CA1340196C

CA1340196C - Immunodiagnostic method for neurocysticercosis

Info

Publication number: CA1340196C
Application number: CA000611731A
Authority: CA
Inventors: Banda V. Ravi Kumar; Vepa Suryanarayana; Vasanthapuram Ravi; Akepati Chandramukhi
Original assignee: Astra AB
Current assignee: AstraZeneca AB
Priority date: 1988-09-19
Filing date: 1989-09-18
Publication date: 1998-12-15
Anticipated expiration: 2015-12-15
Also published as: GB8920931D0; AU617526B2; GB2223757A; AU4142989A; GB2223757B

Abstract

Disclosed is the use of excretory/secretory (E-S) protein antigens of Cysticercus cellulosae in immunodiagnosis of neurocysticercosis. The antigens are substantially free of cross-reactivity with tuberculous meningitis antigen. Also disclosed are methods for obtaining such antigens and methods for diagnosing active human neurocysticercosis using the antigens.

Description

13~0196 Background of the Inventlon Fleld of Inventlon:
Cystlcercus cellulosae, the larval form of the tapeworm Taenla sollum can lnfest human braln, leadlng to serlous neurologlcal syndromes collectlvely descrlbed as Neurocystlcercosls (NCC). Dependlng on the lntra cranlal locatlon of cysts of Cystlcercus cellulosae the followlng cllnlcal presentatlons are often seen.
Locatlon Clinlcal sYmptom Sequelae Braln Parenchyma Epllepsy Space occupylng Motor or leslon (SOL) Sensory deflclt Psychosls Encephalltls Menlnges Chronlc Menlngltls Hydrocephalus Cranlal nerve palsles Motor & Sensory deflclt Ventrlcles SOL Hydrocephalus All the cllnlcal symptoms descrlbed here can be caused by many aetlologlcal factors maklng lt dlfflcult to dlagnose the actual dlsease responslble for the symptoms. A common mlstake ls to dlagnose neurocystlcercosls as a Mycobacterlum tuberculosls lnfectlon of the nervous system. Dlagnosls of neurocystlcercosls ls made based on radlologlcal examlnatlon of the skull. Dead calclfled cysts appear as dense radloopaque leslons on the X-ray of the skull, while the llve cysts appear as hypodense or hyperdense leslons on computerlzed tomography (CAT-SCAN~.

