Polyclonal Antibody Preparation Research Articles

123 Polyclonal antibody preparations from avian origin did not reduce plasmatic haptoglobin concentrations but increased ruminal NH3-N in beef steers during transition from forage to high-grain diets

Abstract This study investigated the effects of feeding an avian-derived polyclonal antibody preparation (PAP; CAMAS, Inc.) against Streptococcus bovis, Fusobacterium necrophorum, and lipopolysaccharides (40, 35, and 25% of the preparation, respectively) on plasmatic haptoglobin, ruminal short chain fatty acids (SCFA), and ammonia-N (NH3-N) concentrations of beef steers during a 21-d step-up adaptation to a high-grain diet. Eight ruminally cannulated Angus crossbred beef steers (658 ± 79 kg BW) were randomly assigned to treatments in a crossover design to be transitioned from a diet containing bermudagrass hay [Cynodon dactylon (L.) Pers.] plus 0.45 kg/d of molasses with 0 (CON) or 3 g/d of PAP (PAP) to a high-grain diet. Transition consisted of three 7-d steps of increased inclusion of cracked corn (35, 60, and 80% diet DM for STEP1, STEP2, and STEP3, respectively). On each transition d and 7 d after STEP3 (STEP3-7d), ruminal fluid samples were obtained every 3 h for 24 h. Blood samples were collected on d 0, 1, and 3 relative to each transition for haptoglobin determinations. Haptoglobin plasmatic concentrations increased (P = 0.03) on d 2 and 3 vs. d 1 during STEP2 and on STEP3 compared to STEP1 and STEP2 (P = 0.01). Steers receiving PAP had greater (P < 0.01) ruminal NH3-N concentration in STEP1; however, there were no effects of treatment on SCFA (P > 0.10). Total SCFA concentrations were affected by the step-up diets (P < 0.01); propionate concentration (Pro) was greater in STEP2 through STEP3-7d vs. STEP1 (P < 0.01), whereas acetate concentration (Ac) and Ac:Pro linearly decreased from STEP1 to STEP3-7d (P < 0.01). Feeding 3 g/d of polyclonal antibody preparations against Streptococcus bovis, Fusobacterium necrophorum, and lipopolysaccharides in a 21-d step-up adaptation to high-grain diets did not affect plasmatic haptoglobin or ruminal SCFA concentrations; however, it increased ruminal NH3-3 concentrations.

124 Polyclonal Antibody Preparations from Avian Origin Increased Fiber Digestibility of Beef Cattle Receiving a Backgrounding Diet

Abstract This study investigated the effects of feeding an avian-derived polyclonal antibody preparation (PAP; CAMAS, Inc.) against Streptococcus bovis, Fusobacterium necrophorum, and lipopolysaccharides (40, 35, and 25% of the preparation, respectively) on plasmatic haptoglobin and glucose concentrations (Exp. 1), and apparent total tract digestibility (Exp. 2) of beef cattle consuming a backgrounding diet. In Exp. 1, Angus crossbreed heifers and steers (n = 90; 373 ± 62 kg BW) were randomly assigned to receive a common ad libitum diet (76% TDN, 15.9% CP, DM basis) with the addition of 1 (PAP1), 3 (PAP3), or 0 g (CON) of PAP daily. Plasmatic concentrations of glucose and haptoglobin were measured on d 0, 14, 28, 42, and 56. In Exp. 2, 25 Angus crossbreed steers (390 ± 65 kg BW) were used in a completely randomized design. Steers were housed in 3 pens to receive the same diet and treatments from Exp. 1. Feed intake was measured using GrowSafe feed bunks, and indigestible neutral detergent fiber was used as the internal marker. In Exp. 1, a day effect was detected for plasmatic haptoglobin and glucose concentrations (P ≤ 0.04). In Exp. 2, no difference in DMI was observed (P = 0.88). Dry matter, organic matter, and acid detergent fiber digestibility in the total tract were reduced (P ≤ 0.05), whereas CP digestibility tended to decrease (P ≤ 0.07) in steers receiving PAP3 vs. CON and PAP1. Apparent total tract starch digestibility was increased (P ≤ 0.02) for PAP1 vs. PAP3. Neutral detergent fiber digestibility was greater (P < 0.01) for PAP1 vs. CON and PAP3. Feeding 1 g/d of a PAP against Streptococcus bovis, Fusobacterium necrophorum, and lipopolysaccharides increased fiber digestibility in backgrounding diets; however, further research is needed to understand the impaired responses on nutrient digestibility when greater doses are provided.

