002ck7519/91%3.00 + 0.00 Pergamon Press pP Society/or Parasirol0g.v

,Q 1991Australian

PURIFICATION OF GNA THOSTOA4A SPINIGERUM SPECIFIC ANTIGEN AND IMMUNODIAGNOSIS OF HUMAN GNATHOSTOMIASIS CHAMNONG

NOPPARATANA,* PRASERT SETASUBAN,* WANPEN CHAICUMPA~~ and PRAMUAN TAPCHAISRI~

*Department of Helminthology and TDepartment of Microbiology and Immunology, Faculty of Tropical Medicine, Mahidol University, Bangkok 10400, Thailand (Received 3 Junuary 199 1: accepted 23 April 199 1) AbStMCt-NOPPARATANA

C.,

SETASUBAN

P.,

CHAICUMPA

W. and

TAPCHAISRI

P. 1991. Purification of

Gnathostoma spinigerum specific antigen and immunodiagnosis of human gnathostomiasis. International Journalfor Parasitology 21: 671-687. Specific antigen of G. spinigerum which has been shown to be a protein with a relative mol. wt of 24,000 (24k) was prepared from the advanced third-stage larvae (L3) obtained from the livers of naturally infected eels. The L3 were ground and extracted with water. Purification procedures involved gel filtration, chromatofocussing and anion exchange column chromatographies, while characterization of the specific antigen was performed by sodium dodecyl sulphateepolyacrylamide gel electrophoresis (SDS-PAGE) and staining, Western blot analysis and isoelectric focussing. The specific antigen which has a pI of 8.5 was used as antigen in the indirect enzyme-linked immunosorbent assay (ELBA) to detect specific antibody in four groups of individuals, namely five parasitologically diagnosed gnathostomiasis patients (group 1); 15 clinically diagnosed gnathostomiasis patients (group 2); 136 patients with other parasitic infections (group 3); and 25 normal healthy parasite-free controls. Sensitivity, specificity and predictive values (positive and negative) of the assay were 100%. INDEX KEY WORDS: Gnathostoma spinigerum; nematodes; diagnostic antigen purification; gel filtration; isoelectric focussing; anion-exchange column chromatography; enzyme-linked immunosorbent assay; immunodiagnosis; human gnathostomiasis.

INTRODUCTION HUMAN gnathostomiasis

have shown that patients with parasitologically confirmed gnathostomiasis had serum IgG as well as IgE responses against the crude somatic extract of advanced third-stage larvae (L3) of the parasite. Due to the use of crude antigens, the assays were also positive for some patients with other parasitic infections (Suntharasamai, Desakorn, Migasena, Bunnag & Harinasuta, 1985; Dharmkrong-at, Migasena, Suntharasamai, Bunnag, Priwan & Sirisinha, 1986; Soesatyo, Ratanasiriwilai, Suntharasamai & Sirisinha, 1987; Tada, Araki, Matsuda, Araki, Akahane & Mimori, 1987; Maleewong, Morakote, Thamasonthi, Charuchinda, Tesana & Khamboonruang, 1988). As with most parasites, little is known about specific immunogen(s) of Gnathostoma spinigerum. Our preliminary work on the analysis of the crude L3 water extract revealed that the preparation was highly complex, comprising more than 40 polypeptides. Among them, 20 components were immunogenic in humans as shown by SDS-PAGE and Western blot analysis using sera of parasitologically confirmed gnathostomiasis patients. The relative mol. wt of the antigenic proteins ranged from 13 to 150k (Nopparatana, Tapchaisri, Setasuban, Chaicumpa & Dekumyoy, 1988). Our recent study on Western blot analysis and immune complexes formation between

by a nematode named Gnathostoma spinigerum is prevalent in Thailand and many Asian countries. Man acquires the disease by consuming raw meat containing infective larvae of the parasite. The common clinical features include an intermittent cutaneous or subcutaneous migratory swelling that may persist for many years. In some cases, migration of the larvae to the central nervous system results in intracranial haemorrhage or eosinophilic meningoencephalitis which can be fatal (Daengsvang, 1986). However, the clinical features are not readily distinguishable from other parasitic diseases namely angiostrongyliasis, trichinosis and cutaneous larva migrans caused by other parasites (Beaver, 1969; Bhaibulaya & Charoenlarp, 1983; Punyagupta, 1978; Kazacos, 1986). Currently, definite diagnosis of h&an gnathostomiasis can only be made following a recovery of the parasite from the infected host which is rarely successful. Attempts have been made to diagnose this disease by immunological methods which detected antibodies. However, the tests have been hampered by the complex and crossreactive nature of the antigens used. Studies by ELISA caused

