NL8601642A - New potato root eel-worm proteins - and antibodies useful for determn. of eel-worm populations in soil - Google Patents

Abstract

Type-specific thermostable proteins isolated from eggs, larvae or adult potato root eelworms (Globodera rostochiensis and G. pallida) are new. Also claimed are polyclonal and monoclonal antibodies to such proteins. G. rostochiensis proteins have molecular wts. of 20.8 (+-) 1.0, 20.6 (+-) 1.0, 18.0 (+-) 1.0 and 18.0 (+-) 1.0 kD and isoelectric points of 5.2 (+-) 0.1, 5.3 (+-) 0.1, 6.0 (+-) 0.1 and 5.7 (+-0.1 respectively. G. pallida proteins have molecular wts. of 21.0 (+-) 1.0, 20.5 (+-) 1.0, 17.0 (+-) 1.0 and 17.0 (+-) 1.0 kD and isoelectric points of 5.32 (+-) 0.1, 5.4 (+-) 0.1, 5.8 (+-) 0.1 and 5.6 (+-) 0.1 respectively. The antibodies may be labelled, e.g. with enzymes. Determn. of eelworm populations is effected by isolating an eelworm protein homogenate from a soil sample, opt. heat-treating the homogenate and removing denatured proteins, and analysing the prod. using the antibodies, e.g. by the sandwich ELISA method.

Description

* _ ί VO 8271 * Title:

Species-specific proteins of the nematodes Globodera rostochiensis and Globodera pallida (potato cyst nematodes); mono- and polyclonal antibodies with specificity for these proteins; use thereof in a method for determining the nematode populations in soil samples.

Potato fatigue caused by potato cyst nematodes (nematodes) is a serious threat to potato cultivation. Two closely related types of potato cyst nematodes are known. Globodera rostochiensis 5 (Wollenweber) Behrens and Globodera pallida (Stone) Behrens, which were not distinguished until 1973 and were classified as Heterodera rostochiensis. Both species can be divided into a number of pathotypes defined by their insensitivity to certain resistance genes crossed into the potato. Eight pathotypes are currently known, five of which are for G5. rostochiensis (R02-R05) and three for G. pallida (Pai ~ Pa3). Large areas are contaminated with both types of nematodes, which can hardly be distinguished from one another in a morphological manner. Potato cultivation is therefore legally bound by strict crop rotation schemes, so that the available land area can only be used sub-optimally. If it is possible to determine quickly and reliably in soil samples whether it concerns a G. rostochiensis or a G. pallida infection, and, in the case of mixed populations, the percentages of both species, the grower can be specifically advised to advise G. rostochiensis -resistant or to grow G. pallida-resistant potato varieties. This makes it possible to make better use of the existing or still to be developed potato varieties, and the suitable cultivation soils, and the use of nematicides can be reduced.

Identification of G_. rostochiensis and G. pallida 86 0 1 64 2 'i -2- based on morphological features, as stated, is not quite possible. Reasons for this are intraspecific variations and interspecific overlapping of morphological features and the influence of environmental factors such as temperature and food availability on some morphological features. Partly because of this, morphological research is a time-consuming matter, which does not qualify for routine large-scale use.

The use of different electrophoresis techniques has also been proposed to distinguish the two types and sometimes even pathotypes. By Wharton et al, "Concepts in Nematode Systematics" from Stone, Platt and Khalil, eds. Academy Press, New York, London, (1983), pp. 235-248 it has been described that differences in isoenzyme patterns can be used, and by Fleming and Marks, Ann. appl.

Biol. 103, (1983), 277-281, G. rostochiensis and G. pallida have been distinguished based on their phosphoglucomutase isoenzyme distribution. However, both this method and other electrophoresis methods, such as polyacrylamide disc-gel electrophoresis, isoelectric focusing, and two-dimensional polyacrylamide slab-gel electrophoresis, which can distinguish populations of potato cyst nematodes, are too cumbersome to be suitable for large-scale routine studies. to be. Moreover, the reproducibility of the protein extraction procedures and the stability of the proteins, which are crucial when the test systems are based on their quantitative determination, are unsatisfactory.

In practice, samples of contaminated soil are therefore usually tested in a greenhouse using a series of test plants with different resistance properties, as described by Kort et al, Nematologica 23, (1978), 333-339, although this method too is very cumbersome and time consuming.

35 There is therefore a great need for an f-1 -λ / ts <5 «3 *>, ^ A y * h tii £ Λ * -3- simple and rapid test method for determining soil contamination by potato cyst nematodes of the species G. rostochiensis and G. pallida with which a distinction can be made between both 5 species on the basis of biochemical or serological characteristics.

The invention meets this need by characterizing some species-specific proteins, which, thanks to a special thermostability, can be easily isolated from protein homogenates of eggs, larvae and adult nematodes and have suitable properties for a serological test. This one. proteins found by analysis of egg protein homogenates and of second stage larvae of representatives of both nematode species using one- and two-dimensional sional gel electrophoresis. In addition to a large amount of identical proteins, a number of homologous proteins were found with small differences in electrophoretic mobility and isoelectric point. Some of these were thermostable and these thermostable species-specific proteins, which are present in relatively large and comparable amounts in both species, could be purified and used to produce both polyclonal and monoclonal antibodies, which are part of a serological soil analysis kit 25 have proved useful.

By the term "thermostable proteins" here is meant proteins which, after heating a protein homogenate, remain in solution after centrifugation for a period of up to 30 minutes after centrifugation, i.e. not "denaturing".

The invention primarily relates to proteins from the potato cyst nematodes species Globodera rostochiensis and Globodera pallida, which are species-specific and thermostable and have been isolated from eggs, larvae or adult copies of the nematodes.

In particular, the invention relates to such proteins from eggs or larvae of the potato cyst nematodes species Globodera rostochiensis, selected from S601642 -4- («) the polypeptide having a molecular weight of 20.8 ± 1.0 kD and an iso- electric point of 5.20 _ + 0.10; (b) the polypeptide having a molecular weight of 20.6 ± 1.0 kD and an isoelectric point of 5.30 ± 0.10; (C) the polypeptide having a molecular weight of 18.0 ± 1.0 kD and an isoelectric point of 6.00 ± 0.10; (d) the polypeptide having a molecular weight of 18.0 ± 1.0 kD and an isoelectric point of 5.70 0.10; as well as such proteins from eggs or larvae of the potato cyst nematodes Globodera pallida, selected from (a) the polypeptide having a molecular weight of 21.0 +. 1.0 kD and an isoelectric point of 5.32 _ + 0.10; (b) the polypeptide having a molecular weight of 20.5 + 1.0 kD and an isoelectric point of 5.40 _ + 0.10; (c) the polypeptide having a molecular weight of 17.0 + 1.0 kD and an isoelectric point of 5.80 + 0.10; (d) the polypeptide having a molecular weight of 17.0 + 1.0 kD and an isoelectric point of 5.60 0.10.

The invention also relates to polyclonal and in particular monoclonal antibodies with specificity for the species-specific and thermostable proteins of the potato cyst nematodes species Globodera rostochiensis and Globodera pallida. First of all, these include antibodies, in particular monoclonal antibodies, with specific affinity for species-specific and thermostable proteins of either nematode species. In addition, antibodies are also included which cross-react with species-specific and thermostable proteins of both nematode species.