Preparation of swine ISG15 polyclonal antibody and its application in classical swine fever virus

Preparation of swine ISG15 polyclonal antibody and its application in classical swine fever virus

Expression, purification and polyclonal antibody preparation of the Schistosoma japonicum SjGrpE protein

To investigate the biological properties of Schistosoma japonicum SjGrpE protein, and to express and purify the recombinant SjGrpE protein and test its immunogenicity. The amino acid composition, molecular weight, hydrophilicity and hydrophobicity, transmembrane region, signal peptide, localization, phosphorylation site, ubiquitination site, glycosylation site, secondary and tertiary structures and B cell epitopes of the SjGrpE protein were predicted using bioinformatics analyses. The SjGrpE gene was amplified using PCR assay using S. japonicum cDNA as a template, double enzyme-digested and linked to the pET28a vector to yield the recombinant plasmid pET28a-SjGrpE. The recombinant plasmid pET28a-SjGrpE was transformed into Escherichia coli BL21, and then IPTG was employed to induce the expression of the target protein, which was purified by nickel ion affinity chromatography. After mice were immunized with the recombinant SjGrpE protein, mouse sera were collected, and the polyclonal antibody against the SjGrpE protein was characterized. SjGrpE protein, which was identified as a hydrophilic protein, was predicted to have a molecular weight of approximately 24.3 kDa without transmembrane regions or signal peptides, and locate in the mitochondrion. SjGrpE protein contained 18 phosphorylation sites and 2 ubiquitination sites, but had no glycosylation sites. In addition, SjGrpE protein contained 5 B-cell epitopes. The full length of SjGrpE gene was approximately 660 bp. The recombinant pET28a-SjGrpE plasmid was successfully generated, and the recombinant SjGrpE protein was obtained following the affinity chromatography, which stimulated mice to secrete high-titer antibodies. The recombinant SjGrpE protein has been successfully prepared and this recombinant protein has a high immunogenicity, which provides a basis for evaluating its value as a vaccine candidate for S. japonicum infections.

Preparation of PrP-specific Polyclonal Antibody via Immunization of PRNP-knockout Mice with Recombinant Human PrP Protein

ObjectiveThe definite diagnosis of human and animal prion diseases depends on the examination of special pathological changes and/or detection of PrPSc in the brain tissues of suspected cases. Thus, developing methods to obtain PrP antibody with good specificity and sensitivity is fundamental for prion identification. MethodsWe prepared a PrP-specific polyclonal antibody (pAb P54) in a PRNP-knockout mouse model via immunization with recombinant full-length human PrP protein residues 23–231. Thereafter, we verified that pAb in Western blot, immunohistochemistry (IHC), and immunofluorescent (IFA) assays. ResultsWestern blot illustrated that the newly prepared pAb P54 could react with recombinant PrP protein, normal brain PrPC from healthy rodents and humans, and pathological PrPSc in the brains of experimental rodents infected with scrapie and humans infected with different types of prion diseases. The electrophoretic patterns of brain PrPC and PrPSc observed after their reaction with pAb P54 were nearly identical to those produced by commercial PrP monoclonal antibodies. Three glycosylated PrP molecules in the brain homogenates were clearly demonstrated in the reactions of these molecules with pAb P54. IHC assay revealed apparent PrP deposits in the GdnCl-treated brain slices of 139A-infected mice and 263K-infected hamsters. IFA tests with pAb P54 also showed clear green signals surrounding blue-stained cell nuclei. ConclusionThe newly prepared pAb P54 demonstrated reliable specificity and sensitivity and, thus, may have potential applications not only in studies of prion biology but also in the diagnosis of human and experimental rodent prion diseases.