3 To whom all correspondence

should

be addressed. 617

618

C. NOPPARATANA, P.

SETASUBAN,

W. CHAICUMPA and P. TAPCHAISRI

1.5

.

1.0

5 $ 0.5,-

le0

I

I

I

I

I

I

I

I

I

I

5

10

15

20

25

30

35

40

45

50

Fraction

number

FIG. 1. Elution pattern of crude L3 antigens (S,_,, in figure). The bar indicates the fraction

from Sephadex G-200 column, S,-S, collected for further purification (S,).

OD,,, = optical density at 280 nm. crude L3 extract and sera of four groups of individuals, i.e. parasitologically confirmed gnathostomiasis, clinically diagnosed gnathostomiasis, other parasitic infections and normal parasite-free controls, showed that the specific L3 component was a 24k protein (Tapchaisri, Nopparatana, Chaicumpa & Setasuban, 1991). In the present report, we purified the 24k protein from the crude water extract of L3. The purified antigen was used for detecting specific antibody in the patients’ sera. MATERIALS

AND METHODS

Preparation of crude L3 water extract. Advanced thirdstage larvae of G. spinigerum were collected from the livers of naturally infected eels purchased from local markets. The larvae were washed several times with normal saline solution (NSS) and once with distilled water, then lyophilized. The dried worms were ground in an alumina paste made in triple distilled water (TDW) containing 0.1 rnM each of phenylmethylsulphonylfluoride (PMSF), tosyl-amide-2-phenylethylchloromethyl ketone (TPCK) (Sigma Chemical Company, U.S.A.) and 10 mM-EDTA. The following steps of the antigen preparation were performed at 4’C unless otherwise stated. After the alumina was removed by low speed centrifugation (250 x g, 5 min), the supernate was centrifuged at 10,000 x g for 30 min. The supernate and the sediment were collected separately. A small volume of TDW was added to the sediment and the preparation was subjected to ultrasonication at 20 kHz for 10 min, then centrifuged at 10,000 x g for 30 min. The sediment was re-extracted one more time, all of the supernates were pooled, dialysed against TDW overnight, then lyophilized (dried crude L3 extract). Sephadex G-200 column chromatography. The crude L3

extract (47.2 mg in 3.5 ml Tris-HCl buffer pH 7.4) was applied onto a Sephadex G200 column equilibrated with 20 mM-Tris-HCl buffer pH 7.4. The proteins were eluted with the same buffer and 50 drop fractions were collected. Each fraction was monitored for protein content at 280 nm by a DU-30 spectrophotometer (Beckman, U.S.A.). The optical densities were plotted against the fraction numbers in a paper. Protein peaks were then obtained. Fractions which constituted the same protein peak were pooled, dialysed against TDW at 4°C overnight and lyophilized. Chromatofocussing column chromatography. Isolation of

the required antigenic component from the crude L3 extract was done by chromatofocussing column chromatography. The dried crude L3 extract was dissolved in a small amount of 25 mt+imidazole-HCl buffer pH 7.4 and dialysed against three changes of the same buffer at 4°C overnight. The preparation was applied to a C16/40 column of a polybuffer exchanger PBE 94 (Pharmacia, Uppsala, Sweden) and equilibrated with polybuffer 74 (Pharmacia LKB, Uppsala, Sweden). A linear pH gradient was set from 7.4 to 4.0. The flow rate was 60 ml h ’ and the pH increment per 5 ml fraction was 0.03 units. Optical densities of the fractions were determined at 280 nm. As for fractions of the G-200 column, the optical densities of the fractions were plotted against the fraction numbers to construct protein peaks. Then fractions of the same protein peak were pooled and dialysed against saturated ammonium sulphate to remove the polybuffer while concentrating the antigens. Anion exchange column chromatography. Protein profiles from Sephadex G-200 and chromatofocussing column chromatographies which contained the 24k protein were pooled and applied to a column of DE 52 (Whatman) equilibrated with 20 mt+Tris-HCI buffer pH 7.4. A linear salt gradient was set from 20 mM-Tris-HCl buffer pH 7.4 to 0.5 M-NaCl using an IS ultragradient mixer. Elution of