The invention further relates to an analysis kit for determining the potato cyst nematode populations in soil samples, which antibodies with specific affinity for species-specific and thermostable proteins of the nematode species Globodera rostochiensis and / or antibodies with specific affinity for species-specific and thermostable proteins of the nematode species Globodera pallida. Depending on the nature of the 1101 Bit ► * -5 method for which the analysis kit is intended, these antibodies may optionally carry a detectable label, such as preferably an enzyme label or also a radioactivity label, etc. Suitable labels known per se to those skilled in the art, as well as methods of coupling the labels to antibodies.

According to a preferred embodiment, the analysis kit also comprises antibodies which cross-react with species specific and thermostable proteins of the potato cyst nematodes 10 species G. rostochiensis and G. pallida.

If desired, not the species-specific antibodies, but these cross-reacting antibodies are appropriately labeled.

Finally, the invention also relates to a method for determining the potato cyst nematode populations in soil samples, wherein a protein homogenate of potato cyst nematodes obtained from a soil sample, optionally after thermal treatment and removal of denatured proteins, in a manner known per se, e.g.

The sandwich ELISA method is tested using species-specific antibodies to species-specific and thermostable proteins of the nematode species (Rostochiensis and / or species-specific antibodies to species-specific and thermostable proteins of the nematode species).

According to a sandwich ELISA method, micro-titer plates are "coated" with an antibody, cross-reacting with the species-specific proteins, after which two limited dilution series are made with a protein homogenate of potato cysts extracted from a soil sample. Subsequently, it is incubated with a species-specific antibody to which an "enzyme label" is attached, and the absorbance of the reaction product of the enzyme is measured for each dilution. Then it is determined which dilutions give values that lie in the linear part of the reaction curve, the reaction curve being the relationship; Λ λ Λ / Λ 3 01/1 ο 4 ά «% -6- indicates between the amount of" labeled "antibody and the amount of reaction product expressed in O.D. values.

From these values, the amount of protein (= antigen) 5 present for each species in the sample can be calculated. The amount of protein is a measure of the contamination expressed in eggs per 100 ml of soil. The average amount of antigen present per egg is assumed. Such a test IQ can of course also be set up reciprocally, in which the species-specific antibody is attached to the plate and the binding of species-specific proteins from extracts of potato cyst nematodes from soil samples is detected with a cross-reacting antibody labeled with, for example, peroxidase. A condition with regard to the antibodies to be used is that they do not react with the same epitope, or inhibit each other in a different way (steric interactions, etc.).

By means of a competitive ELISA method 20 it is easy to determine whether or not antibodies recognize the same antigen determinant on the antigen. Most preferably, high affinity antibodies are used, e.g., 10? or higher.

It has been established experimentally that both useful polyclonal and useful monoclonal antibodies can be produced. Conventional (polyclonal) antisera against the species-specific and thermostable proteins of the two nematode species generally appear to have a strong cross-reactivity, which is consistent with the observation that upon controlled cleavage of the relevant proteins with a porotease both identical and different peptides in the proteins are found. The hybridoma technique can overcome the problems of cross-reactivity and standardization that arise with conventional antisera. Mouse spleen cells, immunized with purified

Sfö1S42 * 7 protein preparations are fused with mouse myeloma cells.

Among the many hybridomas that produced nonspecific antibodies, some were found to react exclusively with only one of the species-specific homologous proteins in an ELISA assay.

The protein homogenate required for the test can be prepared in a manner known per se, for example, by a "French-press" method or by similar methods.

If desired, the protein homogenate is worked up by a thermal treatment, which essentially leaves the thermostable proteins undisturbed but denatures the remaining proteins, eg at 100 ° C "au bain marie" for 15-15 minutes, and removing the denatured proteins, eg by centrifuging and isolating the supernatant containing the thermostable proteins.

The invention will be explained in more detail with reference to the experimental part below.

20

Example I.

Research on proteins from second stage larvae of potato cyst nematodes.

25

The study used twelve populations of rostochiensis and (? Pallida, which are listed in Table A below. These populations were kept on Solanurn tuberosum ssp. Tuberosum "Eigenheimer", which is susceptible to all pathotypes.

To best characterize these populations, the variation in virulence and morphology was investigated. For the morphological examination, second stage larvae were sacrificed in FP-4 (Netscher et al, 35 Nematologica 15 (1969), 286 and fixed in 4% cold formalin. In connection with the measurements to be performed, S8C1842 u - 8- further treated in the manner described by Seinhorst, Nematologica 4 (1959), 67-69 On average, 20 representatives were measured per population.

To determine the virulence properties, the populations were tested for their ability to reproduce on the differentiating potatoes of the international pathotype scheme of Kort et al, Nematologica 23 (1978), 333-339. Solanum tuberosum ssp. andigena was replaced by the commercially available variant Saturna with the resistance gene H1 derived from CPC 1673. In Petri scale experiments, Solanum vernei hybr. 62.33.3 replaced by the Darwina variant. Solanum kurtzianum hybr. 60.21.19 and Solanum multidissectum hybr. P55 / 7 15 were omitted from the test. Virulence properties were measured by inoculating fifty second stage larvae (two per root tip) on roots of sprouts grown in Petri dishes with water agar (Mugniery et al., Sci. Agron. Rennes 1976, 217-220) . The number of females that appeared on the roots of the differentiating potatoes was expressed as a percentage of the number of females on the Eigenheimer sensitive to all pathotypes. This percentage, the relative number of females, was used to indicate the number of virulent genotypes in a population. In a number of cases, the virulence properties in populations were determined based on the Pf / Pi values on the differentiating potato varieties, and expressed as a percentage of the Pf / Pi value on the host sensitive to all pathotypes.

For this purpose, the potato plants were seeded with 25 cysts (Pi) and grown in pots (diameter 8 cm, volume 150 ml) filled with sandy loam. The final population densities (Pf) were determined after a two month growth period.

35 In most combinations identified as compatible according to the international pathotype scheme, 3 5 A 1 SA 0: Öl * -9-, the number of females developing on the differentiating potato varieties was strikingly low compared to the number developing on the host sensitive to all pathotypes (see Table A). For example, 5 the relative number of females produced on Solanum vernei hybrid 62.33.3 by Pa3 populations Nos. 9, 10 and 11 did not exceed 10%. These populations are therefore apparently mixtures of individuals exhibiting differences in their ability to overcome resistance. Furthermore, populations designated as virulent for the same differentiating potato species were found to show differences in their ability to overcome resistance. The relative number of females that developed on Solanum tuberosum ssp. andigena by the 15 populations Nos. 3 and 6 was resp. 30% and 84.6%.

a 360 184 2 -10-

TABEIi A

Geographic source and virulence properties in the Globodera rostochiensis (Ro) and G. pallida (Pa) populations relative number of females

Pop. Pathotype and source S.tuberosum S.vernei S.vernei

No. ssp.andiqena hybr. hybr.

hybr. CPC 1673 58.1642 / 4 62.33.3 1. Ro-jy Wageningen 0 1 ± 0 / 2c 0 2. Ro-j ^ Weert 0 1 ± 1.3C 0

3. Ro3, Hoogeveen 30.0 + 1.5 2.2 + 2C 0.5 ± 0.4C

4. Ro ^, Hardenberg 1.4 + 0.1 0.3 + 0.2 ° 0 5. Ro ^, Emmen 0 10 + 5C 0 6. ROr, Harmerz, German- 84.6 ± 3.7 30 ± 9C .15.9 ± 1.7 country d * cl 7. PaGlarryford , - - 10.4 ± 2.5c

Northern Ireland

8. Pa2, New Leake, -d -d 3.3 ± 2C

England 9. Pa3, Calishead, -d -d 6.7 + 0.7

England 10. Pa3, Far 0er, -d -d 9.3 + 0.7

Denmark 11. Pa-, Frenswegen, - - 5.7 ± 0.6

Germany 12. Pa2, Veendam -d -d 1.5 + 0.5 a As indicated in the original collections b Number of females produced on differentiating potato varieties, as percentages relative to the quantities of females on Solanum tuberosum susceptible to all pathotypes ssp. tuberosum "Eigenheimer", + standard deviations c Data from pot experiments d Not determined 8801 842 -11-

The morphological properties of the second stage larvae are listed in Table B. Together with the color of the females, the twelve populations could be classified into rostochiensis (1-6) and G. pallida 5 (7-12). The morphometric properties of the second stage larvae are variable and overlap between the two species. The body length and tail length are not suitable for species identification and differ markedly from the values described for rostochiensis and 10 G. pallida.

