Production of anti-Cryptosporidium polyclonal antibodies and standardization of direct immunofluorescence for detecting oocysts in water Produção de anticorpos policlonais anti-Cryptosporidium e padronização da imunofluorescência direta para a detecção de oocistos na água

Introduction: The production of anti-Cryptosporidium polyclonal antibodies and its use in direct immunofluorescence assays to determine the presence of Cryptosporidium in water are described in the present work. Methods: Two rabbits were immunized with soluble and particulate antigens from purified Cryptosporidium oocysts. The sera produced were prepared for immunoglobulin G extraction, which were then purified and conjugated with fluorescein isothiocyanate (FITC). Slides containing known amounts of oocysts were prepared to determine the sensitivity of the technique. To test the specificity, slides containing Giardia duodenalis cysts were prepared. Results: The conjugate was successfully used in water samples experimentally contaminated with Cryptosporidium oocysts, and it was possible to detect up to five oocysts/spot, corresponding to contamination of 250 oocysts/mL. Conclusions: The three immunizations performed in the rabbits were enough to produce antibodies against Cryptosporidium, the standard direct immunofluorescence assay permitted the detection of five oocysts in 20% of the samples, and no cross-reaction with Giardia duodenalis cysts occurred.

The direct immunofluorescence assay (DIF) for detecting Cryptosporidium was developed in the 1980s and showed advantages in relation to the technique used up until that point, which involved floating with a zinc sulfate solution and Lugol staining 1 .When compared with acid fuchsin staining, the immunofluorescence assay technique shows greater sensitivity and specificity 2 .
One of the problems in using DIF in water samples is the presence of a large number of organic and inorganic substances capable of binding to antibodies and producing a false-positive result 3 .This problem can be minimized by establishing a purifying stage; the purpose of which is to eliminate the particles that can bind to marked antibodies producing fluorescence that could confuse the less-experienced technician.
This technique and others that use immunofluorescent methods are not able to identify the Cryptosporidium species, the genotype and specific hosts 4 , but are still very important in epidemiological studies and in the evaluation of the water following treatment.Additionally, methods 1,622 and 1,623 of the American Environment Protection Agency 5,6 , developed for monitoring Cryptosporidium in water, require a final stage that involves fluorescent monoclonal antibodies capable of staining the membrane of Cryptosporidium oocysts.
Considering the high cost for performing methods 1,622 or 1,623, the proposal of the present work was to produce polyclonal antibodies that can be used in immunofluorescence assays in water samples from impounding or treated water, aimed at determining contamination levels by Cryptosporidium oocysts.

Antigenic extract preparation
Cryptosporidium parvum oocysts were purified from calf feces by sucrose gradient as previously described 7 .A total of 10 8 oocysts diluted in 10mL of phosphate buffered saline (PBS), pH 7.2, were disrupted by sonication at 60 Hz (five cycles/min in an ice bath followed by a 1-min rest).The material was observed under phase contrast microscopy to ensure the efficacy of sonication to rupture the membrane.The extract was centrifuged for 30 min at 9,000 x g at 4ºC, and the soluble antigen (supernatant) and particulate antigen (sediment) were separated into two aliquots.The protein concentration was determined by the Bradford method 8 using 1mg/mL bovine serum albumin (BSA) as standard.Absorbance readings were performed using a 595-nm filter.

Immunization
Two male New Zealand rabbits, approximately 7 months old, were used for immunization after they were submitted to an adaptation period of 30 days.
Three subcutaneous immunizations were administered in the scapular area of each rabbit, alternating between the right and left sides.The first dosage of the antigenic extracts contained 1.25mg and 0.85mg of the soluble and particulate antigen, respectively, and complete Freund's adjuvant (Sigma Chemical Company).The second immunization was performed after 15 days, using the antigenic extract in the same concentration described before and incomplete Freund's adjuvant (Sigma Chemical Company).The third immunization was repeated 15 days after the application of the second dosage, using identical concentrations of antigenic extract and adjuvant.Blood collections were performed on days 0, 15, 30, 45, 60, 90, and 120 to determine the antibody titers.The production blood collections were performed on days 45, 60, and 75, and the sera were stored at -20ºC until use.

