The collection of fresh and formalin fixed tissue samples, euthanasia of snakes, and the production of monoclonal antibody was authorized under University of Florida Institutional Animal Care and Use Committee Protocol 201101156.
A total of 112 snakes were used in this study, including 94 boa constrictors, 4 annulated tree boas (Corallus annulatus), 4 ball pythons (Python regius), 2 carpet pythons (Morelia spilota), 2 corn snakes (Pantherophis guttatus), 1 emerald tree boa (Corallus caninus), 4 rainbow boas (Epicrates cenchria), and 1 palm viper (Bothriechis marchi) (). Paraffin embedded tissues of 99 snakes were collected from the repositories of Anatomic Pathology Service and Zoological Medicine Infectious Disease Testing Laboratory, College of Veterinary Medicine, University of Florida (UF)(Gainesville, FL, USA), and additional blocks were kindly provided by Northwest ZooPath (Monroe, WA, USA) and Zoo/Exotic Pathology Services (West Sacramento, CA, USA). Fresh or formalin fixed tissue samples of 13 boa constrictors were collected throughout 2008 to 2011 by veterinarians in private practices or UF Veterinary Hospitals, and transferred to Zoological Medicine Infectious Disease Testing Laboratory, College of Veterinary Medicine, UF for IBD diagnosis. Portions of fresh tissues were fixed for processing into paraffin embedded blocks and immuno-TEM labeling, and the remaining biological materials were stored in an ultra-freezer at -80°C. A pathology report was available for each case with the final diagnosis verified by board certified pathologists based on histological examination of H&E stained sections. For each case, one block with standard tissues (liver, kidney, or pancreas) was selected for IHC staining. If IBD inclusion bodies were not described in the standard tissues, an additional block with tissues having IBD inclusion bodies was stained with IHC.
Summary of the collected samples. Diagnosis of IBD was based on the histological finding in H&E stained tissue sections.
Purification of IBDP
The purification method was modified from a published protocol for isolating human Mallory bodies [16
]. Briefly, approximately 5 g of frozen liver and kidney obtained from a boa constrictor were thawed and separately processed. Each tissue was cut into small portions (approximately 3 mm cubes) and homogenized in homogenization buffer (HB) containing, 250 mM sucrose, 10 mM EDTA, 10 mM HEPES, pH 7.4. The tissue to be homogenized was processed 1-2 g at a time with 5 mL HB, and the homogenate was collected and filtered through two layers of gauze, to remove the larger tissue debris. The homogenate was centrifuged at 1000 x g for 10 minutes. The supernatant was removed and the soft pellets were pooled and re-suspended with equal volume of HB, mixed with 1% Sarkosyl (Teknova, 2S3380) at a 1:1 ratio and incubated in 37°C for 30 minutes with frequent vortexing. The suspension was centrifuged at 14,000 x g for 10 minutes at 4°C, and the supernatant was removed. The pellet was resuspended with equal volume of 1% sarkosyl, and the suspension was subjected to one more round of the above procedure, with incubation followed by centrifugation. After removing the supernatant, the pellet was carefully re-suspended in 1 mL of HB which will be referred to hereafter as “inclusion body preparation” (IB prep). The IB preps were stored at 4°C for future analysis.
Suspensions collected from each protein purification step were diluted 50 fold with water, and 50 µL of each diluted sample was placed in a cytocentrifuge chamber (Biomedical Polymers Inc., BMP-CYTO-S50), and centrifuged for 6 minutes at 800 rpm onto a glass microscopic slide using a Cytospin centrifuge (Shandon, Cytospin 2). The slides were air dried, fixed in 10% neutral buffered formalin (10% NBF) (Fisher Scientific, SF100-20) for 10 minutes, washed with distilled water, and stained with H&E stain.
