Development of an enzyme-linked immunosorbent assay and counterimmunoelectrophoresis for the detection of Cryptosporidium parvum copro-antigens

Development of an enzyme-linked immunosorbent assay and counterimmunoe~sctroCs)rorssi+ for the detection of Crypfempuridium prvum copro-antigens K El-Shewyl,

P C Kibsey*,

W M Wenmanl

‘Division of infectious Diseases, Department of Pediatrics, University of Alberta; *Department of Medical Microbiology, Caritas Hospital Group, Edmonton, Alberta, Canada

Summary Diagnosis of intestinal parasitic infections is still mainly dependent upon the cumbersome, insensitive technique of stool microscopy. Alternatively, detection of parasite antigens that are shed in stool denotes infection regardless of symptomatology, physical integrity or excretion pattern of the parasite. Cryptosporidium copro-antigen (CCA) was detected in human and calf stool using enzyme-linked immunosorbent assay (ELBA) and counterimmunoelectrophoresis (CIE). Fifty-nine stool samples (25 samples positive for Cryptosporidium parvum by microscopy, 17 uninfected and 17 positive for other parasites) were tested against monospecific antibody raised against 20 kDa CCA. CCA was detected by ELISA in all Cryptosporidium microscopy positive samples, while CIE demonstrated CCA in 23 out of 25 positive samples. No crossreactions were observed when faecal samples harbouring other parasites were screened by the two techniques. CCA remained detectable after freezing, boiling or upon polyvinyl alcohol preservation. ELISA and CIE together with the developed monospecific antibody may represent new, reliable and confirmative immunodiagnostic tools for cryptosporidiosis. Key

words:

Serodiagn.

Copro-antigens, Immunother.

Cryptosporidium

parvum,

Infect.

1994, Vol. 6, 82-86,

Disease

ELISA,

Introduction Cryptosporidium sp. is a coccidian parasite that infects the mucosal epithelium of a variety of animals including humans. It causes acute self-limited diarrhoea in immunocompetent hosts, and severe gastrointestinal symptoms in immunodeficient patients’JJ. Diagnosis depends upon identification of Cryptosporidium oocysts in the stool using various concentration and staining techniques4,5, although serological and

Received: 24 February 1994 Accepted: 25 February 1994 Correspondence and reprint requests to: WM Wenman, Department of Pediatrics, 2C3.00 Walter Mackenzie Centre, University of Alberta, Edmonton, Alberta, Canada T6G 2R7. 0 I994 Butterworth-Heinemann Ltd 0888~~786/94/02WX2-05

CIE

July

immunofluorescent detection methods are now also feasible+*. Low sensitivity is associated with methods which depend upon the detection of oocysts9; for example, intermittent or atypical oocyst excretion together with inadequate concentration procedures may lead to inaccurate resultslO. In addition, detection of Cryptosporidium antibodies does not differentiate between past and recent infection. There is a need for more sensitive and accurate diagnostic tools for parasite antigen(s) that might be secreted and/or excreted by the organism or from one of its developmental stages. In previous work we identified and isolated an 18-20 kDa Cryptosporidium copro-antigen (CCA) from calf stool that was infected with Cryptosporidium parvuml’. In the present study we attempted to establish enzyme-linked immunosorbent assay (ELISA) and

El-Shewy

et a/.: ELKA

counterimmunoelectrophoresis (CIE) systems, using monospecific antibody raised against these CCA for coprodiagnosis of cryptosporidiosis.

Materials

and methods

Sample collection and preparation

Stool samples used in this study were collected from the following sources: (a) calf stool samples collected from 17 diarrhoeic calves infected with C. parvum, and from 11 uninfected healthy calves from Brown’s Feed Farm, Clive. Alberta, Canada; (b) human stool samples from eight patients infected with C. parvum, and from six noninfected healthy individuals, collected from Caritas Hospital Group, Edmonton, Alberta, Canada; and (c) 17 stool samples from patients harbouring parasites other than C. parvum, collected from Mansoura University Hospital, Egypt. Three consecutive samples every 48 h were collected from each patient as well as from calves. The stool samples were examined repeatedly directly and after concentration for the presence of C. parvum and other parasitesA. Modified Kinyoun’s acid fastJ. and trichrome stainsI were used for staining. After microscopic examination, positive and negative samples were kept frozen during handling and shipping, until stool eluate was prepared by mixing 1 g stool with an equal volume of distilled water, centrifuging at 1500 rpm for 5 min, and removing the supernatant (stool eluate). One half of this eluate was maintained at -20” C. and the other was preserved as described below.