»'3 ^? Λ ^ ^: 0 λ / -12-

TABLE B

Morphological properties of second stage larvae of Globodera rostochiensis (1-6) and G. Pallida (7-12).

Pop. Stylet, body distance b tail flap Styletc a length, / aia length, yumD median bulbus length, um ° cusps no * J * to excretion _porus, / am_

1. 21.0 + 0.6 453 + 19 31.6 + 2.0 50.0 + 2.9 16 R, 9R-P

2. 20.9 + 0.5 438 + 18 32-5 + 3.5 50.0 + 4.4 14 R, 1R-P

3.20.7 ± 0.6 420 + 22 30.6 + 2.8 47.5 + 2.7 20 R, 5R-P

4.20.8 ± 0.6 436 + 19 33.5 + 2.1 48.8 + 2.5 9 R, 5R-P,:

5. .21.4 + 0.9 449 + 18 32.6 + 2.9 45.5 + 2.4 19 R, 1R-P

6.21.0 + 0.5 442 + 20 29.3 + 3.2 48.1 + 3.7 14 R, 1R-P

7.24 + 1.1 459 + 30 36.3 ± 4.0 49.014.8 17 P, 4R-P

8.24 + 0.8 448 + 19 38.812.448.9 + 3.5 20 P

9.23.6 + 0.9 449 + 25 37.4 + 2.8 50.213.8 18 P, 2R-P

22.0 + 0.5 418 + 17 35.2 + 2.6 47.3 + 2.5 20 P

11.22.8 + 0.8 459 + 19 37.2 + 3.0 49.2 + 3.7 25 P

12.23.3 + 0.8 458 + 18 39.0 + 4.3 51.1 + 2.7 19 P, 1R-P

a Numbers refer to the populations in Table A b Distance 1 standard deviations c Numbers of individuals with basal stylet cusps rounded (R) and pointed front (P) as described for G. rostochiensis Rostoch and G. pallida Epworth by Stone, Nematologica 18 (1972) , 591-606; RP intermediate.

fK 9 *. A ί ƒ ft i 04 c -13-

The twelve populations were further investigated with different types of electrophoresis. To this end, second-stage larvae were lured from cysts with potato-root diffuse. The larvae were collected every two days and stored at 4 ° C. Four days after baiting, viable larvae were allowed to pass through a cotton wool filter and about 40,000 individuals were concentrated by centrifugation (1,000 xg), rinsed three times with 20 ml of 10 mM tris-HCl, pH 7.4 and finally suspended 10 in 200 µl 10 mM tris-HCl, pH 7.4. The nematodes were homogenized on ice in a 2 ml glass Potter homogenizer with a tight fitting Teflon plunger (clearance about 25 µm) at 1,700 rpm, fifteen times for 20 seconds at 10 second intervals.

By this treatment, the nematodes were broken open completely as revealed by control with a preparation microscope.

For polyacrylamide gel electrophoresis of native proteins, the nematodes were homogenized in 10 mM 20 tris-HCl, pH 7.4. The homogenate was centrifuged at 105,000 x g for 10 minutes. Protein was determined in the supernatant by the method of Bradford, Anal.

Biochem. 72 (1976), 248-254 by measuring the shift in extinction of Coomassie Brilliant Blue at 595 nm after reaction with protein. The supernatant was made with 10% glycerol (v / v) and 0.01%. Bromphenol blue treated and used directly for electrophoresis.

Native polyacrylamide gel electrophoresis was performed at 4 ° C in a slab gel device (model 220, 30 Bio-Rad, Richmond, California 94804) with 3 mm slots, a 3% acrylamide stacking gel and a 7% acrylamide separation gel (thickness 1.5 mm). The current was 10 mA for 30 minutes and further 20 mA. The buffer system was essentially as described by Laemmli, Nature 227 35 (1970), 680-685, but omitting the sodium dodecyl sulfate (SDS). The positions of the protein bands ** λ ii 1 λ Λ ''> W ΐ · '- -14-

»« I

were expressed as Rf values, calculated by dividing the migration distance of the protein by the migration distance of bromophenol blue. The bromophenol blue used as a marker was assigned a value of Rf = 100.

By such electrophoresis of native proteins, a clear distinction between the two species could be observed. Specific protein bands were found at Rf 35 for G. rostochiensis; 59.5; 65; 10 and 75; whereas for G pallida specific protein bands were found at Rf 24; 55; 59; 76; and 78. The intensities of different species-specific protein bands (eg, Rf 35; 65; and 76) were found to vary with repeated experiments, probably due to a combination of small variations in the extraction procedures (eg, the time between the homogenization and electrophoresis) and instability of the native proteins.

In some cases, species-specific protein bands were even found to be completely absent (eg, Rf 35 in the case of populations 3 and 6), although they were present in duplicate experiments. No consistent differences could be detected between populations of either nematode species. By storing native protein samples at -80 ° C, many of the proteins were found to deteriorate in quality, which was reflected in diffuse bands upon electrophoresis.

Sodium dodecyl sulfate polyacrylamide gel electrophoresis experiments were also performed. To this end, the nematodes were homogenized in 10 mM tris-HCl, pH 7.4, 5% 2-mercaptoethanol (v / v). The homogenate was centrifuged at 105,000 x g. Sixty µl supernatant was saturated with 64 mg urea and stored at -80 ° C until use. Before electrophoresis, 20 µl of 10 mM tris-HCl, pH 7.4, 572-mercaptoethanol (v / v) was added to the thawed sample. After centrifugation at 105,000 x g and determination of the protein concentrations, an equal §50 1 642-15 volume volume of buffer was added with the following composition: 10% (w / v) glycerol, 5%. (v / v) 2-mercaptoethanol, 2.3% (w / v) SDS and 0.01% bromophenol blue (w / v) in 62.5 mM tris-HCl, pH 6.8.

SDS polyacrylamide slab gels were prepared according to

Laemmli with a 6% acrylamide stacking gel and a 12% acrylamide separation gel (thickness 1.5 mm). Electrophoresis was performed at 20 mA for 30 minutes and further at 35 mA. Apparent molecular weights were estimated using phosphorylase B, bovine serum albumin, ovalbumin, carbonic anhydrase, soybean trypsin inhibitor and lysozyme as reference proteins (BioRad low molecular weight standard solution).

When comparing the SDS denatured protein, 15 profiles of £. rostochiensis populations and G. pallida populations, it was found that there were four distinct protein bands specific for the former and seven for the latter nematodes. Subtle quantitative differences were also observed in diffuse bands.