G. spinigerum antigen

purification

679

94

67

30 24~ 20

FIG. 2. SDS-PAGE and Coomassie blue staining of L3 antigen preparations. Numbers at left indicate mol. wt ( x 103). A: MW markers; B: crude L3 extract; C: S,; D: S,; E: S,; and F: S,. proteins was carried out at a rate of 50 ml h-’ and 50 drop fractions were collected. Optical densities of the fractions were determined by spectrophotometry at 280 nm. Fractions of the same protein peak were pooled, dialysed against TDW at 4°C overnight and lyophilized. Serum specimens. A total of 18 1 serum specimens were used. They were five samples from parasitologically confirmed gnathostomiasis patients (group 1); 15 clinically diagnosed gnathostomiasis patients (group 2); two angiostrongyliasis patients, nine patients with trichinosis, 16 patients with paragonimiasis, 10 patients each with schistosomiasis and opisthorchiasis, 12 patients each with taeniasis and hookworm infection, eight patients with strongyloidiasis, three patients with invasive amoebiasis, 17 patients with malaria, 31 patients with mixed parasitic infections and one patient each with sparganosis, cysticercosis, filariasis, ascariasis, capillariasis and trichuriasis (group 3); and 25 normal healthy, parasite-free

controls, 14 from Thailand and 11 from Sweden (group 4). Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot analysis. SDS-PAGE was used for separation of the crude L3 extract and other antigen preparations. The method of Laemmli & Favre (1973) was followed throughout. Western blot analysis was performed as described previously by Burnette (198 1) with modifications. The antigens were fractionated by SDS-PAGE and elcctroblotted onto nitrocellulose paper. The nitrocellulose sheet was submerged in blocking solution (2% bovine serum albumin [BSA] in PBS) for 30 min, washed in PBS containing 0.05% Tween 20 and 0.2% gelatin to remove excess BSA, and then allowed to react with the patient’s serum at dilution 1:200 for 1 h at 25°C on a rocking platform. After washing, the sheet was put in a solution containing 5 x lo5 cpm ml ’ of ‘251protein A. Staphylococcal protein A (Phannacia, Uppsala, Sweden) was radiolabelled with ‘*‘I by the iodogen method

C. NOPPARATANA. P. SETASUBA~. W. CHAICUMPAand P. TAPCHAISRI

680

- 8.0

0

10

20 30

40

50

60

70 80

Fraction FIG. 3. Elution pattern fractions were collected.

90 100 110 120 130 140 150 1 1 mol If’ number NaCl

of crude L3 antigens from chromatofocussing column. Eight i.e. C,-C, (C, *,,, in figure). ---: pH. The bar indicates the fraction collected for further purification (C,).