and C/E detect

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the appropriate wells. The plates were washed three times for 3 min each in PBS pH 7.2 containing 0.05% Tween 20, and shaken dry. Diluted anti-20 kDa monospecific antibody or preimmune serum (1 : 25 serum in PBS-Tween 20) was added 100 u1 well-l and incubated at 37” C for 1 h. After washing as above. 100 ul well-l of anti-rabbit conjugated antibody (Sigma Chemical Co., St. Louis. MO) (1 : 1000 of conjugate in PBS + Tween 20) was added for 1 h at 37” C. Following washing, the plates were again incubated for 30 min with 100 ul wellll of p-nitrophenyl phosphate (Sigma) (1 mg ml-l diethanolamine buffer, pH 9.8, with 0.5 mM MgCI,). The reaction was stopped with 100 ul 2.5 M NaOH. The plates were read on a Titertec Multiscan micro-ELISA reader (Flow Lab. Inc.). Test interpretation: for interpretation of ELISA a cut-off value was determined by calculating the mean optical density (OD) value plus 2 standard deviations of C. parvrm negative samples collected from calves or humans uninfected or infected with parasites other than C. parvum”Processing of stooi samplesfor CIE

Positive and negative stool eluates were treated with SDS sample buffer (5% vollvol) 2-mercaptoethanol, 10% (vol/vol) glycerol, 2% (wtivol). SDS and 0.0625 M tris base) before processing in CIE as follows: 1 vol eluate plus 1 vol SDS sample buffer (5% 2-mercaptoethanol, 10% glycine and 2% SDS), boiled for 5 min, then cooled and diluted 1 : 6 in electrophoresis buffer (30 g tris base, 144 g glycine 1-l. 1 : 5 strength used as working solution). The 1 : 6 dilution was the highest to produce clearly defined precipitation.

Preparation of monospecific anti-20 kDa antibody

Positive stool eluates were analysed by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDSPAGE) according to Laemmlir3. A 20 kDa protein band was electroeluted and concentrated using the method mentioned previouslyliJ4. Pathogen-free 2.5 kg New Zeaiand white female rabbits were immunized with concentrated 20 kDa CCA. Rabbits received four booster doses of 1 ml antigen mixed with 1 ml Freund’s incomplete adjuvant by intramuscular and subcutaneous injections in different sites, with 10 days between injections. Two weeks after the last injection, the rabbits were bled and the collected sera were tested against purified 20 kDa antigen by immunoblot15. Performance of ELBA

The ELISA described by Voller et al.lh was performed as follows: 96 flat bottom well microtitre plates (Flow Lab. Inc., McLean, VA, USA) were coated in triplicate with 100 1.11of stool eluate mixed with 50% foetal calf serum and diluted in 0.05 M carbonate buffer, pH 9.6. After overnight incubation at 4” C, 100 u1 of 2% bovine serum albumin (BSA) in phosphate buffered saline (PBS) pH 7.2 was added to

Performance of CIE

Ten ml of 1% agarose gel (Type I low EEO, Sigma), in electrophoresis buffer, was poured on precleaned 75 x 50 X 1 mm microscope slides (Fisher Scientific, Pittsburgh, PA, USA). Wells of 5 mm diameter separated by 1 cm distance were punched in the gel. Whatman no. 1 filter papers were used as wicks. Wells were oriented parallel to the electric field. Those closest to the anode were filled with 20 1.11of either monospecific antibody or control non-immune serum. The cathodal wells were filled either with purified 20 kDa CCA, fresh stool eluates or eluates treated with SDS sample buffer and boiled or unboiled. Electrophoresis was performed at 70-90 V constant current for 60 min. Slides were inspected for precipitation between the cathode and anode wells immediately and after overnight incubation at 4” C. No additional staining procedure was performed to enhance visibility. Effect of preservation

The ability of both ELISA and CIE to detect CCA in preserved stool eluates was tested using positive stool

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eluates preserved in polyvinyl alcohol (PVA), 10% formalin, sodium formalin acetate (SAF), and 2.5% (vol/vol) potassium dichromate (K2Cr,0,) (1 vol sample + 2 vol preservative). The preserved samples were processed as before for ELISA and CIE testing at different time intervals, through 2 weeks from the start of preservation.