Evaluation of liquid and powdered forms of polyclonal antibody preparation against Streptococcus bovis and Fusobacterium necrophorum in cattle adapted or not adapted to highly fermentable carbohydrate diets.

ObjectiveFeed additives that modify rumen fermentation can be used to prevent metabolic disturbances such as acidosis and optimize beef cattle production. The study evaluated the effects of liquid and powdered forms of polyclonal antibody preparation (PAP) against Streptococcus bovis and Fusobacterium necrophorum on rumen fermentation parameters in ruminally cannulated non-lactating dairy cows that were adapted or unadapted to a high concentrate diet.MethodsA double 3×3 Latin square design was used with three PAP treatments (control, powdered, and liquid PAP) and two adaptation protocols (adapted, unadapted; applied to the square). Adapted animals were transitioned for 2 weeks from an all-forage to an 80% concentrate diet, while unadapted animals were switched abruptly.ResultsInteractions between sampling time and adaptation were observed; 12 h after feeding, the adapted group had lower ruminal pH and greater total short chain fatty acid concentrations than the unadapted group, while the opposite was observed after 24 h. Acetate:propionate ratio, molar proportion of butyrate and ammonia nitrogen concentration were generally greater in adapted than unadapted cattle up to 36 h after feeding. Adaptation promoted 3.5 times the number of Entodinium protozoa but copy numbers of Streptococcus bovis and Fibrobacter succinogens genes in rumen fluid were not affected. However, neither liquid nor powdered forms of PAP altered rumen acidosis variables in adapted or unadapted animals.ConclusionAdaptation of cattle to highly fermentable carbohydrate diets promoted a more stable ruminal environment, but PAP was not effective in this study in which no animal experienced acute or sub-acute rumen acidosis.

SUN-357 RABBIT ANTI-THYMOCYTE GLOBULIN INDUCTION THERAPY IN RENAL TRANSPLANTATION

Rabbit anti-thymocyte globulin (rATG) is a preparation of polyclonal antilymphocyte antibodies. It is used not only as an induction therapy to decrease delayed function or rejection of the graft, but also to reverse rejection, with slightly higher rates of complications such as infections and malignancy. The purpose of this study is to review the experience on the use of thymoglobulin as induction treatment in patients with renal transplantation in Cuenca, Ecuador. A retrospective study was conducted in a series of cases (June-September 2019) in the Departament of Nephrology. Universe consisted of 10 patients who underwent renal transplantation from cadaveric donors. All patients received Thymoglobulin induction (daily dose 1,5mg/Kg; 4 patients for 3 days and 6 patients for 5 days), tacrolimus (initial dose 0,075mg/kg per day; if renal function allowed it, dose was adjusted at day five after surgery, increasing it to 1mg/kg per day), mycophenolate (2g/day), and methylprednisolone (inicial daily dose 250mg intravenous for 3 days, then progressive decrease until reaching 20mg daily at first month of follow-up). Leukocyte, lymphocyte, platelet, hemoglobin, urea and creatinine levels were evaluated at five, ten and thirty days after transplantation. Patients age ranked between 20-60 years old (average of 38) and where predominantly female (70%). Hemodialysis (60%). Hypertension was the first cause of renal disease (30%) and cold ischemia time was between 5 to 10 hours. There was an average 50% histocompatibility between donor and recipient. Peak panel-reactive antibody presented in 4% cases. Marked lymphopenia and anemia were evidenced at 5 days with recovery at 10 and 30 days after transplantation. None showed platelet disease. Recovery of renal function became evident from day 5 (1.6mg/dl) presenting at 30 days an improved function of 1.27mg/dl. Infection rate was 50%. In this study, the use of thymoglobulin dose for 3 to 5 days does not represent a risk for infections, so it is considered effective and safe.