These bands are probably composed of different polypeptides showing small differences in molecular weight. The intensities of these bands have been found to vary with repeated experiments and are therefore not suitable for species identification. Especially the main protein bands of 31 kD; 20.7 kD; and 18 kD for G. rostochiensis and 31.5 kD; 21 kD, 20.5 kD; and 17 kD for G. pallida, could be used to distinguish between these two species. The 20.7 kD protein bands; 18 kD; 21 kD; 20.5 kD; and 17 kD were found to stain gray with silver, while the majority of proteins gave a brown or reddish brown color. Silver staining was done by a modified method from Oakley et al, Anal. Biochem. 105 (1980), 361-363.

In a number of experiments, the proteins were stained with Coomassie Brilliant Blue R250. The staining solution contained 0.2% Coomassie Brilliant Blue, 50% (v / v) methanol J-1 '. f V? V V J J la -16- and TL (v / v) acetic acid. The gels were decolorized in TL (v / v) methanol and TL (v / v) acetic acid. The protein profiles, obtained upon staining with Coomassie Brilliant Blue, again showed that the aforementioned species specific proteins are among the major components in terms of amounts of the total soluble protein fraction.

With one-dimensional SDS electrophoresis no intraspecific differences could be found, neither qualitatively nor quantitatively. Urea-saturated protein-10 samples were allowed to stand at -80 ° C for at least two years

are stored without detectable changes in protein patterns.

The 20.7 kD and 18 kD protein bands, specific for Q. rostochiensis and the 21 kD protein bands; 20.5 kD; and 15 17 kD, specific for G_. pallida, were found to be thermostable and were partially purified. To this end, second stage larvae were homogenized in 10 mM tris-HCl, pH 7.4 and the crude homogenate was heated at 100 ° C in bain marie for 10 minutes. Most of the proteins (about 98%) were precipitated at 105,000 x g by centrifugation. The thermostable proteins remained in solution. After saturation with urea, they could be stored at -80 ° C.

A comparison of the SDS protein patterns of the total soluble protein fraction of second stage larvae with that of the partially purified thermostable species-specific proteins showed that the heat treatment had resulted in approximately 50-fold purification of the species-specific proteins (molecular weights 30 of 20.7, 18, 21, 20.5 and 17 kD, respectively). SDS electrophoresis of 5 µg heat-treated protein showed four to five other distinct thermostable protein bands common to both nematode species. The species-specific thermostable proteins showed a high affinity for silver staining. Using 10 ng protein / * 5 5 Λ ** 4 2 w * t -i * -17- in heat-treated homogenate was sufficient to distinguish the specific proteins.

The thermostable proteins were further characterized by a combination of isoelectric focusing (in the first dimension) and SDS polyacrylamide gel electrophoresis (in the second dimension). For these two-dimensional electrophoresis experiments, the nematodes were homogenized in 10 mM tris-HCl, pH 7.4, 5% 2-mercaptoethanol (v / v).

The homogenate (60 µl) was saturated with 64 mg urea and stored at -80 ° C. After thawing, 13 µl medium consisting of 5 µl ampholines with a pH range of 3 to 10, 20 µl ampholines with a pH range of 4-6 (Biolyte, Bio-Rad), 50 µl 20% Nonidet P-40 (Sigma Chemical Company, St. Louis, MO 63178) and 25 µl of 15 2-mercaptoethanol added. The sample was centrifuged at 105,000 x g and used directly for isoelectric focusing.

The isoelectric focusing in the pH range of 4-6 was done essentially according to the procedure of O'Farrell, J. Biol. Chem. 250 (1975), 4007-4021, with the following modifications. The ampholines with a pH range of 5-7 were replaced by ampholines with a pH range of 4-6 and the gels were polymerized in glass tubes (160 mm with an internal diameter of 2 mm). The 25-50 µl samples were applied without pre-focusing. With the iso-electric focusing the following scheme was followed: 30 min 100 V; 30 min 200 V; 15 hours 300 V; and 3 hours 400 V.

In the second dimension, the proteins were separated in SDS polyacrylamide gels in the manner previously described, but with a constant current of 25 mA. Between the isoelectric focusing and the SDS electrophoresis, the gels were equilibrated in 10% (w / v) glycerol, 5% (v / v) 2-mercaptoethanol, 2.3% (w / v) SDS 35 for 10 minutes.

γν -η Λ * 'A

w, 1 * V * - · »

1 V

-18- ίη 62.5 mM tris-HCl, pH 6.8. The isoelectric points (pi) of the polypeptides were determined by measuring the pH profile of the isoelectric focusing gel with a 5 surface pH electrode (Bio-Rad). The standard deviations of these p1 values averaged 0.1 pH unit.

The two-dimensional electrophoresis patterns of total protein and of partially purified proteins not shown here showed that the 20.7 kD protein band specific for (. Rostochiensis 2-0) actually consists of two polypeptides of 20.6 kD and 20, 8 kD existed. Two-dimensional electrophoresis of an equal mixture of thermostable proteins of the two species demonstrated that there are small differences in isoelectric point 15. The (? Rostochiensis specific polypeptides of 20.6 and 20.8 kD have an isoelectric point (pi) at 5.30 and 5.20, respectively. The G. pallida specific thermostable proteins of 20.5 and 21 kD have a slightly higher isoelectric point of 5.40 and 5, respectively, 32. The 20 K rostochiensis specific polypeptide of 18 kD has a pi of 6.00 and the 17 kD polypeptide of G. pallida has a pi of 5.80.

It was noted that the differences between the pl values of the species specific proteins are constant, while the absolute pl values varied slightly between independent measurements.

The species-specific proteins did not show any detectable changes in molecular weight or isoelectric point after the partial purification, showing that the proteins were not affected by the heat treatment. The thermostable proteins showed no intraspecific variation as determined by two-dimensional electrophoresis of all populations.

55 Example II

£ 0 1 3 i? 0 y ^ V. ώ * - ί -19-

Research on proteins from eggs of potato cyst nematodes.

Potatoes of the Eigenheimer variety, which is sensitive to all pathotypes, were inoculated with cysts of pathotype Roi from Globodera rostochiensis or Pa £ from Globodera pallida, and grown in pots in a greenhouse at 18 ° C in daylight for 16 hours. Fresh cysts were extracted and triturated, and the eggs were collected on a 20 µm mesh sieve. Eggs were homogenized with a tissue mortar at 4 ° C in 10 mM tris-HCl buffer, pH 7.4, with or without 5% (v / v) A-mercaptoethanol. Homogenates were centrifuged in an Airfuge (Beekman, Palo Alto, U.S.A.) for 15 minutes at 105,000 x g, and the supernatant was collected. Protein concentrations were determined with Coomassie Brilliant Blue G250, by measuring the extinction shift at 595 nm in a Bradford spectrophotometer.

One-dimensional polyacrylamide slab gel electrophoresis was performed with 3 µg of protein per lane, essentially according to the Laemmli procedure, using a 12 or a 17.5% separation gel and a 6% stacking gel.

At least 5 headbands were found to differ consistently in electrophoretic mobility between Globodera rostochiensis and Globodera pallida in the one-dimensional polyacrylamide slab gel electrophoretic patterns prepared with 3 µg protein from homogenates of each of the two types of cyst nematodes. The proteins responsible for these bands were found to be relatively abundant, and no Globodera populations were found in which these bands were missing. In addition, in all populations studied, the differences in electrophoresis patterns were found to correlate with the taxonomic classification of Globodera rostochiensis, respectively. Globodera pallida.

Some of these species-specific proteins could be purified in an easy and simple manner.

S «C1542

• f V

-20-

When homogenates of both species were heated in a bain-marie for 10 minutes at 100 ° C, followed by centrifugation at 105,000 x g for 10 minutes, the supernatant was found to contain two of the species-specific proteins, as shown by electrophoresis experiments.