- t3401 96 Recently magnetic resonance imaging (MRI) of central nervous system (CNS) has been applied to visualise the live cysts.
Identification of a cyst in any of these imaging procedures largely depends on the size of the cyst and the reaction of the surrounding host tissue. In many developing countries where the disease is prevalent, imaging facilities are not easily accessible, creating an acute necessity for an alternative, simpler method for the diagnosis. Thus, there is a need for an immunodiagnostic test that can distinguish neurocysticercosis from other neurological illnesses. The majority of existing immunological tests aim at detection of antibodies against antigens of the organism being identified. Moreover, many tests have been developed as sero diagnostic tests to be used in epidemiological surveys and these existing tests were not designed for a very specific diagnosis of neurocysticercosis.
The specificity and sensitivity of the test largely depends on the antigen which is used in the test. The present invention describes a method of preparation of antigens of Cysticercus cellulosae that can confer specificity and sensitivity to immunodiagnosis.
In the following description reference is made to various figures, of which:
Figure 1 shows the separation of extracts from cysts by gel filtration;
Figure 2 shows the results of sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of various proteins;
Figure 3 is an autoradiogram of excretory~secretory proteins labelled with 5S-methionine;
Figure 4 is an autoradiogram of 1 I-labelled excretory/
secretory protein immunoprecipitates with CSFs of patients; and Figure 5 is an autoradiogram of S-labelled excretory/
secretory protein immunoprecipitated with CSFs of patients with various neurological disorders.
Antiqen Preparation and Immunodiagnosis of the Prior Art:
Crude extracts of the larvae of T.solium were prepared as total homogenates of a cyst (1), or homogenates of the solid parts of the cysts i.e. bladder wall and the scolex or the bladder wall alone (2). In certain cases vesicular fluid was also used as an antigen (3). The homogenates were usually subjected to a centrifugation to remove cell debris, nuclei etc at 15000-20000 x g for 15 to 30 minutes. The homogenate was extracted with acetone C 3a - 1340~ 96 to remove the ma~orlty of llplds and was used ln a complement flxatlon test (4). The crude or total antlgens were used in many types of lmmunoassays such as passlve haemagglutlnatlon tests (5 &
3), lmmunoelectrophoresls (1) and enzyme llnked lmmunosorbent assays [(ELISA) (1,6 and 7).~
Uslng a total homogenate of the cyst, Kuhn et. al. (8) descrlbed an ELISA and attrlbuted the sensltlvlty of the test to a set of antlgens among the protelns. These antlgens were nelther purlfled nor tested for thelr speclflclty. The total homogenate contalns antlgenlc llplds and carbohydrates ln addltlon to the protelns vlsuallsed and ldentlfled as lmmunogenlc on western blottlng. The contrlbutlon of the non proteln antlgens to the total sensltlvlty of the ELISA was not verlfled. A slngle antlgen ldentlfled as antlgen B exhlbltlng the propertles of flbronectln was used ln ELISA but the sensltlvlty was not satlsfactory l9).
Total homogenate of the paraslte presents several dlsadvantages as antlgen ln lmmunodlagnosls. (1) It contalns several epltopes havlng lmmunologlcal cross reactlvlty wlth other CNS pathogens (vlde lnfra). (11) It has been shown that several parasltlc helmlnths share cross reactlve antlgens (10). In developlng countrles such as Indla, Mexlco, Brazll, Chlna, Thalland and countrles ln Central Amerlca and Asla where parasltlc lnfestatlon among the populatlon often ls very common, a conslderable number of people harbour serum antlbodles to the cross reactlve antlgens. When such lndlvlduals are affected by dlseases whlch breach the blood braln barrler, the serum antlbodles can enter cerebrosplnal fluld (CSF) and lnterfere wlth dlagnosls of cystlcercosls, glvlng false posltlve results.

~ 13401 96 In vlew of the lack of speclflclty of total homogenates, attempts were made to ldentlfy and purlfy specles and tlssue speclflc antlgens vlz., antlgens derlved from scolex and veslcular fluld. Two polypeptldes of apparent mol wts of about 26,000 and about 8,000 were ldentlfled as specles speclflc antlgens of Taenla sollum. These antlgens reacted ln an lmmunoblot wlth serum obtalned from patlents wlth Taenla sollum lnfectlon and not wlth sera of patlents wlth other helmlnthlc infestatlons (11). The sensltlvlty of these antlgens or speclflclty wlth respect to other related CNS dlsorders ls not yet verlfled. An antlgen purlfled from veslcular fluld uslng a monoclonal antlbody ralsed agalnst total protelns of the veslcular fluld proved very speclflc ln lmmunodlagnosls but lacked the requlred sensltlvlty to be useful ln detectlon of NCC (12). Scolex speclflc protelns of apparent mol wts of about 80,000 and about 10,000 purlfled uslng monoclonal antlbodles conferred 100% sensltlvlty to serodlagnosls of cystlcercosls. It was clalmed that these antlgens were useful to dlscrlmlnate between taenlasls and cystlcercosls. Its usefulness ln dlscrlmlnatlng neurocystlcercosls from other systemlc cystlcercosls was not tested (13).
The dlsadvantage of uslng the total homogenate as antlgen ls further lllustrated by a study conducted at the laboratory of the lnventors (14). Thls study ls summarlsed below:
Cysts were homogenlsed and sonlcated (8x30 second pulses). Homogenates solublllsed ln 1% SDS and treated wlth 1%
mercaptoethanol at 100~C were sub~ected to gel flltratlon on Sephacryl S-300* column and were resolved lnto three fractlons.
*Trade mark -The three peak fractlons obtalned by gel flltratlon were analysed on 10% SDS-PAGE as shown ln Flgure 1. Lane 1:3rd peak Lane 2:2nd peak, Lane 3: 1st peak. Lane 4:Mr markers (94,67,43,30,20.1 & 14.1 KDa). The Mr ranges of the three fractlons are glven below:
Fractlon I : cyst antlgens of mol.wt ~ 66 K Daltons Fractlon II : cyst antlgens of mol.wt between 25 K-66 K Daltons Fractlon III : cyst antlgens of mol.wt between 14 K-35 K Daltons ELISA, uslng the above three fractlons of cystlcercal antlgens was performed ln the followlng way:
150 CSFs collected randomly from varlous neurologlcal patlents were tested agalnst these three fractlons. Antlgens were coated on 96 well mlcrotltre plates of Dynatech* at concentratlons of 5 yg/ml (100 ylJwell) ln 50 mM blcarbonate buffer pH 9.6 overnlght, quenched wlth 2% BSA and CSFs were tested at 1/25, 1/125 and 1/625 dllutlons. Bound human IgG was revealed uslng rabblt antl-human IgG peroxldase con~ugate (Slgma, 1:1000 dllutlon). Orthophenylene dlamlne was used as chromogen (4 mg/10 ml) and 0.3% H2O2 as substrate.
Table 1 summarlses the results of these 150 samples.