Electrophoresis and Gel Staining
Aliquots of IB preps or crude tissue homogenates were reduced by addition of equal volume of 2X loading sample buffer (25 mM Tris, 4% SDS, 100mM DTT, and 30% glycerol). For western blots, the proteins were reduced by addition of 4X NuPAGE LDS sample buffer (Invitrogen, NP0008) and 10X NuPAGE reducing agent (Invitrogen, NP0004) with 500 mM DTT. Following addition of the reducing buffer, the samples were heated to 95-100 °C for 10 minutes. Next, the total protein concentration of a 1:10 dilution of each reduced and unreduced sample was estimated by standard Bradford Protein Assay (Bio-Rad Laboratories, Inc.), subsequently, the total protein concentration of the IB preps were estimated. The reduced proteins were resolved on a 10% or 4%~12% NuPAGE Bis-Tris gel (Invitrogen) with MES or MOPS buffer (Novex) at 200V constant voltage. For evaluation of protein quality, 5 µg of protein were loaded in each well, and the resolved protein was visualized by SimplyBlue (Novex) stain, using the microwave protocol provided by the manufacturer (Novex).
Electro-elution of IBDP
The resolved 68 KDa IBDP bands were cut from the gels, minced, and packed inside glass tubes of the Model 422 Electro-eluter (Bio-Rad, 165-2976). The protein was electro-eluted in Tris-Glycine buffer without SDS, following the protocol provided by the manufacturer (Bio-Rad).
Several solubilizing reagents were used alone or in combination in an attempt to solubilize the isolated insoluble inclusion bodies (IB preps). The tested solubilizing reagents included, 8-12 M urea, 6 M guanidine hydrochloride (Gu-HCl), 1% Triton-100, 2% octyl beta-glucoside (OBG), 1% dodecyl maltoside (DDM), 2-4% SDS, 20% lithium dodecyl-sulfate (LDS), 1 M DTT, dimethyl sulfoxide (DMSO), bicarbonate buffer and 1% acetic acid. Additionally, a combination of 1 M DTT with 8 M urea, or 6 M Gu-HCl, or 1% Triton-100, or 2% OBG, or 1% DDM, or 20% LDS, or 4% SDS were tested. Thirty microliters of IB prep was placed in a 1.5 mL tube, and centrifuged at 12,000 rpm (15,294 x g) for 20 minutes at 4°C using an Eppendorf 15 Amp Centrifuge Model 5810R (Eppendorf North America, Hauppauge, New York). After centrifugation, the supernatant was removed, and the pellet was resuspended in 30 µL of a solubilizing reagent. For the use of combined reagents, 30 µL of DTT was added into the tube. After thorough vortexing, the tube was maintained at room temperature (RT) for 30 minutes, followed by centrifugation again at 12,000 rpm for 20 minutes. Solubilization was monitored by visually comparing the size of the pellet in the treated sample with that in an untreated sample. If the inclusion bodies were partially solubilized by the reagent, the pellet would appear smaller than the pellet of the untreated sample. If the inclusion bodies were completely solubilized, no pellet would be observed after centrifugation.
Polyclonal and Monoclonal Anti-IBDP Antibody Production
Mouse monoclonal antibody against IBDP was produced using the standard protocol of the Hybridoma and Protein Core Laboratories, UF Interdisciplinary Center for Biotechnology Research (ICBR) [17
]. Modifications were necessary due to the insolubility of IBDP. Briefly, two female Balb/cByJ mice were immunized with approximately 100 µg of isolated inclusion bodies (IB prep of #08-76) diluted in sterile physiologic phosphate buffered saline (PBS) and emulsified in Ribi MPL+TDM adjuvant. The immunogen was administered on day 1, 21, 44, and 192. The test bleeds were collected 11 to 14 days after the second and third immunizations. The presence of anti-IBDP antibodies in the post-immunized serum was determined by western blots, ELISA, and IHC staining. Six days after the fourth immunization, mouse 1 was euthanized and the splenic lymphocytes were collected and fused with mouse myeloma cells to form hybridoma cells [17
]. The cultured media of the growing hybridoma mass cultures were collected and screened for anti-IBDP antibody production by ELISA. The mass cultures that tested positive by ELISA were subsequently tested by western blots. The mass cultures that had the highest direct optical density (OD) reading on 3 or 4 different IB preps were further tested for their reactivity to the 68 KDa IBDP by western blots. The cultures that showed reactivity to IBDP in both ELISA and western blots were grown out, and further cloned by limiting dilution. The cultured media collected from each clone were tested again by ELISA, western blots and IHC staining. The monoclonal antibodies were isotyped by ELISA and IsoStrip test following manufacturer’s protocol. The cultured medium of the final selected hybridoma clone was harvested, and purified through a protein G column (GE Healthcare Protein G Sepharose 4 Fast Flow). The concentration of the purified monoclonal anti-IBDP antibody was determined by Bradford Protein Assay and stored at 4°C for future validation.