Both proteins were found to be present in significant amounts in the heated supernatant, and were found to undergo no appreciable irreversible change up to an incubation time of 30 minutes.

Both proteins were further investigated with two-dimensional gel electrophoresis using iso-electric focusing in the first dimension. The two-dimensional polyacrylamide slab gel electrophoresis was done in the manner described in Example I, using Biolyte 15 4/6 (Bio-Rad, Richmond, U.S.A.) to prepare the first dimension isoelectric focusing gel. The proteins in the gels were visualized using the silver staining technique described in Example I, or with Coomassie Brilliant Blue R250 (Bio-20 Rad).

This two-dimensional gel electrophoresis showed that one of the two bands consisted of two proteins showing small differences in molecular weight and pi, while the other band represented only one protein.

All thermostable species-specific proteins were found to differ in molecular weight and pi between the two types of cyst nematodes, as was also established in Example I. The apparent molecular weights and p1 values of these proteins are listed in Example I.

30 Recent research on protein patterns made with protein homogenates from eggs of both potato cyst nematodes has shown that the low molecular weight protein band (with a molecular weight of 17 kD for G. pallida and 18 kD for G_. Rostochiensis consists of two proteins 35 with a different isoelectric point These proteins were only found in eggs.

ISO 1 6 4 2 -21- - ·?

Pl £. pallida molecular weight 17 kD 5.6 and 5.8 Q_. rostochiensis molecular weight 18 kD 5.7 and 6.0

Conventional rabbit antisera prepared with Globodera pallida or Globodera 5 rostochiensis egg homogenates contain a component that specifically binds to the thermostable proteins of the species from which the material used for immunization was derived, as evidenced by their reaction to a nitrocellulose blot, prepared from a one-dimensional polyacrylamide 10 slab gel separation of these proteins. The transfer of proteins from the acrylamide gels to the nitrocellulose was essentially performed on the by Towbin et al.

Proc. Natl. Acad. Sci. U.S.A. 76 (1979), 4350-4354, in which the procedure to visualize the proteins was slightly modified. To detect a specific binding between the electrophoretically separated cyst nematode proteins and rabbit antisera against Globodera pallida or against Globodera rostochiensis, the nitrocellulose blots were incubated with a 1: 3,000 diluted goat anti-rabbit antiserum for 2 hours at room temperature. conjugated with HRP (horse radish peroxidase,

Bio-Rad). The blots were then treated for color development with a solution of 0.005% (w / v) 4-chloro-1-naphthol in 20 mM Tris.HCl, pH 7.2, containing 500 mM NaCl, 20% (v / v) methanol, and 0.015% (w / v) H 2 O 2. After washing in distilled water to finish the reaction, the blots were dried and immediately photographed.

To obtain rabbit antisera suitable for the detection of cyst nematode proteins on nitrocellulose blots, an amount of the homogenized egg supernatant containing 1 mg protein was mixed with an equal volume amount of electrophoretic sample buffer consisting of 10% ( w / v) glycerol, 5% (v / v) f-mercaptoethanol, 2.3% (w / v) sodium dodecyl sulfate, and 0.01% (w / v) bromophenol blue, in 62.5 mM Tris.HCl, pH 7.4. After heating for 5 minutes at 100 ° C au bain marie, 800184i ƒ -22-, the sample was briefly electrophoresed in a 6% stacking gel, which was slabbed on top of a 140 x 1.5 mm 25% separation gel with a length of 100 mm was placed. Electrophoresis was terminated as soon as the bromophenol blue had reached the separation gel, and the stacking gel was washed in an aqueous solution of 257 (v / v) isopropanol and 10 7 (v / v) ethanol. The gel was then cut and freeze-dried, and the powdered pieces were slurried with 1 ml of phosphate 10 buffered saline (PBS), pH 7.2. Half of this suspension mixed with an equal volume amount of Freund's complete adjuvant (Difco, Detroit, U.S.A.) was injected subcutaneously at various sites in the rabbit dorsal region. The same injection was administered 6 weeks after the first, but this time with Freund's incomplete adjuvant (Difco), and antisera was drained from the animals 14 days later. Despite the electrophoretically different properties of the thermostable proteins of both Globodera species, neither antisera was able to distinguish the two species due to a high degree of cross-reactivity.

Example III

Production of monoclonal antibodies a) Protein isolation

Potato cysts, washed and sieved, are grown overnight in aqua bidest in a 2% solution (w / v). The cysts are then homogenized at a concentration of 3,000 cysts per ml using a special tissue mortar as described by Seinhorst and den Ouden, Nematologica 12 (1966), 170-171. In this homogenization, the larvae and, if the procedure is done correctly, the eggs remain intact. The cyst skins are separated from the rest of the homogen

separated by sieving the suspension over a 150 µm sieve. The egg / larva suspension thus obtained is then centrifuged (approx. 1,000 x g, 5 min.), Washed three times with bidest (10 x the volume of suspension 5 at a time), and washed once with 10 mM Tris / HCl pH

7.4. After the last wash, the pellet is taken up in 10 mM Tris / HCl pH 7.4 (add about 3 x the pellet volume to buffer). The egg / larva suspension can then be homogenized in 2 ways: 10 1) Using a tissue mortar (close-fitting Teflon potter) in a 2 ml glass tube (minimum clearance) - Homogenization time 10 x 20 sec. with 10 sec. intermittently, on ice. (For quantities up to 5 ml).

15 2) Using a "french press". The egg / larva suspension is frozen in a 50 ml plastic centrifuge tube. The frozen suspension is then passed through the press 4 times (for quantities larger than 10 ml).

20

After the homogenization, the (optionally thawed) suspension is aliquoted in 1.5 ml aliquots into 1.5 ml reaction vessels and heated for 10 minutes (100 ° C, "au bain Marie"). The suspensions from the reaction vials are then combined in a 15 or 30 ml (depending on the amount) centrifuge tube, and centrifuged at 15,000 x g for 20 minutes.

The supernatant obtained consists largely of a few proteins, which are different in both types of potato cyst nematodes. This preparation may be further processed before use as an antigen, or stored at -20 ° C in a frozen or dry-frozen state. This preparation also serves as an antigen in the assays that "screen" hybridoma clones for specific antibody production.

b). Protein determination 86 0 12 ^ 2 * -24-

The protein content is determined according to Bradford by measuring the shift in extinction of Coomassie Brilliant Blue G-250 at 595 nm, after reaction with protein.

5 c) Conjugation of Antigen to KLH

required:. - Hemocyanin from Keyhole Limpets (Sigma H 2133) (= KLH) 10 - Protein from potato cyst nematodes, frozen dry from a 10 mM Tris-HCl buffer, pH 7.4, and dissolved in water at a concentration of 4 mg / ml. This solution is dialyzed overnight against SPDP buffer at 4 ° C.

- 0.1 M Sodium phosphate buffer pH 7.5 / 0.1 M NaCl (SPDP buffer).

15 - SPDP (3- (2 Pyridyldithio) propionic acid N-hydroxy succinimide ester) (Pharmacia); 40 mM solution in ethanol.

- Pd-10 columns (Pharmacia).

- Dialysis tubing (diameter 6 mm, cut off 10,000 D).

DTT (dithiothreitol).

