- Protoscolices antigen production:
Deposit of hydatid fluid from camel lung cysts containing protoscolices was exposed to freezing and thawing 3 times and use 10 times of its volume of 0.15 M phosphate buffer saline (PBS) to wash, pH 7.2 then sonicated by 150 W ultrasonic disintegrator and the sonicate was sedimented at 10000 rpm for 30 minutes, the supernatant was split into aliquots and stored at - 20℃ [9].
- Purification of pAg by DEAE-Sephadex A 50 chromatography:
Removal of cross-reactive proteins and host components from Echinococcus antigens is a mandatory to be done by purification [10]. DEAE Sephadex A 50 chromatography has been used to purify the antigens of camel hydatid cysts based on their charges thus the DEAE group maintains positive charges that are neutralized by negative chloride charges [11]. Anion exchange chromatography was done by adding 0.5 gm DEAE Sephadex A 50 powder (Pharmacia, Uppsala, Sweden) to 200 ml of 0.5 M Tris-HCL buffer adjusted at pH 7 slowly for 1-2 days at 4℃ that this Sephadex was swollen to 22 ml beads. In a 30x2.5 column, the swollen beads suspension was poured using a glass rod to avoid air bubbles trapping, then the swollen beads were covered by binding buffer (20 mM Tris-HCI) and the hydatid antigen sample was dialyzed versus the binding buffer and allowed to penetrate the beads which were then washed by eluting buffer (20 mM Tris-HCL/150 mM NaCl) and the serum sample protein was calculated by collecting 10 fractions each is 2 ml and by using spectrophotometer (Perkin Elmer Lambda 1 A), absorbance at 280 nm for each fraction was measured and peak fractions of high absorbance were pooled together and the protein content was estimated by BioRad protein assay, USA [12], [13].
Indirect ELISA was used to assess the reactivity of pAg according to [14] and the absorbance was measured at 492 nm using ELISA reader (BioRad microplate reader, Richmond co., Wilmington, USA), measurement of the cutoff point for positivity was an indicator for mean optical density reading.
- Immunization of a rabbit and construction of ppAb
A New Zealand white male rabbit free from parasitic infections, purchased from Rabbit Research Unit, College of Agriculture, Cairo University. The experiment duration was 4 weeks under standard laboratory care at 21°C, 16% moisture, filtered drinking water with additional salt (1cm/5 liter) and vitamins (1cm/10 liter). Diet contains 15% protein, 3% fat and 22% fiber. A rabbit was immunized by i.m injection of a priming dose of 1 mg purified pAg mixed with a similar volume of complete Freund’s adjuvant (Sigma), then a first booster dose of 0.5 mg pAg was given two weeks after priming dose and two booster doses of 0.5 mg pAg at weekly intervals [15]. Three days after the last immunization, the blood sample was taken and centrifuged at 4000 rpm for 15 min. and serum IgG ppAb was fractionated, then kept at -20℃. Protein content was estimated by Bradford dye-binding procedure thus using the color change of Coomassie Brilliant Blue G-250 dye by reference to a standard curve consisting of known concentrations of purified protein by using a protein assay kit (Bio-Rad, Richmond, CA, USA) [12]. The rabbit was exposed to euthanasia by I.V injection of phenobarbital sodium after the experiment to end the animal’s suffering due to severe pain during experiment and it was not allowed to be re-used it again.
- Isolation of rabbit ppAb
As a result of polar and ionic groups of proteins, their solutions form hydrogen bonds in water and on the addition of highly charged ions such as ammonium of sulfate, they compete with proteins to bind water; this removes water molecules from proteins being precipitated [16]. Saturated ammonium sulfate solution (NH4)2SO4 was mixed with rabbit serum, centrifuged for 20 min. at 3500 rpm at 4℃, the supernatant was discarded and after repeated precipitations, the final precipitate was dissolved in 0.01 M PBS, pH 7.2 and (NH4)2SO4 was dialyzed against 0.01 M PBS, pH 7.2 for 72 hours to obtain pure protein [16]. Protein content in isolated IgG ppAb after ammonium sulfate treatment was determined by the Bradford dye-binding procedure [12].