Titering sera samples
The immunoenzymatic assay (enzyme-linked immunosorbent assay, ELISA) was used to determine the kinetics of anti-Cryptosporidium antibody production.The protocol adopted for the experiment followed that proposed by Frost et al. 9 Briefly, flat-bottom plates (Immulon 2, Dynatech Laboratories, Inc.) were adsorbed with soluble antigen or particulate antigen at four different concentrations (250, 500, 1,000, and 1,500ng) diluted in a carbonate buffer (0.05 M, pH 9.6).The rabbit sera were diluted 1:100, 1:200, 1:400, 1:800, and 1:1600 in incubation buffer (PBS; 0.25% casein; 0.05% Tween 20).The rabbit anti-IgG immunoglobulin conjugated to the enzyme peroxidase (Sigma Chemical Company, A-0545) diluted at 1:2500 and 1:5000 was added, and the enzymatic activity was revealed using orthophenylenediamine solution (OPD) diluted in substrate buffer (4mg of OPD+10.5mL of citrate buffer pH 5.0+4µL of H 2 O 2 ).The reaction was interrupted using 50L of 5% H 2 SO 4 .The negative control of the reaction was performed using the serum collected from the same rabbit on day 0 of the experiment, prior to inoculation with the antigenic extract.The serum collected on day 45 of the experiment was used as a positive control, in accordance with the proposal of Fernandes et al. 10 The absorbance readings of the ELISA test were performed in the microplate reader (BIORAD Model 550) using a 492nm wavelength filter.

Glycoprotein extraction
The extraction of glycoproteins was performed according to method proposed by Penha 11 .A pool of sera collected on days 30, 45, and 60 was centrifuged at 3,700 x g for 5 min at 4ºC.The supernatant was harvested, and 5% dextran was added in the proportion of 1.25mL of dextran to 25mL of serum, drop by drop, under stirring at room temperature.The mixture was maintained for 30 min at room temperature, stirring every 10 min.An 11.1% CaCl 2 solution was added, drop by drop, under agitation in an ice bath, up to a final concentration of 1%.The mixture was allowed to stand for 30 min in the ice bath and was then centrifuged at 14,400 x g for 15 min.The supernatant was separated, and the same volume of PBS pH 7.2 was added.Twice the volume of saturated ammonium sulphate (pH 7.6) was added, and the mixture was left under slow magnetic stirring for 1h at room temperature.Following precipitation of immunoglobulins G, the sample was centrifuged for 30 min at 14,800 x g at 4ºC.
Dialysis was performed using a membrane (Sigma, Dialysis Sacks) that permits a 12kDa exclusion limit for protein retention and ammonium sulfate release.The process was performed for 3 days in a flask with two liters of PBS pH 7.2, with changes every 12h.The immunoglobulins were centrifuged at 3,700 x g for 15min at 4ºC to remove impurities.The dialyzed material was centrifuged at 1,330 x g for 15min at 4ºC to separate the immunoglobulins.Proteins were quantified in a Metrolab 1,700 Spectrophotometer, with UV rays, at 260nm and 280nm.
Fluorescein isothiocyanate conjugation The fluorescein isothiocyanate (FITC) was dissolved in carbonate/bicarbonate buffer and added, drop by drop, to the immunoglobulin suspension in the proportion of 1.6μg of FITC to 1mg of protein, at room temperature under slow magnetic stirring.The mixture was maintained under slow magnetic stirring for 2h, taking care to avoid foaming.
The sample was then submitted to molecular exclusion chromatography in a Sephadex G 25 column (Sigma Aldrich).After hydrating the Sephadex with ultrapure water, it was washed five times with borate saline and transferred to a gel filtration column.The volume of the column was estimated respecting the proportion of 20mL of gel for 1mL of conjugate.The initial fraction (conjugate) was separated and dialyzed in borate saline for 48h, centrifuged at 1,330 x g for 10min at 4ºC, aliquoted and stored at -20ºC.