described by Paus, Bormer & Nustad (1982). The ‘251-protein A was allowed to bind the antigen-antibody complexes on the nitrocellulose sheet at 25°C for 30 min. The sheet was washed to remove unbound “‘I-protein A and dried on a filter paper. After the paper dried, autoradiography was performed by placing the nitrocellulose on a paper screen and exposing it at - 70°C to Kodak X-O mat RP film with an intensifying screen. The film was developed according to the instructions of the manufacturer after 18-24 h of exposure. Isoelectricfocussing (ZEF). Analytical IEF of the protein components was performed in thin-layer (0.7 mm) polyacrylamide gel cast in a flat-bed electrophoresis unit FBE (Pharmacia, Uppsala, Sweden). The method described by Righetti & Chellemi (1987) was used with modifications. The gel (5% T, 3% C) containing 2% ampholine pH 3.5-10.0 (Pharmacia LKB) and 4 M-urea was cast on a glass plate. After complete polymerization of the gel, the plate was placed in an FBE 3000 flat-bed electrophoretic apparatus (Pharmacia, Uppsala, Sweden). One molar phosphoric acid and 1 M-NaOH were used as an anolyte and catholyte, respectively. The electrofocussing was initially performed at 700 V for 1 h. Each protein sample to be electrofocussed was applied (l&15 ~1) on a piece of 5 mm x 7 mm Whatman no. 3 filter paper which was placed 2.5 cm from the anode. Electrofocussing was continued with 1000 V for 3 h. The gel was then removed for silver staining (Oakley, Kirsch & Morris, 1980). The pH gradient of individual protein was worked out by comparing its location with locations of proteins of the standard p1 calibration kit (pH 3.0-10.0) run concurrently in the same gel. Indirect enzyme-linked immunosorbent assoy ( ELISA j

Each well in a microtitre plate was coated with 100 ~1 of appropriate antigen at appropriate concentration made in carbonate-bicarbonate buffer pH 9.6. The plate was incubated at 37°C for I h then overnight at 4°C. After the excess antigen had been removed by washing with the PBS Tween 20 (PBST), the wells were blocked with 1% BSA made in PBST at 37°C for I h. Excess BSA was removed then 100 ~1 of the serum sample to be tested at dilution 1:320 was added to the appropriate well. The last wells in each plate had PBS added to serve as blanks. The antigen-antibody reaction was allowed to occur at 37°C for 1 h, then washed with PBST. Rabbit anti-human IgG-horseradish peroxidase conjugate (Dakopatt, Denmark) at dilution I: 1000 was added to each well and incubated at 37°C for I h. After the unbound conjugate was removed by washing, 100 ~1 of freshly prepared peroxidase substrate was added to each well. The plate was kept at room temperature in the dark for 30 min, then the reaction was stopped by adding 50 ~1 of 1 N-NaOH. The optical densities of each well were determined at 405 nm by an ELISA reader (Uniskan II, Labsystem). The precision of the assay was evaluated by testing, on different occasions. the same positive serum (a pool of the four parasiteconfirmed serum samples) for 30 times. The optical densities were determined and per cent coefficient of variation (% CV) was calculated from the formula: Standard

deviation x 100.

Mean of optical densities The % CV was found to range from 5.4 to 6.3 when different preparations of antigens were used which indicated high reproducibility of the assay (Palmer & Cavallaro, 1980).

G. spinigerum antigen purification

ABCDEFGHI

681

J

FIG. 4. SDS-PAGE and Coomassie blue staining of crude L3 extract and its chromatofocussing profiles. Numbers at left indicate mol. wt (x IO’). A: MW markers; B: crude L3 extract; C-J: C,-C,, respectively. Statistical analysis. The sensitivity, specificity and the predictive values were calculated using the method of Galen (1980). RESULTS

The crude L3 extract containing 47.2 mg proteins was applied onto a Sephadex G-200 column equilibrated with 20 mM-Tris-HCJ, pH 7.4. Four protein profiles namely S,, S,, S, and S, were obtained (Fig. 1). The protein contents of the four profiles were 13.9, 5.6, 5.6 and 16.7 mg, respectively. Thus the total protein recovery was 88.6%. The 24k protein was confined to the S, profile as revealed by SDS-PAGE and Coomassie blue staining (Fig. 2). Another batch of crude L3 extract containing 38.8 mg proteins was subjected to chromatofocussing

column chromatography. Eight peaks of proteins namely C,-C, were obtained (Fig. 3) and the protein recoveries of the peaks were 5.4, 1.0, 8.9, 8.0, 6.7, 1.8, 3.0 and 2.9 mg, respectively. The total protein recovery was 97.1%. The 24k protein was not adsorbed to the column and was found in the exclusion peak, i.e. C, (as checked by the SDS-PAGE and Coomassie blue staining). The C, also contained 18 more proteins with mol. wts ranging from 13 to 63k (Fig. 4). The S, profile of Sephadex G-200 column chromatography and the exclusion peak (C,) of the chromatofocussing which contained the 24k protein were pooled, dialysed against TDW overnight at 4°C and lyophilized in two equal aliquots. One aliquot (5.5 mg) was used for other experiments, i.e. SDS-PAGE, Western blotting, IEF, while another aliquot (5.5 mg)