- Labeling of ppAb with HRP
IgG ppAb molecules are conjugated to HRP enzyme exploiting its glycoprotein nature thus the saccharide residues of the enzyme are oxidized by sodium periodate giving rise to aldehyde groups which react with the IgG molecule amino groups which in turn lead to the production of Schiff bases being reduced to produce high molecular weight conjugate [17]. According to [18] labeling was done on two successive days; on the 1st day, 5 mg of HRP was dissolved in 1.2 ml distilled water (dist. H2O) then 0.2 ml sodium periodate was added for 20 min. at room temperature and dialysis against 1 mM sodium acetate buffer, pH 4 at 4℃ overnight to give solution (A), 5 mg/ml of IgG ppAb was dialyzed against 0.02 M carbonate buffer, pH 9.6 at 4℃ overnight to give solution (B). In the 2nd day, solution (A) was mixed with the solution (B) at room temperature for two hours and 0.1 ml of sodium borohydride was added to the new mixture which finally dialyzed against 0.01 M of PBS, pH 7.2 at 4℃ overnight and the resulting conjugate was stored at – 20℃ till used.
- Synthesis of antibody-AuNPs conjugates
- Source of AuNPs: AuNPs (40 nm/particle) were in the form of gold HCL solution with a concentration of 3.08x108 particles/ml, they were purchased from ( Tech. Egypt Co., 6 October, Egypt).
- Method of loading of IgG ppAb on AuNPs: according to [19], all glasswares were soaked with Aqua Regia (PanReac, Germany) for overnight and in the next day rinsed with tap water, the hydrogen tetrachloroaurate III (HAuCl4) (Sigma, Aldrich, Germany) solution was vortexed and an amount of 250 ml of it was added in a round bottom flask, condensed for 1 hour and refluxed using a sand bath then 25 ml of 38.8 mM sodium citrate were added and the flask was left to reflux again for 15 min. and stored in a dark place. Functionalization of 30 ml of AuNPs solution with 45 µl of 1mM mercaptoundecanoic acid (MUA) in ethanol (Sigma, Aldrich, Germany) for overnight at 4 ℃ and the AuNPs concentration was determined in the solution was determined before and after functionalization by UV/vis spectrophotometry using Beer’s law [20]. Conjugation of ppAb with AuNPs-MUA; 5 ml of AuNPs-MUA was added to 2 ml of ppAb in presence of N-hydroxy Succinimide/1-Ethyl 3-Dimethylaminopropyl Carbodiimide (NHS/EDC) as cross-linkers (Fluka, Germany) in order to obtain powerfully built AuNPs-MUA conjugates, this was achieved by addition of 5 ml of ppAb-AuNPs conjugates to 5 ml of a mixture of (5 mM sodium phosphate buffer = pH 7, 1.2 mM NHS and2.8 mM EDC) then left overnight at 4℃ so as to elicit electrostatic binding between ppAb and AuNPs-MUA [21].
- Quantitation of protein after conjugation of ppAb with AuNPs
Based on the Bradford dye-binding procedure, quantitation of protein content in ppAb-AuNPs conjugates was done by preparing standards of protein samples in the form of serial dilutions of Bovine Serum Albumin (BSA) with dist. H2O as (BSA: 1, 5, 10, 15, 25, 50 and 80 µl against dist. H2O: 99, 95, 90, 74, 50 and 20 µl respectively), then preparing standards of ppAb-AuNPs conjugates samples in the form of conjugates with dist. H2O as (conjugates 3, 10, 15 and 20 µl against dist. H2O: 97, 90, 85 and 80 µl respectively) and then 5 ml of diluted dye (phosphoric acid and methanol) was mixed with both preparations and the absorbance of color was measured at 595 nm for each standard tube being planned on standard curve for protein content measurement calculation.