The resolved protein gel was transblotted onto a nitrocellulose membrane using standard protocol of the iBlot dry blotting system (Invitrogen). The membrane was blocked, washed, incubated, and developed following previously described method [17
]. Briefly, after blocking and washing, each lane of the membrane was separated by a Fast Blot-Developer manifold (Pierce, 88040), and incubated with mouse serum in a dilution of 1:100 or 1:500 for 1 hour. For hybridoma mass culture and clone screening, each lane was incubated with undiluted cultured medium. The blot was washed, and incubated with alkaline phosphatase-conjugated rabbit-anti-mouse antibody (Sigma, A1902) at a dilution of 1:1,000 for one hour. After washing, the blot was colorometrically developed by incubation with BCIP/NBT alkaline phosphatase substrate (Sigma, B5655) following instructions of the manufacturer.
Flat bottom 96 well assay plates were coated with IB prep that was diluted in optimal concentrations (10, 20, 30, 40 µg/mL) with bicarbonate buffer. The IB preps isolated from liver and kidney of 2 IBD positive boa constrictors (#08-76, #08-122) were used as coating antigens on separate plates. The general ELISA procedure was the standard protocol used in Hybridoma and Protein Core Laboratories, UF ICBR [17
]. Briefly, the plates were coated by incubation with IBDP overnight at 4°C, washed, blocked, and incubated with diluted mouse serum or undiluted cultured medium of the hybridoma cells, and incubated for 1 hour at RT. After washing, the wells were incubated with a 1:1000 dilution of alkaline phosphate conjugated rabbit anti-mouse IgG antibody (Sigma, A1902) for 1 hour at RT. For antibody subtyping, a conjugated anti-mouse IgG antibody (Sigma, A3438) or a conjugated anti-mouse IgM antibody (Sigma A9688) were used as the secondary antibodies. Subsequently, the wells were washed again and developed with para-nitrophenyl phosphate substrate (PnPP; Sigma, N2765) for 1 hour at RT. The direct OD values of each well were recorded with the absorbance at 405 nm, and compared against the baseline reading, which were the OD reads of wells coated with fresh cell culture medium.
Immuno-Transmission Electron Microscopy
Fresh liver and kidney tissues collected from three IBD+ boa constrictors were submitted for examination using TEM. The tissues were fixed with electron microscopy grade 4% paraformaldehyde (4% PF), 1% glutaraldehyde in 1X PBS, pH 7.24. The samples were washed in PBS pH 7.24, subsequently water washed and dehydrated in a graded ethanol series (25%, 50%, 75%, 95%, 100%, 100%), infiltrated in Lowicryl HM20 acrylic resin (Electron Microscopy Sciences, Hatfield, PA, USA) and UV cured at -10°C for 48 hours. Cured resin blocks were trimmed, thin sectioned and collected on Formvar coated Ni 400 mesh grids (Electron Microscopy Sciences, Hatfield, PA, USA). Ultrathin sections were immuno-labeled at RT as follows; the grids were treated with 200 mM NH4Cl in high salt Tween-20 (HST) for 20 minutes, rinsed in HST, incubated 1 hour with blocking solution (1.5% BSA, 0.5% cold water fish skin gelatin, 0.01% Tween-20 in HST, pH 7.2), and incubated with immunized mouse 1 serum in 1:100 dilution, or the purified anti-IBDP MAB in 1:10 dilution overnight at 4°C. For negative controls, the grids were incubated with non-related mouse IgG antibodies (ICBR-EMBL private stock). The following day, the grids were washed three times in PBS, and incubated for 1 hour at 21°C on 18 nm colloidal gold affinity-purified goat anti-mouse IgG (Jackson ImmunoResearch, West Grove, PA, USA) diluted 1:30 in PBS. Subsequent washes in PBS and distilled water, post-stained with 2% aq. uranyl acetate and Reynold’s lead citrate. Sections were examined with a Hitachi H-7000 TEM (Hitachi High Technologies America, Inc. Schaumburg, IL, USA) and 2k x 2k digital images acquired with a Veleta camera and iTEM software (Olympus Soft-Imaging Solutions Corp, Lakewood, CO, USA).