*Trade mark + ~ ~ ~
o o ++ ~ , ~ , , , , ~ q~
, ~ ~ o + ' ~
4 + ~ I ~) I I I I ~a o o + I ~ u~ ~ I I I C a~
o ~r e ~,, o + ~ ~ _I ~ I I I V
.,, + ~ ~
~~ H ~1 0 U
0 + ~ ~ ~ I I I I
S~ +
+ 0 O
+ I ~1 ~ ~ L _I
~ O
C CO I
O + l ~ l .,1 + I ~ U~ Ul I I I ~ 0 ~11 + nl CU ~J
+ ~'~
+
+ ~O U~ ~ I I I I ~ ~
c b ~ ~ 0 ~. +
o lu r + .
O ' ~ ~10 o .1 ~ ~ o ~ ~e~
Z ~ ~ o ~o ~ U) 0 ~ ~: CU o ~
0 0~ f ~ r ~
C C o u~
o J e ~
Z ~ ~ ~ ~ I ., o U ~U
' 40 ~ 0 ~0 I t~ ~~nCO ~ O
~~ E~ e :~ ,~ h. ~ . ~ ~ ~
CO"C ~~ r J ~~ 0 ~
r~ o ~ r~
U ~ ~~ -r ~ CO 0 ~
0 ~ r _1 ~4 .C ~ O
~., ~O _, - , , u ~ a o 1~ 0 ~ C~ U
~ U O :~ ~ ~ C
U O ~~ ~I CqJ~ ~ ~0 P~ + CU 0 f' C ~ cU + J~ + ~ + ~ ~U
o ) ~,~ ,/ r' I ~u ~ + ~~
J ) ~~
04 ~: ~u + U ~.
,~ O +
~n z ~1 ~ ~ ~ + + t) .. ~ .. . .

13~0196 , .

Followlng concluslons were drawn from thls data.
Though the test was sensltlve enough to recognlse all cases of neurocystlcercosls, the maxlmum false posltlve results were glven by the cases of tuberculous menlngltls. Therefore, the test ls not useful wherever both dlseases are prevalent.
Reactlon of tuberculous menlngltls CSFs with cyst antlgens was shown to be due to cross-reactlvlty between mycobacterlal antlgens and the cyst antlgens.
1. Mycobacterlum tuberculosls sonlcates lnhlblted the blndlng of cystlcercosls posltlve CSFs to cyst antlgens but dld not totally abollsh the reactlon.