Oocyst detection by direct immunofluorescence
To determine the sensitivity of the direct immunofluorescence (DIF) using the polyclonal antibodies, the slides were adsorbed with 1,000, 500, 100, 50, 10, 5, and 1 oocyst/spot, in quintuplicate, diluted in PBS, pH 7.2.As each spot was filled with 20µL, these amounts are equivalent to 50,000 to 50 oocysts/mL.Slides containing 1,000, 500, 100, and 10 of Giardia duodenalis cysts were prepared, also in quintuplicate, aimed at evaluating the specificity of the technique.Following the addition of oocysts, the slides were left standing at room temperature for 15 min for the oocysts to sediment and then incubated at 37ºC for 1h, or until completely dry.Subsequently, the slides were placed in light-proof containers and frozen at -20ºC until use.
Immediately before use, the slides were thawed at 37ºC for 10 to 15 min and submitted to three washes with PBS pH 7.2.The conjugate diluted in Evans's blue was added to the slides pure or in the following dilutions: 1:1, 1:2, 1:4, 1:8, 1:16, 1:32, 1:64, and 1:128.The slides were incubated in a moist chamber for 30 min in the dark.After performing three washes with PBS, pH 7.2, the slides were incubated at 37ºC for 10 min to dry and were examined under an Olympus BX4TF fluorescence microscope.

Ethical considerations
All procedures related to managing the animals and material collection were approved by the Ethics Committee on Animal Experimentation of the Federal University of Parana (CEEA/ UFPR), protocol no.23075.029322/2007-47,certificate 250.The amount of protein obtained from 10 8 oocysts was 2.5mg/mL and 1.7mg/mL for the soluble and particulate antigens, respectively.The production kinetics of anti-Cryptosporidium-specific antibodies was tested by the ELISA technique, in which 500ng of protein/mL as antigen, 1:100 dilution of the serum, and 1:2,500 dilution of the conjugate were used.The cutoff point was determined by estimating the average absorbance of the negative controls plus two standard deviations, corresponding to 0.100.Thus, samples with absorbance equal to or higher than 0.100 were considered positive.The rabbit sera collected during the experiment were titered, and all showed levels of absorbance higher than 0.100, verifying the production of anti-Cryptosporidium antibodies (Figure 1).
The purified and FITC-conjugated anti-Cryptosporidium polyclonal antibodies tested in DIF assays detected oocysts in all replicates that contained 1,000 to 100 parasites (Figure 2).Two out of the five replicates containing 50 to 10 oocysts were positive, only one replicate with five oocysts was positive, and no sample containing only one oocyst was positive (Table 1).
No cross-reaction with Giardia duodenalis cysts was observed in any of the slide spots.Identification of Cryptosporidium in water is impaired by the low number of oocysts and by adverse environmental factors that may alter their morphology 1 .Thus, techniques that can concentrate and correctly identify the parasites are required.Methods 1,622 and 1,623 of the American Environment Protection Agency 5,6 are considered a reference for detecting Cryptosporidium in water.Among the several steps required to perform these methods, the final morphological identification involves the use of differential interference contrast microscopy, 4'-6-diamino-2-phenylindole (DAPI) staining, and immunofluorescence using a commercial kit.In Brazil and other developing countries, poly-or monoclonal antibodies against Cryptosporidium are not produced by national companies.Thus, the importation of immunofluorescence kits is costly for sanitation companies to monitor the parasites in water.Considering these aspects, the present work standardized a direct immunofluorescence reaction using anti-Cryptosporidium polyclonal antibodies produced by immunization in rabbits.These animals were chosen because they present greater volume compared with other animals and, consequently, produce a larger quantity of blood.
The anti-Cryptosporidium antibodies were successfully produced and used in DIF assays, which revealed sufficient sensitivity to detect 5 oocyst/spot or 250 oocysts/mL.
Cross-reactions can occur in direct immunofluorescence tests performed in water samples, as the presence of a large number of fluorescent particles may cause difficulties during the microscopic confirmation of suspect oocysts 3,12,13 .In the present work, the reactivity of the anti-Cryptosporidium antibodies with Giardia duodenalis cysts was tested, and no cross-reaction was observed.Despite the large morphological difference between oocysts and cysts, the intent of the specificity test was to precisely observe the presence of possible components present in the cysts that might be recognized by the anti-Cryptosporidium antibodies.

FIGURE 1 -
FIGURE 1 -of antibodies production in rabbits immunized with antigenic extract of Cryptosporidium parvum oocysts, evaluated by ELISA performed with soluble and particulated antigen.