The prokaryotic expression, polyclonal antibody preparation, and subcellular localization of the transmembrane protein NS2A of the duck Tembusu virus

Duck Tembusu virus (DTMUV) is a single-stranded, positive-sense RNA arbovirus, belonging to the genus Flavivirus, the family Flaviviridae. As a transmembrane protein, non-structural protein 2A (NS2A) plays an important role in virion assembly, replication complex and antagonizing host immune response. Since NS2A protein contains many hydrophobicaminoacids, it is hard to gain the full-length protein of NS2A for prokaryotic expression. Therefore, to make a deep study, prokaryotic expression and polyclonal antibody preparation of truncated DTMUV NS2A was performed. The truncated NS2A gene (178-450 bp) was obtained, and sub-cloned into the prokaryotic vector pGEX-4T-1 (pGEX-4T-1-NS2A178-450bp). Subsequently, the recombinant GST-NS2A60-150aa protein was successfully expressed in E. coli BL21 (DE3) with the induction by 0.3 mmol/l isopropyl β-D-thiogalactoside (IPTG) for 6 h at 37°C. The GST-NS2A60-150aa protein was extracted from the gel. The BALB/c mice were immunized with the purified recombinant NS2A protein to prepare polyclonal antibodies against the truncated NS2A protein. The titer of the polyclonal antibodies, determined by ELISA analysis, was 1:128,000. The specificity of the polyclonal antibodies (mPAb-DTMUV-NS2A60-150aa) were verified by Western blot analysis. Furthermore, the indirect immunofluorescence (IFA) was performed to explore the subcellular localization of NS2A. NS2A protein was, in the transfected cells, located mainly around nucleus in the endoplasmatic reticulum. Taken together, our study provided a useful tool for the further exploration of the biological functions and molecular mechanism of DTMUV NS2A. Keywords: duck Tembusu virus; non-structural protein 2A; prokaryotic expression; polyclonal antibodies; subcellular location.

416 Polyclonal antibody preparations from avian origin increase mean ruminal pH and reduce rectal temperature of beef steers during transition from forage to high-grain diets

Abstract This study investigated the effects of feeding an avian-derived polyclonal antibody preparation (PAP; CAMAS, Inc.) against Streptococcus bovis, Fusobacterium necrophorum, and lipopolysaccharides (40, 35, and 25% of the preparation, respectively) on dry matter intake (DMI), mean ruminal pH (pH), and rectal temperature (RT) of beef steers during a 21-d step-up adaptation to a high-grain diet. Eight ruminally cannulated Angus crossbred beef steers (658 ± 79 kg of BW) were randomly assigned in a crossover design to be transitioned from a diet containing bermudagrass hay [Cynodon dactylon (L.) Pers.] ad libitum plus 0.45 kg/d of molasses with 0 (CON) or 3 g of PAP (PAP) to a high-grain diet. Transition consisted of three 7-d steps of increased inclusion of cracked corn (35, 60, and 80% of the diet DM for STEP1, STEP2, and STEP3, respectively). On each transition d and 7 d after STEP3 (STEP3-7d), ruminal pH and RT were measured every 3 h for 24 h and feed intake was recorded daily throughout the study using the GrowSafe feed intake monitoring system. Feed intake was not influenced by PAP on transition days (P ≥ 0.44) or during the wk each transition diet was offered (P ≥ 0.57). However, DMI linearly decreased, whereas RT increased from STEP1 to STEP3 (P < 0.01). Steers receiving PAP in STEP2 had reduced RT (P = 0.05). Additionally, steers receiving PAP on STEP3 had greater ruminal pH (P = 0.03), nevertheless, pH was similar between treatments in STEP3-7d (P = 0.39). Feeding 3 g of polyclonal antibody preparations against Streptococcus bovis, Fusobacterium necrophorum, and lipopolysaccharides in a 21-d step-up adaptation to high-grain diets may stabilize ruminal pH and reduce rectal temperature of beef steers. Future studies should characterize the effects of PAP on immune response to elucidate potential modes of action of these feed additives.