2. Thls lnhlbltlon lncreased wlth lncreaslng concentratlons of mycobacterlal antlgens.

3. Rabblt antlserum ralsed agalnst mycobacterlal sonlcates reacted wlth cyst antlgens.
The above observatlons lllustrate that crude antlgens lndeed contaln epltopes that cross react wlth other CNS pathogens.
Cystlcercl are multlcellular organlsms that cannot be phagocytlsed and processed by macrophages or granulocytes or other antlgen presentlng cells unllke the unlcellular mlcroorganlsms.
Therefore, the human lmmune system rarely gets sensltlsed to all the protelns of the parasltes, the more so ln an lmmunologlcally prlvlleged compartment llke CNS. So, the antlgens that are secreted or excreted or shed by these parasltes lnto body flulds of the host (termed as E-S antlgens) are lmmunologlcally very lmportant. E-S antlgens of many parasltlc helmlnths were obtalned by malntalnlng varlous developmental stages of the helmlnths ln vltro ln tlssue culture medla and purlfylng the antlgens from the 13~019~
, growth medla (15). Cystlcercus cellulosae were also malntalned ln vltro wlth serum and tlssue extracts (16). On keeplng these parasltes for short perlods of tlme ln Dulbecco's mlnlmal essentlal medlum (DMEM) or phosphate buffered sallne (PBS) and pulslng these parasltes wlth 14C labelled amlno aclds, lt was shown that they were metabollcally actlve (17). However, only negllglble amounts of radloactlvely labelled protelns were detected ln the medlum. The utlllty of these antlgens ln lmmunodlagnosls was never tested.
Detalls of the Dlsclosure or Inventlon~
The present lnventlon ls based on the premlse that E-S
antlgens of cystlcercus cellulosae obtalned by malntalnlng the parasltes ln vltro may be closely related to the antlgens that come out of cyst as a part of cyst metabollc processes ln vlvo.
Thus they may confer speclflclty and sensltlvlty to the lmmunodlagnostlc tests deslgned to detect antlbodles ln the body flulds of the patlents, such as CSF and serum. The same antlgens or some degraded products of these antlgens may be found ln CSFs of the patlents. Thls would form the basls for deslgnlng lmmunodlagnostlcs based on antlgen detectlon. It ls understood that lt ls posslble to develop polyclonal monospeclflc or monoclonal antlbodles agalnst the E-S antlgens whlch then could be used ln such an antlgen detectlon test. As some of these antlgens may confer protectlve lmmunlty to the host these antlgens may be used for developlng vacclnes to neutrallze the lnfestatlons by cystlcercus cellulosae.
By the present lnventlon we descrlbe a novel method of malntalnlng cystlcercus cellulosae ln serum free cell culture 13~0136 . ~

media for extended perlods of time and using the antlgens elaborated by the parasite into the medium for immunodiagnosis of NCC involving interaction of CSF of the patients with the E-S
antigens. The emphasis here is the use of the antlgens ln ldentifying NCC wlthout any interference by other forms of systemic cysticercosis. The novel method of diagnosis described may also be useful ln dlagnoslng cystlcercus cellulosae lnfestation of pigs intended for human consumptlon.
Source and malntenance of cysts:
Pork muscle lnfested wlth cystlcerci are obtained from an abattolr. Pork muscle is wiped clean of contamlnants on the surface wlth ethanol. Cysts are dlssected out lntact wlthout rupturlng the bladder wall ~lf ruptured the cyst ls dlscarded) and placed ln saline contalnlng Penclllin G 1000 units/ml, Gentamycln 40 ~gs/ml and Amphotericln B 5 ~gs/ml. Cysts are thoroughly washed with sterile saline contalnlng antibiotics as above. They are then washed with serum free medium RPMI 1640 or Eagles minimal essential medium (MEM) wlth Earles salts or Hank's salts. Cysts are placed in RPMI 1640 or Eagles MEM contalnlng Pencillin G 1000 units/ml and Gentamycln 40 ~g/ml at 37~C wlth 5% C02.
Collectlon of E-S antlgen:
The medlum ls changed and dlscarded at least twlce durlng the first 24-60 hours to ensure complete removal of host components. Suitably, the medium can be replaced twice durlng the flrst 48 hours. The medlum is collected at intervals from then on until cysts start evaglnatlng. Suitably, for practical reasons, the medium is collected every 24 hours. Viablllty of the cysts is monitored by bladder wall contractions that can be observed under 13-~019~
., .