Formalin Fixation and Embedding
Fresh tissues including liver, kidney, and pancreas obtained from five euthanized boa constrictors were dissected into approximately 5 mm thick sections, placed in cassettes, fixed in 10% NBF or 4% PF for 48 hours, and finally embedded into paraffin. In order to evaluate the effects of formalin fixation time, freshly obtained liver, kidney, and pancreas of an IBD+ boa constrictor were each cut into 10 sections that were approximately 5 mm thick. One piece of each sectioned liver, kidney, and pancreas were placed in a cassette (ten sets of tissues), followed by fixation in ten identical containers filled with 10% NBF. On Day 2 (48 hours after initial fixation), Day 7, Day 8, Day 9, Day 15, Day 23, Day 32, Day 39, Day 50, and Day 58, one cassette was removed and the tissues were embedded in paraffin. For tissue set of Day 58, only kidney and pancreas were embedded.
Paraffin embedded tissues obtained from the case repositories were embedded in the laboratory of Anatomic Pathology Service in the UF Veterinary Hospital (Lab 1). Using an automated processor (Thermo Electric Corporation, Shandon Excelsior), the fixed tissues were dehydrated in graded ethanol, followed by infiltration of xylene and paraffin. The processed tissues were manually mounted in paraffin blocks. In order to evaluate the IHC staining condition, liver and pancreas of one boa constrictor were fixed, processed, and embedded separately in two laboratories, Lab 1 and the laboratory of Molecular Pathology Core (Lab 2), College of Medicine, UF. The embedding procedures of the two labs can be found in Table S1
All H&E staining were done by an automatic slide stainer (Gemini Varistain, Thermo Shandon, Illinois, IL). For paraffin embedded tissues, the slides were deparaffinized with xylene, and the tissue sections were rehydrated in a graded series of ethanol solutions. The rehydrated tissues were stained with hemotoxylin (Richard-Allan Scientific, 7212) for 2 minutes, incubated with clarifier 2 (Richard-Allan Scientific, 7402) for 30 seconds, followed by incubating with bluing reagent (Richard-Allan Scientific, 7301) for 1 minute, then incubated one minute in 80% ethanol before staining with eosin (Richard-Allan Scientific, 71311) for 1 minute. In between the application of each reagent, the slides were washed with running water. For the cytospin prepared IB preps, the fixed microscopic slides were stained with the above protocol with minor modifications as follow. After staining with hemotoxylin, the slides were incubated with clarifier 2 for 15 seconds. After bluing, the slides were stained with eosin without incubating with 80% ethanol. Finally, the H&E stained slides were dehydrated in a graded ethanol series, dipped in xylene, and coverslipped.
Immunohistochemical staining was used for confirming the reactivity of the antibodies to the IBD inclusion bodies within tissue sections. The staining was performed in Lab 2 using the methods provided below.
Slide preparation and antigen retrieval (AR)
For paraffin embedded tissues, the sections were deparaffinized in xylene, followed by rehydrating in graded ethanol, and finally rinsed with water. The deparaffined tissues were either treated with AR reagents or were not treated with AR reagents. The following AR reagents were evaluated: trypsin (Invitrogen, Digest-All2), Trilogy (Cell Marque), Citra (Biogenex), Dako Target Retrieval Solution, pH 6.0 (DAKO), Dako Target Retrieval Solution, pH 9.0 (DAKO). The trypsin AR was done by incubating the slides for 5 minutes at 37°C. The AR treatments with other reagents were done by incubating the slides for 30 minutes at 95°C. For double AR treatment, the slides were incubated with Trilogy for 30 minutes at 95°C, followed by additional 5 minutes of incubation with trypsin at 37°C.