20 Polyclonal antibody preparations from avian origin improves growth performance of beef cattle during the first fourteen days of the backgrounding phase

Abstract This study aimed to evaluate the effects of feeding avian-derived polyclonal antibody preparation (PAP; CAMAS, Inc.) against Streptococcus bovis, Fusobacterium necrophorum, and lipopolysaccharides (40, 35, and 25% of the preparation, respectively) on growth performance of beef cattle during the backgrounding phase. From d 0 to 56, Angus crossbreed heifers (n = 80; 360 ± 60 kg of BW; 470 ± 26 d of age) and steers (n = 20; 386 ± 65 kg of BW; 465 ± 30 d of age) were blocked by BW and randomly assigned to 1 of 16 concrete-floored pens (108 m2), equipped with 2 GrowSafe (GrowSafe Systems Ltd., Airdrie, Alberta, Canada) feed bunks each. Animals received a common ad libitum diet (76% TDN, 15.9% CP, DM basis) with the addition of 1 (PAP1), 3 (PAP3), or 0 g (CON) of PAP per d. Feed intake was recorded daily and BW were obtained on d -1, 0, 14, 28, 42, 55, and 56, to assess changes in BW, ADG, DMI, and G:F. Based upon orthogonal contrasts (CON vs. PAP1, and PAP1 vs. PAP3), BW and ADG on d 14, and DMI from 0 to 28, and 0 to 42 were greater for PAP1 vs. CON (P ≤ 0.03), whereas PAP3 animals were intermediate (P ≥ 0.20). No differences in final BW, DMI, and ADG from d 0 to 56 were detected among treatments (P ≥ 0.22). In conclusion, feeding 1g of polyclonal antibody preparations against Streptococcus bovis, Fusobacterium necrophorum, and lipopolysaccharides in a backgrounding diet, improved growth performance in the first 14 d of feeding suggesting that feeding these PAP for longer than 14 d may not be necessary. The effects on subsequent feedlot performance when using PAP should be evaluated in future studies

116 Polyclonal antibody preparations from avian origin improves growth performance of beef cattle during the first fourteen days of the backgrounding phase

Abstract This study aimed to evaluate the effects of feeding avian-derived polyclonal antibody preparation (PAP; CAMAS, Inc.) against Streptococcus bovis, Fusobacterium necrophorum, and lipopolysaccharides (40, 35, and 25% of the preparation, respectively) on growth performance of beef cattle during the backgrounding phase. From d 0 to 56, Angus crossbreed heifers (n = 80; 360 ± 60 kg of BW; 470 ± 26 d of age) and steers (n = 20; 386 ± 65 kg of BW; 465 ± 30 d of age) were blocked by BW and randomly assigned to 1 of 16 concrete-floored pens (108 m2), equipped with 2 GrowSafe (GrowSafe Systems Ltd., Airdrie, Alberta, Canada) feed bunks each. Animals received a common ad libitum diet (76% TDN, 15.9% CP, DM basis) with the addition of 1 (PAP1), 3 (PAP3), or 0 g (CON) of PAP per d. Feed intake was recorded daily and BW were obtained on d -1, 0, 14, 28, 42, 55, and 56, to assess changes in BW, ADG, DMI, and G:F. Based upon orthogonal contrasts (CON vs. PAP1, and PAP1 vs. PAP3), BW and ADG on d 14, and DMI from 0 to 28, and 0 to 42 were greater for PAP1 vs. CON (P ≤ 0.03), whereas PAP3 animals were intermediate (P ≥ 0.20). No differences in final BW, DMI, and ADG from d 0 to 56 were detected among treatments (P ≥ 0.22). In conclusion, feeding 1g of polyclonal antibody preparations against Streptococcus bovis, Fusobacterium necrophorum, and lipopolysaccharides in a backgrounding diet, improved growth performance in the first 14 d of feeding suggesting that feeding these PAP for longer than 14 d may not be necessary. The effects on subsequent feedlot performance when using PAP should be evaluated in future studies

The development of indirect and sandwich ELISA-based detection methods for the detection of Campylobacter jejuni using monoclonal and polyclonal antibody preparations

The development of indirect and sandwich ELISA-based detection methods for the detection of Campylobacter jejuni using monoclonal and polyclonal antibody preparations

On the trail of steroid aromatase: The work of Kenneth J. Ryan

On the trail of steroid aromatase: The work of Kenneth J. Ryan

Bacterial expression, preparation and identification of polyclonal antibody against Escherichia coli FtsZ