an lnverted microscope. Evaglnatlon 18 observed from the 6th day onwards. Soon after collectlon of the medlum lt ls centrlfuged at 104,000 x g for 60 mln. to remove any lnsoluble debrls and membrane contamlnants. To the supernatant are added paramethyl sulphonyl fluorlde (2 mM) parachloro mercury benzoate (0.06%) and ammonlum sulphate to a saturatlon of 90% malntalnlng the pH of the solutlon at 7-7.4. The preclpltatlon of protelns by the ammonlum sulphate ls carrled out at 4~C and the preclpltate subsequently collected by centrlfugatlon at 15,000 x g for 30 mlns at 4~C. The preclpltate ls dlssolved ln 50 mM sodlum phosphate buffer pH 7.4 and extenslvely dlalysed agalnst the same buffer wlth several changes for 48 hours. Alternatlvely the membrane fragments can be removed by other methods known ln the art such as membrane flltratlon. Slmllarly, antlgens can be lsolated by other methods such as ultraflltratlon. UV spectrum of the solutlon shows an absorptlon maxlmum at 278-280 nm. Proteln estlmated by the procedure of Schaffner and Welssmann (18) glves 25-35 ~g proteln/cyst/24 hrs, when cysts are malntalned at a paraslte denslty of 15 cysts / 10 ml.
Sodlum Dodecyl Sulfate polyacrylamlde gel electrophoresls (SDS-PAGE) of these protelns stalned wlth coomassle blue R ls glven ln Flgure 2 whlch lllustrates coomassle blue stalnlng of E-S protelns analysed on 7.5 - 15% gradlent SDS-PAGE Lane l:Hlgh Mr markers (116,97.4,66,45 and 29 KDa) Lanes 2 & 3 : Ammonlum sulphate preclpltated E-S protelns of two lndependent preparatlons, Lane 4: Low Mr markers (94,67,43,30,20.1 and 14.4 KDa) 1 3 ~ 6 .. ..

Ma~or proteln bands seen are of apparent molecular welght of about 97,000, 66,000, 50,000, 38,000, 28,000 and an unresolved mlxture of protelns of apparent molecular welghts ln the range of 10,000-14,000 daltons. A number of mlnor bands are also seen.
Evldence for de novo synthesls of B-S Protelns T
Cysts after the flrst 48 hours ln the medlum, were transferred to methlonlne deflclent medlum contalnlng 35S-methlonlne 40 ~ Cltcyst. The cysts and the medlum were taken to varlous tlme polnts. The cysts were dlssected out lnto bladder wall and scolex and each was homogenlsed. The homogenates were analysed on 12% SDS-PAGE and sub~ected to fluorography. The medlum was acetone preclpltated and analysed on 12% SDS-PAGE and sub~ected to fluorography. There was an actlve lncorporatlon of S-methlonlne both lnto bladder wall and scolex protelns, and by 48 hrs several protelns appeared ln the medlum.
Flgure 3 ls an autoradlogram of Excretory/Secretory protelns labelled wlth 35S-methlonlne. Three cysts were lncubated separately ln 1 ml of medlum each contalnlng 40 ~Cl of 35S-methlonlne for 48 hrs. Medlum was collected, sub~ected to ultra-centrlfugatlon and the supernatant was acetone preclpltated. Each lane represents proteln from a slngle cyst.
As ls seen ln Flg. 3 several protelns were actlvely syntheslsed excreted/secreted lnto the medlum as a part of the metabollc actlvlty of the paraslte.
Summary of the Inventlon:
When larvae-contalnlng cysts are malntalned ln vltro ln serum free medla, as a part of metabollc turnover, some proteln 13~0196 antigens appear in the medium either by a process of excretion or secretion. These antigens, collected before the evagination of scolex, represent antigens of larval stages. The antigens, the method for their preparation and their use for diagnosis and possibly prognosis of NCC is claimed as well as their use in production of vaccine against the infestation of Cysticercus cellulosae. The diagnostic method described here may also be used to diagnose cysticercosis in pigs intended for human consumption.
In one aspect, therefore, the invention provides the use of excretory/secretory (E-S) protein antigens of Cysticercus cellulosae in immunodiagnosis of neurocysticercosis, said protein antigens being substantially free of cross-reactivity with tuberculous meningitis antigen, and being obtainable by a) maintaining intact and undamaged cysticerci from infested pork muscle in vitro in serum free cell culture growth medium containing antibiotics, b) removing all the antigens in the first 24-60 hours of the maintenance by repeatedly replacing the medium, c) subsequently collecting the medium at intervals until the ~0 time of evagination of scolex, d) removing any membrane fragments, and e) isolation of the antigens.
In another aspect, the invention provides a substan-tially pure protein antigen excreted/secreted by Cysticercus cellulosae which has an apparent molecular weight of about 97,000 or 66,000 or 50,000 or 38,000 daltons as estimated by SDS-PAGE, being substantially free of cross-reactivity with tuberculous ~ 13 13~ 19~