682

C. NOPPARATANA,P.

SETASUBAN,

W. CHAICUMPA and P. TAPCHAISRI

0.70 t 0.60 -

O”O -0 0

5

10

15

20

25 30 35

40

45

50

55

60

65

70

Fraction number FIG. 5. Elution of pooled S, and C, from DE52 column. Three fractions, D,-D, (D,~,,, in figure) were collected. 0 : Absorbance at 280 nm; --: concentration of NaCl The bar indicates fraction containing the 24k protein.

was dissolved in 20 mM-TrisPHC1 buffer pH 7.4, applied onto the DE52 column and eluted with a linear salt gradient from 20 mM-Tris-HCl buffer to 0.5 MNaC 1. Three peaks, namely D,-D,, of proteins were eluted from the column (Fig. 5). Each protein pool was dialysed against TDW and lyophilized. The protein recoveries of the three peaks were 0.1, 1.3 and 0.5 mg, respectively. The total recovery was 34.5%. SDSPAGE and Coomassie blue staining of the D, profile which was eluted between 0.05 and 0.15 M-NaCl in Tris-HCl revealed that this peak of the DE52 column chromatography contained predominantly the 24k protein and a trace amount of a 17.5k protein (Fig. 6). Table 1 summarized yields of the purification process. The crude L3 extract, the eight protein profiles obtained from chromatofocussing column chromatography ((Z-C,) and the first DE52 profile (D,) were characterized by isoelectric focussing followed by silver staining (Fig. 7). The standard p1 proteins were included in the gel (lanes A, F and M). It was found that the p1 of the crude L3 extract ranged between 4.5 and 9.7 (lane B). The 24k protein had ~18.5 as shown in lanes C (C, profile) and L(D, profile), respectively. Lanes D, E and G-K were proteins of C,, C, and C,C,, respectively. The immunogenicity of the 24k protein in the crude L3 extract, C, and D, profiles was confirmed by Western blot analysis against the serum of one of the five parasitologically confirmed human gnathostomiasis. As shown in Fig. 8, the serum contained a high concentration of specific antibody to the 24k protein. With the use of the same serum, it was revealed that the C, contained two other immunogenic

components, one of which was a 39k protein (this component was also found in the C, SDS-PAGE and Coomassie blue staining; Fig. 4) while another component was a 50k non-protein component (as it was not present in C, SDSPAGE and Coomassie blue staining but could be visualized after Con A and silver staining). The D, contained two immunogenic components which were the 24k protein and the 50k non-protein moiety. The 17.5k protein in the D, fraction was not immunogenic. The crude L3 extract, C, and D, were used as antigens (at concentration 2.5 pg ml-’ carbonatebicarbonate buffer) in the indirect ELISA for detecting specific antibody to G. spinigerum in the four groups of serum specimens. Table 2 shows means (a, standard deviations (s D) and ranges of optical densities at 405 nm of all sera tested at dilution 1:320. When the crude L3 extract was used in the ELISA, the X, s D and ranges of the sera of the 14 normal healthy Thais were 0.069, 0.097 and O-0.292, respectively. These figures were significantly higher than those of the normal healthy Swedes which were 0.008, 0.006 and O-0.018, respectively (P O.OS). Thus sensitivity, specificity and positive and negative predictive values were calculated using the 8 + 4 s. D.of all normal individuals (14 Thais + 11 Swedes) which was 0.101 as the cut-off limit between positive and negative gnathostomiasis. It

was found that all of group 1 (lOO%), nine of group 2 (60%), 14 of group 3 (8.7%) and none of group 4 (0%) were positive. The sensitivity, specificity and positive and negative predictive values were 100, 91.3, 22.2 and 100%, respectively (Table 3). The D, preparation which contained mainly 24k protein and trace amounts of 17.5k protein and 50k non-protein antigen gave the best results of ELISA. When the D, was used as the antigen in the assay, no significant difference was observed on 2, s D. and ranges of optical densities between the normal Thais and the normal Swedes (P > 0.10). Thus the 8 + 4 s D of all of the normal healthy individuals of group 4 which was 0.051 was used as a cut-off limit between positive and negative gnathostomiasis. It was found