To prepare polyclonal antibody (PcAb) against Escherichia coli filamentous thermosensitive protein Z (Ec-FtsZ), the artificially synthesized gene fragment coding Ec-FtsZ was subcloned into pET-22b(+) plasmid, and Ec-FtsZ protein was expressed in E. coli BL21(DE3) cell under an optimal bacterial expression condition. Then Ec-FtsZ protein was purified by HisTrap affinity chromatography, and the GTPase (Guanosine triphosphatase) activity of purified Ec-FtsZ protein was further analyzed by malachite green assay. Subsequently, the purified Ec-FtsZ protein was used to immunize rat subcutaneously for preparation of anti-Ec-FtsZ PcAb. The results of enzyme-linked immunosorbent assay (ELISA), Western blotting analysis and immunofluorescence assay showed that the titer of PcAb was 1:256 000, and PcAb exhibited a perfect antigenic specificity against purified and endogenous Ec-FtsZ protein. All these data indicated that the anti-Ec-FtsZ PcAb is successfully prepared, which can be used for further cellular function study and biochemical analysis of Ec-FtsZ protein in vivo.

Preparation and identification of polyclonal antibody against human cytomegalovirus encoding protein UL23

Human cytomegalovirus (HCMV), a member of the human herpesvirus family, is a common opportunistic virus causing severe ailments and deaths in people with immature or compromised immune systems. UL23 is a virion protein found in the tegument and is expressed in the cytoplasm in HCMV infected cells. However, UL23 is dispensable for viral replication in cultured cells and little is currently known about its function. In order to further study of UL23, polyclonal antibody of UL23 was prepared. UL23 gene fragment was cloned from HCMV Towne by PCR and ligated into pET28a (+). The recombinant plasmid pET28a (+)-UL23 was transformed into E.coli BL21(DE3) to induce expression of the target protein. Then we efficiently purified the recombinant protein affinity chromatography under unique denaturation conditions. Recombinant UL23 protein was used as immunogen to inoculate New Zealand white rabbits and the sera was collected after the fourth immunization. UL23 Polyclonal antibody was purified from antisera using CNBr-activated Sepharose 4 beads. Our UL23 Polyclonal antibody showed specific reaction with UL23 in ELISA, Western-blot and immunofluorescence. More importantly, UL23 Polyclonal antibody could specifically recognize UL23 protein in HCMV infected cells, which laid a foundation for further study of HCMV UL23.

Subpollens delivery of Platanus acerifolia pollen allergen Pla a3 and nucleic acid into lungs and cells

Pollen allergy is a very serious seasonal respiratory disease. However, there has been a lack of understanding how pollen allergens enter the body and act on cells. This study focused on the release, transport and characteristic of Pla a3 allergen of the Platanus acerifolia pollen. Pla a3 protein was purified by prokaryotic expression system for preparation of polyclonal antibody. The distribution and release of Pla a3 protein in pollen were observed by immunohistochemistry. Mice were immunized with purified Pla a3 protein and SPPs, respectively. The pathological examination of mouse lung tissue proved that SPPs, as a fine particle in the range of 0.1-1μm, can enter the deep part of the lung directly through the respiratory tract and led to inflammation. Furthermore, DAPI staining confirmed a certain amount of nucleic acids in SPPs. After incubation with SPPs for 6 h, the Pla a3 mRNA could be detected in A549 cells by PCR. This suggests that nucleic acid wrapped in SPPs could be delivered into A549 cells. These results could provide a new clue and experimental data accumulation for further study on the mechanism of pollen sensitization.

Polyclonal antibodies as a feed additive for cattle adapted or not adapted to highly fermentable carbohydrate diets

ABSTRACT The effect of a polyclonal antibody preparation (PAP) against Streptococcus bovis and Fusobacterium necrophorum on ruminal fermentation and digestion in ruminally cannulated cows was investigated in two 3 × 3 Latin squares in factorial arrangement of treatments 3 × 2 regarding two feed additives (PAP in powder (PAPP) and in liquid (PAPL) presentation) plus control (CON) and two managements of diets (with or without adaptation to highly fermentable carbohydrate diets). Adapted group had greater DMI (p < 0.0001) and DM (p < 0.0001), NDF (p = 0.03) and total carbohydrates (p < 0.0001) apparent digestibility when compared to non-adapted group. PAPL had greater DM (p = 0.02), NDF (p = 0.03) and total carbohydrates apparent digestibility when compared to PAPP or CON. Adapted animals had lower (p < 0.0001) rumen pH when compared to non-adapted animals. Moreover, PAPL group had greater (p = 0.04) rumen pH values when compared to PAPP and CON. PAPL showed potential effect as an additive by increasing apparent digestibility of DM, NDF and total carbohydrates and also for being more efficient to prevent the drop in rumen pH during the peak of fermentation.