20

200 µl SPDP (40mM) is added to 30 mg KLH in 5 ml SPDP buffer, this solution is incubated at room temperature on a "head-over-head" rotor for 30 min. The KLH is separated from the unused SPDP reagent on a PD-10 column collecting 20 fractions of 10 drops. The protein from potato cyst nematodes is also treated with SPDP, adding 100 µl of 40 µM SPDP to 2 mg in 0.5 ml.

Again, separation of free SPDP from protein 30 takes place on a PD-10 column, and 20 fractions of 10 drops are collected. The absorbance of the two columns is measured at 280 nm to determine the protein fractions. These correspond to the first peak of extinction values, the second peak being caused by the extinction that SPDP itself has at 280 nm (free SPDP).

For KLH, according to calculation, 100 to 200 coupling sites are created per molecule 8601642 -> 25. However, for the protein from potato cyst nematodes, calculation of the coupling degree is difficult because of the unknown molar extinction coefficient at 280 nm.

The SPDP coupled to KLH is now activated with DTT (dithiothreitol). DTT is added in solid form to 5 mg of SPDP-treated KLH (approx. 1.2 ml) to a final concentration of 50 mM and incubated for 5 min.

The DTT is removed on a PD-10 column (by the method described above). Now 2.5 mg (about 1.0 ml) of the KLH thus treated is added to 1 mg of SPDP-treated protein from one of the potato cyst nematodes, in about 0.4 ml and the subsequent coupling reaction is followed by the increase in absorbance of the solution at 343 nm. When the absorbance no longer increases at this wavelength (after about 45 minutes), the reaction is stopped by freezing the conjugated protein.

For use as an antigen, the conjugate is precipitated on aluminum hydroxide (alum precipitation).

To this end, the obtained conjugate is dialyzed against a buffer of 10 mM Na / K phosphate, pH 7.2; 0.15 M NaCl (PBS). 2.3 ml of 1 M NaHCO 3 is added to 2.5 mg conjugate in 5 ml of PBS, after which the solution is added dropwise with a total of 5 ml of 10% (w / v) alum (K-Al sulphate) while mixing. This mixture is incubated overnight at 4 ° C, then centrifuged at 700 x g for 15 min. After aspiration of the supernatant, the precipitate can be resuspended in any desired volume of PBS. For use as an antigen, the alum precipitate 30 is resuspended in such a way that 1 ml suspension contains 0.5 mg conjugate (this assumes that no loss of protein occurs during alum precipitation). 50 or 100 µg conjugate is injected intraperitoneally as an alum precipitate suspension per mouse and per injection.

. ^ j * λ -1. ; ·> Λ .¾ 0 y i - 4 -26- d) Immunization protocol 1) Intraperitoneal using an alum precipitate 5

For priming, 50 µg of protein conjugate is injected intraperiodoneally into a Balb / c mouse with a syringe fitted with a G25, 5/8, 16 mm needle. After a minimum of 4 weeks, a "booster" injection is given intraperitoneally with 100 µg protein conjugate. The spleen is isolated three or four days after this injection.

2) Intramiltin injection using an alum precipitate ^ In this case, the a protein conjugate is sprayed only once, as follows:

A Balb / c mouse is anesthetized with ethyl ether and the left ventral side of the animal is shaved with clippers. Usually the location of the 20 spleen is already visible. The skin is then disinfected with 96% ethanol and carefully cut + 7 mm with sharp scissors without damaging the peritoneum. Some space can now be made. between skin and peritoneum, so that the peritoneum can be easily cut, also over a length of + 7 mm, directly above the spleen. The spleen is now partly removed from the abdominal cavity with tweezers, after which the injection needle (G30, 1/2, 13 mm) is inserted into the spleen, in the longitudinal direction of the organ. With a retracting movement, a 50 to 100 µl suspension is slowly injected. The spleen is then returned to its original place.

86 0 1 6 4 2 -27- '*

The peritoneum is sutured with a linen thread (2 knots) and the skin is "stapled" with a staple (Clay Adams, woundclips / autoclips nr 7631, stainless 5 steel, 9 mm). The spleen is isolated three or four days after this surgery. (See Part f: Fusion Protocol.) E) Myeloma Cell Culture 10 The myeloma cell line is used: SP 2/0-Ag 14, SP 2/0 for short. These cells are cultured at least 5 days before a fusion. The cell line is stored in liquid nitrogen in 2 ml ampoules (1 ml cell suspension per ampoule with 5.10 or 1.10 cells 15 in freezing medium f% o (v / v) DMSO (Merck), 40% (v / v) FCS in RPMI 1640 (Roswell Park Memorial Institute) J. Before culturing the cells, one ampoule is rapidly thawed in a water bath at 37 ° C. The ampoule is then centrifuged (5 min, 470 xg) and the supernatant is aspirated. The cell pellet is then resuspended in 10 ml normal culture medium / IU IU / ml penicillin; 7.5 IU / ml streptomycin; 2 mM L-glutamine; 1 mM pyruvate; 5% (v / v) Fetal calf serum (FCS); 5% (v / v) Newborn calf serum (NCS), in RPMI 164θ] or myeloma medium (normal culture medium, supplemented with 20 µg / ml 8-azaguanine) and pipetted into a 75 cm2 bottle (Costar 3275). ° C incubator placed under an 8% (v / v) CO2 atmosphere, the cap is always turned a quarter turn to allow free diffusion with the surrounding air.The culture is inspected every 20 days, the number of living cells being determined each time by diluting a sample 1: 1 with a solution of 0.2% (w / v) trypan blue (Merck) in PBS and counting this suspension in a hemocytometer.

The cell concentration is returned to about 35 2 .105 cells / ml each day by diluting the culture with fresh myeloma medium. The culture is transferred to 150 cm2 bottles (Costar 3150) at least two days before the merger.

S 6 01642 t * -28-

The division time of the cells is 16-24 hours, depending on the growth phase and the condition of the cells.

5 f) Merger protocol

Three or four days after the last immunization, the Balb / c mouse is sacrificed by cervical dislocation.

10 The dead animal is completely disinfected externally with 70% alcohol, and then placed in the flow cabinet on a disinfected preparation table with the belly side up. With the aid of sterile tweezers and scissors, the skin on the ventral side is now cut away without damaging the peritoneum, and so far that the spleen is visible on the left lateroventral side. The peritoneum is then sprayed with 70% alcohol, after which it is cut open along the linea alba at the level of the abdominal cavity, without damaging the organs. By subsequently cutting slightly to the left, the spleen is reached, which is then carefully prepared with tweezers and scissors without damaging other organs. The prepared spleen is then rinsed in a petri dish with RPMI 1640. This procedure is repeated two more times, each time in a clean Petri dish with medium.

After the final rinse, the spleen is triturated on a nylon gauze (mesh 200) using the plunger of a 1 ml injection syringe 20 ml RPMI 1640 + 5% (v / v) FCS.

A sample is taken from the cell suspension and the spleen cell suspension is pipetted into a 50 ml polypropylene centrifuge tip tube. From the myeloma culture 2 .10? cells are pipetted into the same centrifuge tube, after which both cell suspensions are centrifuged (5 min., 470 x g, room temperature). After centrifugation, the supernatant is aspirated from both tubes and the pellet resuspended in 10 ml RPMI 1640. Cells 8 8 ö 1 δ ί - -29-

are centrifuged and washed again. During the first spin step, the sample of the spleen suspension is diluted 50 times in ΙΛ.ΙΛ M NH4CI - 0.017 M Tris / HCl pH

7.2, in which the cells are incubated for 15 to 20 minutes.

In this buffer, the erythrocytes lyse and only the nucleated spleen cells remain which are then counted.