Read ts Microscopy

oocysts were detected in eight humans and 17 calf stool samples, whereas no occysts were detected on repeated examination of the noninfected control samples. Other parasites found in 17 human stool samples were Schistusoma mansoni in six samples, C. parvum

Giardia lamblia, Entamoeba histolytica, Enterobius vermicularis and Hymenolepis nana in two samples each, while Heterophyes heterophyes was detected in a

single sample. Two samples exhibited a mixed infection. one with G. lamblia and Endolimax nana, the other with S. mansoni and Ascaris lumbricoides.

Moreover. a declining ELBA reading was associated with serial dilution of the monospecific antibody (data not shown). CIE

With the use of monospecific anti-20 kDa antibody. CIE demonstrated a well defined single white precipitation arc between the two wells. This pattern was detected in 23 of the 25 positive samples tested (92%) (Figure lb). One human and one calf sample did not show any precipitation on repeated examination. Human samples (Figure lb, no. 5) showed a similar precipitation pattern to calf samples (Figure lb, nos l-4). Sixteen out of 17 (six human and 10 calf) noninfected control samples showed no precipitation (Figure lb, nos 6 and 7), while one exhibited an atypical illdefined precipitation. No crossreaction was detected in samples harbouring other parasites. The precipitation pattern was obtained only when SDS treated samples, with or without boiling, were used as antigens (Figure 2b). However, purified and concentrated 20 CCA yielded a clear precipitation in the absence of SDS (data not shown).

Specificity of anti-20 kDa monospecific antibody

The rabbit antibody proved to be specific as shown by its crossreaction with the 20 kDa CCA in both purified and eluate forms in immunoblots (Figure la).

Figure 1. a, lmmunoblot analysis of purified, concentrated 20 kDa copro-antigen (a), and stool eluate from a calf infected with C. parvum (b). Both were probed with anti-20 kDa monospecific antibody. b, CIE of stool eluates form C. parvum infected human and calf, demonstrating the characteristic CCA precipitation pattern (arrow). The cathodal wells (-) contained calf (I-4). and human stool eluates (5). Control noninfected human and calf samples are represented in wells 6 and 7, respectively. The anodal wells (+I contained monospecific antibody raised against 20 kDa CCA.

ELISA

Direct optical density readings at 450 nm for means of triplicates of positive and negative human and calf eluates are shown in Figures 3 and 4 respectively. A cut-off value was determined by calculating the mean plus 2 SD of negative human and calf samples tested. All confirmed positive human and calf samples had an

Figure 2. Photograph of CIE demonstrating the effect of different preservatives and SDS on CCA. a, Stool preserved for 1 week in 2.5% K,Cr,O, (K); SAD (Sal; saline (S); formalin 10% (F); and PVA (P), (arrow points to the intact precipitation observed with the sample preserved in PVA); b, SDS effect on CCA. Stool eluate without SDS treatment (11, SDS unboiled sample (2), SDS boiled sample.

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et al.: ELlSA

and C/E detect

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85

PVA (Figure 2a). A positive ELISA reading was also detected with samples preserved for 10 days in PVA (data not shown). 0.8 3

0.6

2

0.4

Discussion cut-off

point

0.2

Human stool samples Figure 3. Diagnostic sensitivity and specificity of ELISA for human stool eluates. a, Six uninfected negative samples; b, Eight samples with C. parvum and c, 17 stool samples harbouring parasites other than C. parvum. The cut-off point was determined as the mean absorbance value plus 2 standard deviations g + 2 SD) of 23 human samples negative for C. parvum.

0.81

-1

B

1

Calf stool samples Figure 4. Diagnostic sensitivity and specificity of ELBA for calf stool eluates. a, Eleven uninfected negative samples; b, 17 samples with C. pan/urn. The cut-off point determined as the mean absorbance value plus 2 SD (X + SD) of 11 calf samples negative for C. pan/urn.