meningitis antigen and being obtainable by the method defined above.
In another aspect the invention provides a process for obtaining a substantially pure protein antigen of the invention, which comprises the following steps:
a) maintaining intact and undamaged cysticerci from infested pork muscle ln vitro in serum free cell culture growth medium containing antibiotics, b) removing all the antigens in the first 24-60 hours of the maintenance by repeatedly replacing the medium, c) subsequently collecting the medium at intervals until the time of evagination, d) removing any membrane fragments, and e) isolating the antigens from the collected medium.
In yet another aspect the invention provides a method for detecting neurocysticercosis which comprises: interacting body fluid from a human to be diagnosed with at least one excreted or secreted antigen of ln vitro cultured Cysticercus cellulosae having an apparent molecular weight as determined by SDS-PAGE of about 97,000 or 66,000 or 50,000 or 38,000 daltons or a mixture of said antigens, said antigens being substantially free of cross-reactivity with tuberculous meningitis antigen, and detecting the possible presence of Cysticercus cellulosae infestation.
Modified forms of the antigen could for example be polyethylene glycol linking or cross-linking with other proteins or bifunctional reagents or digesting with proteases. The invention also relates to a method of diagnosing active human NCC

C

13~019~

which comprises interacting CSF from a human to be diagnosed with at least one excreted or secreted antigen of ln vitro maintained Cysticercus cellulosae having an apparent molecular weight as determined by SDS-PAGE of about 97,000 or 66,000 or 50,000 or 38,000 daltons or a mixture of said antigens. The antigen of 38,000 daltons comprises of a partial amino acid sequence valine-glutamic-acid-tyrosine-threonine-cysteine-threonine (VEYTCT).
EXAMPLES
Example 1 and 2 relate to the utility of these antigens in an ELISA system to detect anti cysticercal antibodies in CSF of patients with various neurological disorders. Examples 3 and 4 relate to further identification of important antigens by their molecular weight in immunoprecipitation using patients CSF.
EXAMPLE 1:
Secretory/Excretory proteins collected as described in the disclosure are coated on rigid 96 well microtitre plates of Dynatech at a concentration of 5 yg protein~ml (100 yl/well -~ 14a ,~ ~
~ ,, -13~19~
,~.

overnight at room temperature. The remalnlng proteln blndlng sltes of the plates were quenched uslng 2% bovlne serum albumln tBSA) ln PBS. CSFs dlluted l/25, l/125 and 1/625 ln phosphate buffered sallne contalnlng 0.05% Tween-20* (PBST) and lncubated for 2 hrs at 37~C. After the lncubatlon, wells were washed thrlce wlth PBST. Rabblt antl human IgG peroxldase con~ugate dlluted 1:1000 ln PBST was used ln a volume of 100 ~l/well and lncubated for 2 hrs at 37~C after whlch the wells were washed thrlce wlth PBST. Bound con~ugate was revealed ln 20 mlns uslng orthophenylene dlamlne as chromogen (4 mg/10 ml) and H202 as substrate. The reactlon was stopped wlth 50 ~l of 4N H2S04 and absorbency at 490 nm was determlned ln Dynatech MR 700 ELISA
reader. Any absorbency value greater than 0.10 at a CSF dllutlon of 1/100 was consldered posltlve.
Table 2 contalns the results.

_________________________________________________________________ Dlagnosls No of samples Posltlves _________________________________________________________________ Cystlcercosls Postmortem proven 2 Surgery proven 2 2 Cllnlcally suspected 7 6 Japanese Encephalltls IgM capture ELISA +ve 30 o Tuberculous menlngltls 25 0 Culture proven surgery proven Tuberculous menlngltls 71 2 (elther Ag+ve absorbency -0.15 or antlbody + ve) 490nm *Trade Mark 13~0196 ~ .