Almost every spleen contains about 10 ^ nucleated cells. The cells of one spleen and myeloma cells are then mixed in a ratio of 10: 1, ensuring that the final volume of the suspension is 10 ml.

The cells are centrifuged again (5 min., 470 x g, room temperature), the supernatant is aspirated and the cell pellet is beaten. To the cells, 1 ml of PEG 4000 solution (50% (v / v) PEG 4000 (Merck GA) and 5% (v / v) DMSO in PBS with pH = 7.0) is added over a period of 60 sec. keeping the tube in a beaker with water at 37 ° C. After this addition, the suspension is swirled for 90 seconds, still at 37 ° C, causing a visible clumping of cells.

20 Then, in 30 sec. 3 ml RPMI-1640, then in 30 sec. 10 ml RPMI 1640, and finally in 30 sec. 36 ml of RPMI 1640 added. The fusion mixture is then incubated for 10 min in a 37 ° C oven to allow the newly formed fusion products to stabilize. The suspension is then centrifuged gently (5 min., 100 x g).

The supernatant is aspirated and the pellet resuspended in 45 ml AH medium (normal culture medium supplemented with 13.6 µg / ml hypoxanthine and 1 µg / ml azaserine) to which 10% (v / v) HECS (Costar) has been added. Twice 15 ml of this suspension are each pipetted into a 75 cm 2 bottle (Costar 3275) and incubated at 37 ° C, 8% (v / v) CO2 for 4 days. The remaining 15 ml is further diluted to a final volume of 120 ml, and with the Transplate (Costar) this suspension is distributed over twenty 96-well 35 TC plates (Costar 3596) at 100 μl / well. The outer row of pits is never filled. The pictures then become

StH) 1642

> V

-30- incubated for 7 days at 37 ° C, 8% (v / v) CO CO.

g) Culture of hybridomas 1) The 75 cm bottles 5 days after the fusion, the cell suspension is taken from the culture bottles and centrifuged in a 50 ml pointed tube (5 min., 470 x g). The cell pellet is taken up in 2 ml of freezing medium, after which the resulting suspension is divided into 2 freezing tubes (Costar 2028).

The tubes are placed in the "freezing tray" (Union Carbide) (position 5) after which the "tray" is placed on the nitrogen storage container (Union Carbide). After a few hours (minimum 2), the tubes are placed in a container and immersed in the liquid N..

2) The 96-well TC plates

After 7 days, the cultures are inspected for sterility and cell growth and 100 µl AH medium is added using e.g. the Transplate. After a further 3 to 4 days, a little medium is weighed (+ 50 µl) and 150 µl AH medium is added. After about 14 days, the growth of the cells has usually progressed to such an extent that at least 3/4 of the bottom of the wells is covered with cells.

Testing is then carried out (see part h: testing and selection protocol), and the cells in the wells excepted, except 25, are transferred to a 24-well plate (Costar 3424), with 1 ml in each well normal medium + 10% (v / v) HECS (Costar). The retained cells are used to subclone (see below under 3). After approximately 1 week of culture on the 24-well plate, with the medium half-changed every 2-3 days, there are usually enough cells (bottom at least 3/4 covered) to transfer them to a 25 cm vial. (Costar 3025), containing 8801642 -31-2 ml normal medium + 10% (v / v) HECS (Costar). The first few days, these bottles are placed obliquely with the cap on a 5 ml pipette and when the then available bottom has grown to 1/3 to 1/2 the bottles are laid flat. Until then, the bottles will be viewed every other day and possibly supplemented with medium. From the day the bottles are laid flat, they are checked daily, and if necessary ^ -0 medium is added. Once the cells are observed to grow exponentially, change to daily counting and replenishing medium reducing the cell concentration to 2-105 cells / ml. When there is 7 ml of medium in the vial, 15 and it is necessary to dilute to more than 10 ml, the cells are transferred to a 75 cm2 (Costar 3275). Once in this bottle, some ampoules (containing 2-5.10 ^ cells) are frozen as soon as possible.

3) Subcloning When cells grow in a well (from a 96-well plate, sometimes from a 24-well plate) producing antibodies that react with the antigen, these cells are often subcloned. For this purpose, using trypan blue to determine the number of living cells, 25 and then a sample of the cell culture is diluted so that 10 ml normal medium + 10% (v / v) HECS (Costar) at a concentration of 5 cells / ml is available. From a 96 well TC plate (Costar 3596), 30 wells are then filled with 100 µl (0.5 50 cell / well) medium and 30 wells with 200 µl (1 tbsp / well). The procedure followed afterwards is identical to the procedure described under 2) above.

It should be noted that a clone sometimes has to be subcloned several times, if it is suspected that cells are losing chromosomes or otherwise altering 3 0 0 1 £ 32--W W W W 'dat dat dat dat dat dat dat dat dat dat dat dat dat dat dat dat dat dat dat dat dat dat dat antil antil. 1. 1 dat 1 dat 2 dat 1' 2 dat 2 '1' 1 '1 dat 1 dat 1 terug dat loopt dat terug terug dat terug terug terug terug terug terug antil terug terug terug terug. Antil antil Even when an ELISA result suddenly decreases, this procedure is often followed from a preventive point of view.

h) Test and selection protocol

The testing and selection of hybridomas is done by means of an ELISA test. The procedure is as follows: 96 well ELlSA plates are activated by filling the wells with 100 μl of coating buffer (0.1 M 15 Na 2 CO 3 / NaHCO 3 pH 9.0) + 0.025% glutaraldehyde (BDH), then for 2 incubate in a damp box for hours at 57 ° C.

the plates are washed 5 times with tap water and dried by knocking out the drops 20 with a swishing motion.

Antigen (protein preparation of G. pallida or G. rostochiensis) is diluted to 1 µg / ml in coating buffer and the plates are filled with 100 µl per well. Incubation is done overnight at room temperature in a moist box.

- the plates are again washed 5 times with tap water and dried.

plates are filled with "post-coating buffer" (0.5% BSA (Organon TLeknika) and 0.05% Tween 80 (BDH) in PBS, 30 pH 7.2), 200 µl per well, and incubated for at least one hour, in a damp box, at room temperature.

The plates can now be stored for up to one week at 4 ° C, optionally added to the buffer with 0.02% NaN3 (Merck).

35 - the plates are again washed 5 times with tap water and beaten dry.

the plates are filled with 50 µl per well of PBS + SO1 642 -33-1.1% (v / v) Tween 80 (BDH), then supplemented with 50 µl per well of tissue culture supernatant. For the 96-well plates, this step is done using a "Transplate" 5 (Costar) in the "flow" cabinet. 100 µl of medium is then taken from one plate and tested against both types of antigen so that it can be investigated directly whether specific antibodies against either type of potato cyst nematode are produced.

The plates are incubated in a moist box for 2 hours at 37 ° C.

- the plates are washed 5 times and then beaten dry.

the plates are filled with 100 µl per well of PBS

+ 0.1% (w / v) BSA + 0.05% (v / v) Tween 80 + goat anti-15 mouse IgG (H + L) / Horse radish peroxidase (Bio-Rad, Affinity purified, 1: 3000 diluted). Incubation takes place in a moist box for 2 hours at 37 ° C.

- the plates are washed 10 times with tap water and beaten dry.

Plates are filled with 100 µl per well of

a solution of 0.04% (w / v) orthophenyldiamine (Sigma) -0.04% H2O2 (Lamers and Indemans) in substrate buffer (0.1 M sodium citrate (BDH), 0.2M KH2PO4 (Merck) pH

5.0). The plates are incubated in the dark for half an hour, the reaction is stopped by adding 50 µl 2.5 M H 2 SO 4 (Merck) to all wells.