Primary antibody incubation
The prepared slides were washed with tris-buffered saline (TBS), and blocked with Sniper blocking reagent (Biocare Medical, BS966) for 15 minutes at RT. After washing again with TBS, the blocked tissues were covered with diluted mouse serum in antibody diluent (Invitrogen, 00-3218) or with medium collected from the growing hybridoma clones, and incubated overnight at 4°C. For antibody validation, the slides were covered by anti-IBDP MAB in a specific dilution (1:1,000, 1:2,000, 1:5,000, 1:10,000, 1:20,000), and incubated for 1 hour at RT, or overnight at 4°C.
Detection system and counter staining
After washing, the paraffin embedded tissues were incubated with 3% peroxide in methanol for 10 minutes. The slides were rinsed with water and washed by TBS before covering the tissue with HRP conjugated goat-anti-mouse antibody (Biocare Medical, MHRP520) for 30 minutes at RT. After washing, the HRP-conjugated secondary antibody was visualized by development with diaminobenzidine (DAB; Vector Laboratory, SK-4100) or VECTOR NovaRED (Vector Laboratory, SK-4800) according to the manufacturer’s protocol. For the standardized IHC staining, all slides were stained using NovaRED HRP chromagen. The tissues were counterstained with hematoxylin, dehydrated in graded ethanol, placed in xylene, and cover slipped by Cytoseal XYL (Thermo Scientific).
Automated staining machine
For the tissues to be stained automatically, after AR and blocking, the slides were applied onto the automated staining machine (Autostainer Plus, DAKO), in which the washes and incubation of the primary and secondary antibodies were performed. Subsequently, the slides were developed manually using the procedures described above.
Using light microscopy, the intensity of the IHC stain was given one of the four scores: 0 (no staining), 1 (faint, barely visible), 2 (moderate), 3 (strong). For each IHC staining run, a positive control slide (IHC score 3) and negative control slides (IHC score 0) were stained parallel to the slides to be scored. The negative control of each sample was a duplicated slide that stained with a commercial non-specific mouse IgG instead of anti-IBDP antibody.
For evaluating the effect of storage time in paraffin and cross reactivity of the anti-IBDP MAB, the IHC staining results were interpreted as either IHC stain positive or negative. A positive IHC stain (IHC+) was defined by a visible staining pattern (IHC score 1 to 3) compared to the negative control (IHC score 0). A negative IHC stain (IHC-) was defined by no visible staining pattern (IHC score 0) compared to the negative control (IHC score 0).
IHC Diagnostic Performance Evaluation and Statistics
In this study, samples with a positive or negative diagnosis for IBD by examination of the H&E stain (current gold standard) were classified as IBD+ or IBD-, respectively. The sensitivity, specificity, positive predictive value (PPV) and negative predictive values (NPV) of the IHC test, compared to H&E, were calculated following standard procedures [18
]. Sensitivity was defined as the proportion of samples classified as H&E stain positive that tested positive by using the IHC test. Specificity was defined as the proportion of samples classified as H&E stain negative that tested negative by using the IHC test. Positive predictive value was defined as the probability that given a positive IHC test, the sample actually tested positive by using H&E stain. Negative predictive value was defined as the probability that given a negative IHC test result, the sample actually tested negative by using H&E stain. Positive and negative predictive values were calculated using prevalence estimates ranging from 5% to 80% with the calculated sensitivity and specificity of the IHC test determined by this study.
In order to assess the effect of storage time in paraffin, the paraffin embedded tissues were divided into two groups: Group 1. tissue samples embedded within the time period 1990-2000, and Group 2. tissue samples embedded within the time period 2001-2011. The sensitivity and specificity of the IHC test in Group 1 and Group 2 were compared using the Fisher Exact X2
test, following standard procedures [19