OD higher than the cut-off point with a sensitivity and specificity of 100%. There were no false positives. as six human and 11 calf noninfected control samples had an OD lower than the cut-off point (Figures 3a and 4a). In addition, no crossreaction was observed while screening the stool eluates of patients possessing other parasites (Figure 3~). Effect of preservation

ELISA samples PVA. A with CIE

rind SDS treatment

and CIE were unable to detect positive in any preservatives, with the exception of well defined arc of precipitation was detected of stool eluates in after 1 week preservation

The most common method for diagnosis of intestinal parasites is still by microscopic examination of stool, despite its recognized insensitivity”-“‘. Unlike traditional methods of diagnosis, detection of parasite antigens that are shed in stool denotes parasite persistence independent of symptomatology. physical integrity or excretion pattern. Recently, diagnosis of intestinal amoebiasis and giardiasis by detection of copro-antigens has been reported to be a practical diagnostic procedure’“-‘-‘. In this study we report the feasibility of diagnosing Cryptosporidium infection by detection of CCA. This strategy circumvents the problems of variability in oocyst excretion and low sensitivity of the commonly used diagnostic procedures’.“‘. In a previous study we identified and isolated an 18-20 kDa CCA”. Monospecific antibody raised against 20 kDa CCA was used in this study to detect C. parvunz infection in human and bovine stool, using ELlSA and ClE. The detected CCA proved to be unique and specific to C. parvum infection. Neither ELISA nor CIE exhibited crossreaction with stool samples positive for eight other intestinal parasites. In addition. human and calf positive samples showed a similar pattern of CIE precipitation, although with different intensities. Anti20 kDa antibody proved to be specific for CCA in stool, as shown by a declining ELISA reading in association with serial antibody dilution. In immunoblots this antibody crossreacted with 20 kDa CCA in both purified and eluate form, excluding the possibility of nonspecific reaction. Although the ELISA developed in this study detected CCA in all confirmed positive samples with a sensitivity and specificity of 100%. CIE failed to detect CCA in two positive samples. This may be due to antigenic degradation by bacterial flora or more likely. these two samples contained low concentrations of antigen sufficient only to be detected by the more sensitive ELISA. Neither technique demonstrated samples: although these CCA in non-Cryptosporidium samples did not include a coccidial parasite they did represent some of the most common human intestinal parasites. Reproducibility of both assays was demonstrated by an acceptable low coefficient-of-variation, even with the use of different batches of antibody. At the same time, a consistent pattern of precipitation was always obtained on repetition of CIE. Interestingly, positive stool samples did not show visible precipitation in CIE except after SDS sample buffer treatment. Apparently SDS in the sample buffer resulted in mobilization of antigenic contents from the positive faecal matter. and enhanced movement of the mobilized CCA. Purified and concentrated 20 kDa

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antigen showed precipitation without SDS; however, this antigen was treated with SDS during its preparation”. SDS sample buffer is usually used to enhance protein mobility in an electric field by adding negative charges through its reducing effectZJ. In the current experiment, bromophenol in the sample buffer was used as an indicator for the electrophoresis front. Apparently, CCA retains its antigenic structure under conditions prevalent in the host intestinal tract due to different pH and digestive proteolytic enzymes. In addition, CCA remained detectable by ELISA and CIE after boiling, freezing or upon PVA preservation. The antigenicity of CCA was lost upon preservation in K2Cr202, SAF and formalin. This may be due to denaturation of antigen protein by formalin. In a similar study addition of formalin to positive stool samples invalidated ELISA for detection of copro-antigens in intestinal amoebiasis??. Although K2Cr20, is usually used to conserve infectivity of C. parvum oocysts5J5, it failed to maintain the antigenicity of the detected CCA. ELISA and CIE assays described in this study, used with monospecific antibody, proved to be highly sensitive, reproducible and specific for CCA. With further refinement of CCA, possibly including use of monoclonal antibodies, ELISA and/or CIE could become practical and reliable immunodiagnostic tools for cryptosporidiosis. We are presently evaluating the sensitivity and specificity of ELISA compared to microscopy for the diagnosis of C. parvum infections. Acknowledgements

We are grateful to Dr R Kilani (Microbiology and Public Health, University of Alberta, Edmonton, Alberta, Canada) for her technical advice. We thank Dr M Hegazi (Mansoura University, Egypt) and Dr LM Makhlouf (Suez Canal University, Egypt) for providing stool samples. We also thank Mr A Williams (Brown’s Feed Farm, Clive, Alberta, Canada), for providing calf stool samples. References

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