G. spinigerum antigen

685

purification

ABCDEFGHIJKLM FIG. 7. Isoelectric focussing A, F and M : pI protein

TABLE

and silver staining of L3 antigen preparations. markers; B : crude L3 extract; C-E and G-K

Numbers at left indicate pl : C,-C, and C,&; L : D,.

ELISA

~-~ENSITIVITY.S~EC~F~C~TYANDPOSITIVEANDNE~;ATIVEPREDICTIVEVALUESOFTHE

L3

cut-off Antigen

OD

Crude L3 extract C, D,

0.457 0.354 0.102 0.051

EXTRACT,C,

AND

D,

Sensitivity (%)

WHENTHECRUDE

WEREUSEDASTHEANTIGENS

Specificity (%)

+ Predictive value (X)

- Predictive value (%)

100

96.9

44.4

100

100 100 100

90.7 91.3 100

21.1 22.2 100

100 100 100

OD = optical density.

1 (loo%), seven of group 2 (46.7%) none of groups 3 and 4 were positive. The sensitivity, specificity and the positive and negative predictive values were all 100% (Table 3). that and

all of group

DISCUSSION Eighty-six milligrammes of the crude L3 extract was applied to the Sephadex G-200 and chromatofocussing column chromatography, 11 mg of the pooled S, and C, fractions was obtained. When half of the pool (5.5 mg) was subjected to the DE52 anion exchange column chromatography, only 34.5% of the protein sample was recovered. Low protein yield was due to

the fact that most of the proteins in the S, (data not shown) and some of the proteins in the C, fraction (Fig. 7) were negatively charged. Their p1 values were less than the pH of the buffer (pH 7.4) used for equilibrating and eluting the proteins. These negatively charged proteins thus bound firmly to the anionic cellulose. The phenomenon favoured the elution of the positively charged proteins (p1 > pH) including the 24k moiety which has a pI at 8.5. The D, peak of the DE52 column, besides containing the 24k protein, was found to have another protein with the relative mol. wt 17.5k, when the preparation was subjected to SDS-PAGE and Coomassie blue

686

C. NOPPARATANA,

P.

SETASUBAN, W. CHAICUMPA and P. TAPCHAISRI

FE. 8. Reactivities of the serum of parasitologically confirmed gnathostomiasis patient against crude L3 extract (A), C, (B) and D, (C) in Western blot analysis. Numbers at left indicate mol. wt ( x IO’).

staining. However, Western blot analysis revealed that the 17Sk protein was not immunogenic in human as it did not react with the serum of the parasitologically confirmed gnathostomiasis. Instead, D, was found to contain a component located at the 50k region which did not show up in the SDS-PAGE and Coomassie blue staining but did appear in the Western blotting and could be revealed by Con A and silver staining. Thus the component was a carbohydrate antigen. The yield of the D, from DE52 column chromatography of 5.5 mg of pooled S, + C, was only 100 pg. Thus it was only 0.23% of the original crude L3 extract. Owing to this low D, yield, several batches were then prepared and pooled. The pool of D, was

used in SDS-PAGE, Western blot analysis, electrofocussing and indirect ELISA. It was found that the ELISA was more sensitive than the Western blot analysis. Seven of the 15 patients of group 2 who were clinically diagnosed as gnathostomiasis (they presented with intermittent cutaneous migratory swellings and had a history of eating raw meat) gave positive reaction in ELISA though the serum dilution used in the test was as high as 1:320, while only five of them were positive by Western blot analysis (at serum dilution 1:200). Although parasites could not be recovered from those of group 2 whose ELISA were negative, the possibility exists that their intermittent cutaneous migratory swellings might be