- Patients sampling
Blood samples from human cases were collected from Benha University Hospital, Benha Teaching Hospital, Kasr Eleni Hospital, Pediatrics Abo-Elresh Hospital, Ain Shams University (Al-Damerdash) Hospital, and Theodore Bilharz Institute Hospital during the period from March 2016 to October 2017 and manipulated in the immune-parasitology department of TBRI, Giza Province, Egypt. Samples were divided into 3 groups; group 1 (hydatidosis group), group 2 (cross-reactivity group) and group 3 (control group).
- Group 1: blood samples from patients having hydatidosis proved by U/S, CT scan, IHAT and surgical excision of the cyst being confirmed by microscopic examination of hydatid cyst wall and sand.
- Group 2: blood samples from patients infected by nana, E. vermicularis, and F. gigantica, they are the patients who were negative to investigations in group 1 but stool analysis showed their previous parasitic infections.
- Group 3: blood samples from apparently normal individuals; workers and official personnel of the hospital who showed negative investigations and stool analysis.
Samples each of 5 ml were allowed to clot for 2 hours at room temperature and serum was separated and kept at – 20℃ until used.
Data collection sheet was done by collecting data about age, sex, residence, and occupation. Clinical presentation (symptoms & signs) and personal hygiene (washing hands, vegetables & contact with dogs) data were recorded in addition to data collected from patients’ sheets as cyst size, treatment, IHAT laboratory result thus titer above 1/160 was considered positive.
- The Detection of pAg in patients’ serum samples by sandwich ELISA
Coating of polystyrene microtiter plates wells (96-flat bottom wells, M 129A Dynatehc, Telangana, India) with 100 µl of a 20 µg/ml concentration of purified IgG ppAb which were incubated at room temperature for overnight, then washing 3 times with 0.1 M PBS/T, and pH was maintained at 7.4. Free sites in wells were blocked with 200 µl/well of BSA, pH at 7.4 for 2 hours at 37℃, then washed with buffer 3 times. Tested serum samples, each of 100 µl/well were added to wells and incubated for 2 hours at 37℃, then wells were washed 3 times. IgG ppAb conjugated with HRP in PBS/T (1/10 µg/ml) was added by 100 µl/well for 1 hour at room temperature, then wells were washed 3 times with washing buffer. Substrate solution was added by 100 µl/well for 30 min. in the dark at room temperature, then 50 µl/well of 8N H2SO4 was added to stop the enzyme-substrate reaction and then the absorbance was measured at 492 nm by ELISA reader (Bio-Rad microplate reader, Richmond, CA, USA).
- Detection of circulating pAg in patients’ serum samples by nanogold sandwich ELISA
Coating of polystyrene microtiter plates wells colored white for optical absorbance, was done with 100 µl of a 20 µg/ml concentration of purified IgG ppAb conjugated with AuNPs representing a capture antibody and diluted in 0.1 M carbonate buffer, pH 9.6 (Sigma, UK) for overnight at room temperature, then washed 3 times with 0.1 M PBS/T, pH 7.4. Blocking of wells was done by 100 µl/well 0.1 BSA for 2 hours at 37℃, then washed 3 times with PBS/T. Tested serum samples were pipetted into wells as 100 µl/well of each and incubated for 2 hours at 37℃, then washed 3 times. The remaining steps as above.
- Analysis of data
Chi-square test (X2-value) and Fisher exact test (FET) were used for inter-group comparison of categorical data. ROC curve was used to assess the validity of techniques thus including diagnostic sensitivity, specificity and predictive values [22]. Probability (P-value) was detect to know significance of the results as: if P > 0.05 (this was represented as non-significant difference), if P ≤ 0.05 (significant difference) and if P < 0.01 (highly significant difference). Accuracy was detected as the percentage of agreement between both sensitivity and specificity. Sensitivity (%) = (No. of true +ve results/No. of +ve results + No. of false –ve results) x100, specificity (%) = (No. of true –ve results/No. of –ve results + No. of false +ve results) x 100, positive predictive value (%) = No. of true +ve results/No. of true +ve results + No. of false +ve results) x100 and negative predictive value (%) = No. of true -ve results/No. of true –ve results + No. of false –ve results) x100.