An effective strategy for preparation of a polyclonal antibody with an addition of carbon chain of ciprofloxacin

In this study, we synthesized amino propyl ciprofloxacin (CPLX-NH2) as a ciprofloxacin (CPLX) derivative. Moreover, the immune antigen CPLX-NH2-BSA and coating antigen CPLX-NH2-OVA were prepared vi...

Prokaryotic Expression of H1N1 Influenza A Virus Haemagglutinin Protein Globular Domain (HA1)

Abstract Background: Influenza viruses are a significant cause of morbidity and mortality .The influenza virus pandemics, 1918, 1977, and especially the most recent one, A/H1N1/2009, made evident the need for generating recombinant Influenza H1N1 antigens which are essential to develop both basic and applied research programs. Among influenza virus proteins, haemagglutinin (HA) is a major surface antigen of influenza virus, thus it is highly topical in influenza research and vaccine engineering programs. Alternatively, expression of fragments of the HA (HA1 and HA2) proteins in prokaryotic systems can potentially be the most efficacious strategy for manufacture of large quantities of influenza vaccine in a short period of time. Methodology: The gene encoding the HA1 protein from the influenza A/Puerto Rico/8/34 was amplified by PCR, then cloned into pTZ57R/T cloning vector. The fidelity of HA1 open reading frame was confirmed by bidirectional sequencing, then sub-cloned into pET28a prokaryotic expression plasmid, and proteins containing HA1 N-terminally fused to His-Tag were produced in Escherichia coli BL21 through IPTG inducing. The accuracy of the expression was confirmed by running time coursed fraction samples taken before and after the IPTG induction in SDS-PAGE, Western blot analysis was also used for confirmation of the recombinant protein. Conclusion: The HA1 protein produced here could be considered and evaluated as a protective antigen which its immunogenicity potential needs to be assessed in animal models along with proper control groups. Moreover, it could be subjected for polyclonal antibody preparation, which, in turn, may be used as an essential material in western blot analyses, as well as in other immunological applications, such as ELISA, immunocytochemistry, immunohistochemistry, and other immunological and serological studies. Key words: Influenza A, H1N1 , Hemagglutinin , Prokaryotic Expression

IMMUNODIAGNOSIS INFEKSI Aeromonas hydrophila PADA IKAN

Aeromonas hydrophila causes a disease that often infects fish and is known as Motile Aeromonas Septicaemia (MAS), Hemorrhagi Septisemia, Ulcer disease or Red-Sore disease. The aims of this study were to develop polyclonal antibody of Aeromonas hydrophila in the rabbits to confirm the diagnosis of Aeromonas hydrophila in the fish by immunohistochemistry staining method. Preparation of polyclonal antibodies was performed on the rabbits used to Aeromonas hydrophila bacteria that have been tested biochemically by intravenous and intraperitoneal injection. Doses of Aeromonas hydrophila bacteria were 109 CPU/ml of 0.5 ml at first injection, 1 ml at second injection, 2 ml at thirth injection and 3 ml at fourth injection. Blood serum collection was performed at week 5 after injection from an ear and intracardial vein. The result of antibody titer was 28 = 1024 which measured by tube test. Furthermore, polyclonal antibody was used to immunohistochemistry staining with 400x dilution. The results of the staining showed that an immunopositive reaction in the liver, skin,lien, gill, kidney, and heart of fish to Aeromonas hydrophila antibody. The research conclution was polyclonal antibody from rabbit can be used to accurately confirm the diagnosis of Aeromonas hydrophila based on antigen and antibody reaction.