The above table shows that thls antlgen ls superlor to the crude antlgen and confers speclflclty to lmmunologlcal reactlon.
~XAMPL~ 2 This example descrlbes a double bllnd study of the dlagnostlc value of excretory-secretory antigens ln whlch the deslgn of the test ellmlnates the blas of the lnvestlgator performlng the test.
37 CSF samples obtalned from patlents wlth varlous neurologlcal dlsorders wlth conflrmed dlagnosls were glven serlal numbers from 1 to 37 by an unblased observer other than the lnventors. The samples were allquoted lnto three sets. Two lndependent laboratorles of the lnventors recelved the CSF samples and two persons ln each laboratory l.e. a total of 4 persons conducted the ~LISA lndependently as descrlbed ln detall ln example 1. All four lnvestlgators tabulated the results and comrnunlcated the results to the unblased observer. The results of all four lnvestlgators were ldentlcal and are glven ln table 3.

I~Ol9o TA~LB 3 __________ _______________________________________________ Neurocysticercosis 3 3 Spinal anaesthesia (Normal controls) 8 0 Post Operative meningitis (culture proven) 5 0 Japanese Rncephalitis 7 0 Pyogenic Meningitis (Pneumococcal) 3 O
Cryptococcal Meningitis (Culture proven) 1 0 Tuberculous Meningitis (Culture proven) 2 0 Thoracic cord compre~sion a o Cranio Vertebral Junction Anomaly 1 O
Prolapsed Intervertebral di~c 1 O
Motor neuron disease 1 0 Ataxic neuropathy 1 0 Hemiplegia 1 0 Pseudobulbar palsy 1 0 _______________________________________________________ The above results indicate that excretory-secretory antigens can confer 100~ sensitivity and 100~
specificity to a diagnostic test designed to detect antibodies in CSFs of patients.

1 3 ~ 3 6 r Secretory/excretory antlgens obtalned as descrlbed ln the lnventlon were labelled wlth 125I uslng the Iodogen method.
The labelled protelns were lncubated wlth CSFs from known neuro-cystlcercosls, tuberculous menlngltls and varlous other neurologlcal dlsorders. Immune complexes were brought down wlth rabblt antl human IgG, carrler human IgG and maklng the whole solutlon to 2.5% polyethylene glycol. The preclpltates were analysed on a gradlent SDS-PAGE of 7.5%-15% whlch was sub~ected to autoradlography as shown ln Flgure 4 whlch ls an autoradlogram of 125I labelled secretory/excretory protelns lmmunopreclpltated wlth CSFs of patlents. CSF (30 ~l) ln l: 3 dllutlons was lncubated wlth E-S protelns ~3xlO cpm) and lmmune complexes were preclpltated as stated ln the text. Lane 1: Control-No CSF:Lanes 2,3&4 CSF from neurocystlcercosls, Lane 5: Tuberculous menlngltls, Lanes 6, 7 & 8: other neurologlcal dlsorders.
Slgnlflcantly, protelns of apparent molecular welght of about 97 kDa, 66 kDa, 50 kDa and 38 kDa were brought down ln lmmunopreclpltatlon.

E-S protelns labelled wlth 5S-methlonlne by malntalnlng the cysts ln vltro ln methlonlne deflclent medlum contalnlng 35S-methlonlne were lmmunopreclpltated wlth CSF antlbodles as mentloned ln example 2. The fluorography of SDS-PAGE of the lmmunopreclpltates ls shown ln Flgure 5.
Flgure 5 ls an Autoradlogram of 35S - labelled E-S
protelns lmmunopreclpltated wlth CSFs of patlents wlth varlous neurologlcal dlsorders. Lanes 9 and 5 represent lmmuno-~ 13~019~

19preclpltates from CSFs of NCC patlents.
As ln Example 3, 97, 66 and 50 kDa protelns are lmmunopreclpltated here also.

.. ... ... .. . . . .

