al. (1987)



showed some o the r p rob lems concern ing M A b s with b inding to plates and abi l i ty to be conjuga ted .

High D A S - E L I S A readings ob ta ined with single M A b PVA-151 used both as the cap ture and the conjugate showed that after the first s tep sufficient numbers of free ep i tops remain on the P V A antigen for b inding of con juga ted ant ibodies . Add i t iona l ly , M A b PVA-151 or PVA-328 used as first an t ibody and PVA-151 as conjuga te gave higher sensi t ivi ty than o the r M A b s or their AP-con juga te s in our exper iments . F rom our results we se lec ted M A b PVA-151 as the best M A b , not only fox" absorp t ion of the virus from infective sap but also in the function of a conjugate in D A S - E L I S A . This M A b recognizes a several isolates of P V A and therefore seems to be specific for the ep i tope c o m m o n to these isolates. M A b PVA-151 has a wide spec t rum of react ivi ty and might be useful in rout ine diagnost ic tests.

Acknowledgements

We thank Rena t a Hadfimkovfi and Eva Hanusovfi for technical assistance.

References

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Bahrani, Z., J.L. Sherwood, M.R. Sandborn & G.C. Keyser, 1988. The use of monoclonal antibodies to detect wheat soil-borne mosaic virus. Journal of General Virology 69: 1317-1322.

Boonekamp, P.M., H. Pomp & G.C. Gussenhoven. 1990. Production and characterization of monoclonal antibodies to potato virus A. Jottrnal of Phytopatholo,gy 128:112-124.

Clark. M.F. & A.N. Adams, 1977. Characteristic of the microplate method of enzyme-linked immunosorbent assay for detection of plant viruses. Journal of General Virology 3"4: 475-483.

12efovsk,'i, N., M. Filigarov,'i, H. Branigovfi, P.Z~ik & P. Dedir 1991. Some factors influencing purification of potato virus A (PVA). Acta Virologica 35:469-471.

Galfr6, G. & C. Milstein, 1981. Preparation of monoclonal antibodies: strategies and procedures. Methods in Enzymology 73: 3-46.

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Martin. R.R. & R. Stace-Smith, 1984. Production and characterization of monoclonal antibodies specific to potato leaf roll virus. Canadian ,lournal of Plant Pathology 6: 206-209.

Mowat, W.P., 1985. Simplified enzyme immunoassay for plant virus detection and identification. Report of the Scottish ( ' top Rerearch Institute for 1984. p. 188.

Regenmortel, M.H.V. van, 1984. Monoclonal antibodies in plant virology. Microbiological Sciences 1: 73-78.

Sherwood, J.L., M.R. Randborn & G.C. Keyser, 1987. Production of monoclonal antibodies to peanut mottle virus and their use in enzyme-linked immunosorbent assay and dot- immunobinding assay. Phytopathology 77:1158-1161,

S6ber, J,, L. J~irvektilg, I. Toots, J. Radavsky, R. Willems & M. Sarma, 1988. Antigenic characterization of potato virus X with monoclonal antibodies. Journal of General Virology 69: 1799-18(17.

Stace-Smith, R. & J.H. Tremaine. 1970. Purification and composition of potato virus Y. Ph vtopathology 60:1785- 1789.

Steinbuch, M. & R. Audran. 1969. The isolation of lgG from mammalian sera with the aid of caprylic acid. Archives o.f Biochentistt3, and Biophysics 134: 279-284.


reactivity of monoclonal antibodies to potato virus a in various types of enzyme-linked...

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