G. spinigerum antigen purification the larva migrans due to some other parasites. With the limitation in numbers of the parasitologically confirmed gnathostomiasis patients (group 1) and the sera for testing cross-reactivity (group 3), however, our ELISA using D, as the antigen was presented with 100% sensitivity, specificity and predictive values. In addition, at the the serum dilution 1:320, the cut-off optical density for positive reaction of ELISA was 0.051. This end point could be monitored by eye as the colour was clearly different from the negative reaction which had no background colour of the serum at the dilution used. This is particularly useful for most diagnostic laboratories in the developing parts of the world where public health problems due to parasitic infections are real and where the ELISA readers are not readily available. Acknowledgements-The study was financially supported in part by a Mahidol University Research Grant. The authors thank Professor Dr Santasiri Sornmani, Dean of the Faculty of Tropical Medicine, Mahidol University, Bangkok, for his enthusjasm, encouragement and support by endorsing the maior part of the financial support. Thanks are due also to Dr Marianne Faehlmann, Kalkar Hospital, Sweden, for the supply of normal Swede sera. Efficient typing of the manuscript by Miss Thanya Nirantasook is acknowledged.

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BURNETTE W. N. 1981. Western blotting: electrophoretic transfer of proteins from sodium dodecyl sulfatepolyacrylamide gels to unmodified nitrocellulose and radiographic detection with antibody and radioiodinated protein A. Analytical Biochemistry 112: 195-203. DAENGSVANC~ S. 1986. Gnathostoma spinigerum and human gnathostomiasis: a brief review. In: The 25th Anniversary of the

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NOPPARATANAC., TAPCHAISRIP., SETASUBANP., CHAICUMPA W. & DEKUMYOYP. 1988. Antibody responses in human gnathostomiasis. Southeast Asian Journal of Tropical Medicine and Public Health 19: 2 19-224. OAKLEY B. R.. KIRSCH D. R. & MORRIS N. R.

1980. A simplified ultrasensitive silver stain for detecting proteins in polyacrylamide gels. Analytical Biochemistry 105: 361363. PALMER D. F. & CAVALLAROJ. K. 1980. Some concepts of quality control in immunoserology. In: Manual of Clinical Immunology, 2nd edition (Edited by ROSE N. R. & FRIEDMAN H.), pp. 1078-1082. American Society for Microbiology, Washington, D.C. PAUS E., BORMER0. & NUSTAD K. 1982. Radioiodination of proteins with the iodogen method. In: Radioimmunoassay and Related Procedures in Medicine, Proceedings of an International Atomic Energy Agency Symposium, Vienna, Austria. PUNYAGUPTA S. 1978. Clinical manifestation of eosinophilic meningitis (Gnathostoma spinigerum). Southeast Asian Journal 9: 278.

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RIGHETTI P. G. & CHILLEMI F. 1978. Isoelectric focusing of peptides. Journalof Chromatography 157: 243-251. SOESATYOM. H. N. E., RATANASIRIWILAI W., SUNTHARASAMAI P. & SIRISINHA S. 1987. IgE responses in human gnathostomiasis. Transactions of the Royal Society of Tropical Medicine and Hygiene 81: 799-80 1. SUNTHARASAMAI P., DESAKORNV., MIGASENAS., BUNNAG D. & HARINASUTAT. 1985. ELISA for immunodiagnosis of human gnathostomiasis. Southeast Asian Journal of Tropical Medicine

and Public Health 16: 274-279.

TADA I., ARAKI T., MATSUDA H., ARAKI K., AKAHANE H. & MIMORI T. 1987. A study on immunodiagnosis of gnathostomiasis by ELISA and double diffusion with special reference to the antigenicity of Gnathostoma doloresi. Southeast Asian Journal of Tropical Medicine and Public Health 18: 444448. TAPCHAISRIP., NOPPARATANAC., CHAICUMPAW. & SETASUBAN P. 1991. Specific antigen of Gnathostoma spinigerum for immunodiagnosis of human gnathostomiasis. International Journal,for Parasitology 21: 3 15-3 19.