Claims

1. The use of excretory/secretory (E-S) protein antigens of Cysticercus cellulosae in immunodiagnosis of neurocysticercosis, said protein antigens being substantially free of cross-reactivity with tuberculous meningitis antigen, and being obtainable by a) maintaining intact and undamaged cysticerci from infested pork muscle in vitro in serum free cell culture growth medium containing antibiotics, b) removing all the antigens in the first 24-60 hours of the maintenance by repeatedly replacing the medium, c) subsequently collecting the medium at intervals until the time of evagination of scolex, d) removing any membrane fragments, and e) isolation of the antigens.

2. The use according to claim 1 wherein antigens of apparent molecular weights of about 97,000, 66,000, 50,000 and 38,000 are utilized.

3. The use according to claim 1 wherein antigens of apparent molecular weights of 66,000 and 38,000 are utilized.

4. The use according to any one of claims 1, 2 and 3 wherein the immunoassay is designed to detect antibodies in a body fluid of a patient.

5. The use according to claim 4 wherein the body fluid is cerebrospinal fluid (CSF) or serum.

6. The use according to any one of claims 1, 2 and 3 wherein the immunoassay is designed to detect antigen in a body fluid of a patient such as CSF and serum.

7. The use according to claim 6 wherein the body fluid is cerebrospinal fluid (CSF) or serum.

8. A protein antigen obtainable by secretion or excretion by Cysticerus cellulosae which has an apparent molecular weight of about 97,000 or 66,000 or 50,000 or 38,000 as estimated by SDS-PAGE, being substantially free of cross-reactivity with tuberculous meningitis antigen, and being obtainable by a) maintaining intact and undamaged cysticerci from infested pork muscle in vitro in serum free cell culture growth medium containing antibiotics, b) removing all the antigens in the first 24-60 hours of the maintenance by repeatedly replacing the medium, c) subsequently collecting the medium at intervals until the time of evagination of scolex, d) removing any membrane fragments, and e) isolation of the antigens.

9. A protein antigen obtainable by secretion or excretion by Cysticerus cellulosae which is a glycoprotein of apparent molecular weight of about 97,000 or about 50,000 as estimated by SDS-PAGE, being substantially free of cross-reactivity with tuberculous meningitis antigen, and being obtainable by a) maintaining intact and undamaged cysticerci from infested pork muscle in vitro in serum free cell culture growth medium containing antibiotics, b) removing all the antigens in the first 24-60 hours of the maintenance by repeatedly replacing the medium, c) subsequently collecting the medium at intervals until the time of evagination of scolex, d) removing any membrane fragments, and e) isolation of the antigens.

10. A protein antigen obtainable by secretion or excretion by Cysticercus cellulosae which is a glycoprotein of apparent molecular weight of about 38,000 comprising a partial amino acid sequence of valine-glutamic acid-tyrosine-threonine-cysteine-threonine, (VEYTCT), said antigen being substantially free of cross-reactivity with tuberculous meningitis antigen, and being obtainable by a) maintaining intact and undamaged cysticerci from infested pork muscle in vitro in serum free cell culture growth medium containing antibiotics, b) removing all the antigens in the first 24-60 hours of the maintenance by repeatedly replacing the medium, c) subsequently collecting the medium at intervals until the time of evagination of scolex, d) removing any membrane fragments, and e) isolation of the antigens.

11. The use of a protein antigen according to any one of claims 8, 9 and 10, in the preparation of vaccine against infestation of Cysticercus cellulosae.

12. A process for obtaining a protein antigen as defined in any one of claims 8, 9 and 10 comprising the following steps:
a) maintaining intact and undamaged cysticerci from infested pork muscle in vitro in serum free cell culture growth medium containing antibiotics, b) removing all the antigens in the first 24-60 hours of the maintenance by repeatedly replacing the medium, c) subsequently collecting the medium at intervals until the time of evagination of scolex, d) removing any membrane fragments, and e) isolation of the antigens.

13. A method for diagnosing active human neurocysticercosis which comprises: interacting CSF from a human to be diagnosed with at least one excreted or secreted protein antigen according to any one of claims 8, 9 and 10 of in vitro cultured Cysticercus cellulosae having an apparent molecular weight as determined by SDS-PAGE of about 97,000 or 66,000 or 50,000 or 38,000 daltons or a mixture of said antigens, said antigens being substantially free of cross-reactivity with tuberculous meningitis antigen, and detecting the possible presence of Cysticercus cellulosae infestation.