Extinction is measured in an ELISA reader (Easy-Reader, SLT lab instruments, Austria) at 492 nm.

30

This test is performed for the first time _ + 2 weeks after merger and then approximately every week as a control.

i) RESULTS

So far, two hybridoma clones (WGP 2 and WGP 3) have been isolated producing antibodies 8001842

* · V

-34- react only with thermostable protein from £. pallida. In addition, various clones are available that react with thermostable protein from both jG. pallida as G. rostochiensis.

5 The specificity of the hybridoma clones WGP 2 and WGP 3 has been demonstrated using

1) ELISA

2) Immuno-blotting 3) Rosette test 10 ad 1) Absolute specificity was demonstrated even with slight dilution of the ascites fluid. ad 2) Performed with 1: 1 diluted cell culture supernatants on native gel blots. In all cases, the bands dyed turned out to be those bands which corresponded to an Rf of 59.5 and 65 in the case of G. rostochiensis and those of an Rf of 55 and 59 in the case of £. pallida. Naturally, the hybridoma clones WGP 2 and WGP 3 reacted with bands specific for £. pallida. However, all aspecific hybridoma clones available hitherto reacted with said protein bands of both species.

ad 3) Sheep red blood cells (SRBC) were "coated" with heat stable protein from £. pallida or G_. rostochiensis and incubated (at 4 ° C) with cells of the hybridoma clones. The antibodies located on the cell membrane bind to the antigen on the SRBC. Since a red blood cell is many times smaller than a hybridoma cell, a rosette of (small) red blood cells will arise around a (large) hybridoma cell. In the case of a hybridoma clone producing specific antibody against G. pallida protein, rosettes will only be formed when G. pallida protein "coated" SRBC are added. Ascites have been produced in Balb / c mice from both specific and nonspecific hybridoma-35 clones. The anti-'8 Q 0 1 O 4 2 λ · -35- bodies were purified from these ascites fluids using a hydroxylapatite column connected to an HPLC. For this purification, elution was performed with phosphate buffers (pH 6.8) in a gradient from 10 to 400 mM.

Fractions were collected and checked for antibody activity with an ELISA and then concentrated.

These purified antibodies can be used in a practical test to determine the nematode populations in soil samples.

Ascites of hybridoma clone WGP 2 is purified on a hydroxylapatite column (Bio-Rad) and labeled with "horse radish peroxydase" (Boehringer) according to the two-step glutaraldehyde method as described by Avrameas et al, Immunochemistry 8, 1175 (1971). The concentration of labeled antibody was 0.77 mg / ml. On a "coated" ELISA plate (described in Example III h), a 1: 1 dilution series was made of cell culture supernatants of the nonspecific reactive hybridoma clones WGR 1 and WGR 8 with both types of potato cyst nematodes. The wells 20 then contained 100 µl of diluted supernatant with a highest dilution of 128 times. 100 µl of a 5000 or 10000 diluted (labeled) WGP 2 solution was then added to all wells. The microtiter plate with both antibodies was then incubated for 2 hours in a moist box at 37 ° C, after which the plate was washed 10 times with tap water and beaten dry. The plate was filled with substrate solution (see example III h) and finally measured on the ELISA reader.

The result was that, relative to the blank 30 (WGP 2 only), a marked stimulation of the binding of the labeled specific WGP 2 antibody had occurred as shown in the table below: WGR 1 WGR 8 2 ^ WGP 2 5000x 133-215% 250 -289% (dilution 128x to lx) WGP 2 100x 180% 180% (for all dilutions) 830 1 64 2 -36-

This seems to indicate that some nonspecific clones stimulate binding to the specific clone WGP 2.

This stimulation can be useful in setting up a practical test in which the idea then goes to the following design: Two microtiter plates are "coated" with an unspecific reacting clone and are then incubated in duplicate with samples. One image is developed with a non-species-specific anti-Globodera monoclonal antibody conjugate that does not compete with the monoclonal antibody already present on the image, the other image with a conjugate of the WGP 2 monoclonal antibody. The expected reaction pattern is labeled second antibody is then as follows: a) in the case of a sample containing rostochiensis, a second antibody which is unspecific but does not compete with the first antibody will give positive reaction. A second antibody specific for G. pallida will not react.

b) in the case of a sample that G_. pallida, a second antibody which is nonspecific will give a normal reaction. A second antibody specific for G. pallida will give a reaction that is positive and enhanced by the first antibody to about 200%.

In this design, the second antibody that is nonspecific is used as a reference point for the second antibody specific for (?. Pallida.

86 0 164 2

Claims (9)

1. Proteins from the potato cyst nematodes species Globodera rostochiensis and Globodera pallida, which are species-specific and thermostable and have been isolated from eggs, larvae or adult copies of the nematodes. Proteins from eggs or larvae of the potato cyst nematodes Globodera rostochiensis according to claim 1, selected from (a) the polypeptide having a molecular weight of 20.8 + 1.0 kD and an isoelectric point of 5.20 + 0, 10; (b) the polypeptide having a molecular weight of 20.6 + 1.0 kD and an isoelectric point of 5.30 + 0.10; (C) the polypeptide having a molecular weight of 18.0 + 1.0 kD and an isoelectric point of 6.00 ± 0.10; (d) the polypeptide having a molecular weight of 18.0 + 1.0 kD and an isoelectric point of 5.70 + 0.1. Proteins from eggs or larvae of the potato cyst nematodes Globodera pallida according to claim 1, selected from (a) the polypeptide having a molecular weight of 21.0 ± 1.0 kD and an isoelectric point of 5.32 + 0 , 10; (b) the polypeptide having a molecular weight of 20.5 ± 1.0 kD and an isoelectric point of 5.40 ± 0.10; (C) the polypeptide having a molecular weight of 17.0 -1 + 1.0 kD and an isoelectric point of 5.80 860 1 54 2 Λ Υ -38- +0.10; (d) the polypeptide having a molecular weight of 17.0 ± 1.0 kD and an isoelectric point of 5.60 ± 0.1. Polyclonal and monoclonal antibodies with specificity for species specific and thermostable proteins according to claim 1 of the potato cyst nematodes species Globodera rostochiensis and Globodera pallida. Polyclonal and monoclonal antibodies with specificity for species specific and thermostable proteins according to claim 1 or 2 of the potato cyst species Globodera rostochiensis. Polyclonal and monoclonal antibodies with specificity for species-specific and thermostable proteins according to claim 1 or 3 of the potato cystal species Globodera pallida. Analysis kit for determining the potato cyst nematode populations in soil samples, comprising antibodies according to claim 5 with specificity for species-specific and thermostable proteins of the potato cyst nematodes, species Globodera rostochiensis and / or antibodies according to claim 6, with specificity for species-specific and thermostable proteins of the potato cyst nematodes species Globodera pallida, which antibodies carry a label, e.g. enzyme label, if desired. Analysis kit according to claim 7, also comprising antibodies which cross-react with species specific and thermostable proteins of the potato cyst nematodes species Globodera rostochiensis and Globodera pallida, which antibodies optionally carry a label, eg enzyme label. 9. A method for determining the potato cyst nematode populations in soil samples, wherein a protein homogenate of potato cyst nematode extracted from a soil sample, optionally after thermal treatment 55 and removal of denatured proteins, in a manner known per se, e.g. The sandwich ELISA method is tested using antibodies according to claim 5 and / or claim 6. "A i λ Λ? i. · *