Tratamientos y bioterapias para COVID-19 / Therapeutics and biotherapies for covid-19

INTRODUCCIÓN: Un nuevo brote de coronavirus surgió en 2019 en Wuhan, China, causando conmoción en el sistema sanitario de todo el mundo; el Comité Internacional de Taxonomía de Virus lo denominó SARS-CoV-2, agente causante de la enfermedad COVID-19.El espectro de gravedad de la enfermedad es muy amplio: la mayoría de los pacientes no presentan gravedad, pero otros pueden desarrollar neumonías, y la insuficiencia respiratoria aguda es la causa más frecuente de mortalidad. Objetivo: analizar y desarrollar las distintas alternativas terapéuticas aportadas por la Biotecnología para tratar los síntomas de aquellos pacientes con COVID-19. Metodología: se realizó una revisión de la bibliografía disponible, a partir de enero de 2020 en PubMed, acerca de los tratamientos que se encuentran aún en ensayos clínicos y aquellos que cuentan con aprobación bajo uso de emergencia para la enfermedad COVID-19. También se realizaron búsquedas a través de Google y Google Académico para publicaciones de organismos de Salud en referencia a políticas de salud establecidas para la terapéutica durante dicha pandemia. Resultados: este trabajo aborda las nuevas alternativas terapéuticas para COVID-19 derivadas de la Biotecnología, que se encuentran tanto en uso como en etapas de ensayos clínicos comprendidos dentro del segmento de los biofármacos y las bioterapias. Se incluye un breve resumen del estatus regulatorio de entidades de salud, el mecanismo de acción de dichas terapias y características generales de cada uno. Se incluyen novedosas bioterapias que se empezaron a implementar para afrontar la pandemia. Conclusiones: la pandemia de coronavirus está poniendo a prueba el sistema sanitario internacional, para brindar soluciones tanto desde el diagnóstico y prevención como para el tratamiento de la población a fin de disminuir la mortalidad. Esto incluyó, obviamente también, al área de la Biotecnología aplicada a la salud, que ha aportado en los tres aspectos mencionados; el presente trabajo se centra en las respuestas de tipo terapéutico que ha brindado y que están comercializadas o en fases clínicas. (AU)

INTRODUCTION: A new coronavirus outbreak emerged in 2019 in Wuhan, China, causing a shock to the healthcare system around the world; the International Committee on Taxonomy of Viruses named it SARS-CoV- 2, the infectious agent of the COVID-19 disease. The spectrum of severity of the disease is very wide, most patients are not serious, but others can develop pneumonia, with acute respiratory failure being the most frequent cause of mortality. Objective: to analyze and develop the different therapeutic alternatives provided by Biotechnology dedicated to Health, to treat the symptoms of those COVID-19 patients who require it, and thus reduce mortality.Methodology: a review of the available literature from January 2020 in PubMed of the treatments that are still in clinical trials and those that have been approved under emergency use for the disease COVID-19 was performed. Searches were also carried out through Google and Google Scholar for publications of Health organizations in reference to health policies established for therapeutics during the mentioned pandemic. Results: this work addresses the new therapeutic alternatives derived from Biotechnology, which are both in use and in stages of clinical trials, to treat patients who developed COVID-19 included within the segment of biopharmaceuticals and biotherapies. A brief summary of the regulatory status of health entities, the mechanism of action of said therapies and general characteristics of each one is included. Innovative biotherapies that began to be implemented to face the pandemic are included. Conclusions: The coronavirus pandemic has driven the international health system to the test, to provide solutions both from the diagnosis, prevention and treatment of the population to reduce the mortality of patients. This obviously also included the area of Biotechnology applied to health, which has contributed in the three aspects mentioned. The present work focuses on the therapeutic responses that it has provided and that are commercialized or in clinical phases. (AU)

Codon Optimization, Soluble Expression and Purification of PE_PGRS45 Gene from Mycobacterium tuberculosis and Preparation of Its Polyclonal Antibody Protein.

Studies have demonstrated that PE_PGRS45 is constitutively expressed under various environmental conditions (such as nutrient depletion, hypoxia, and low pH) of the in vitro growth conditions examined, indicating that PE_PGRS45 protein is critical to the basic functions of Mycobacterium tuberculosis. However, there are few reports about the biochemical function and pathogenic mechanism of PE_PGRS45 protein. The fact that this M. tuberculosis gene is not easily expressed in E. coli may be mainly due to the high content of G+C and the use of unique codons. Fusion tags are indispensable tools used to improve the soluble expression of recombinant proteins and accelerate the characterization of protein structure and function. In the present study, His6, Trx, and His6-MBP were used as fusion tags, but only MBP-PE_PGRS45 was expressed solubly. The purification using His6-MBP tag-specific binding to the Ni column was easy to separate after the tag cleavage. We used the purified PE_PGRS45 to immunize New Zealand rabbits and obtained anti- PE_PGRS45 serum. We found that the titer of polyclonal antibodies against PE_PGR45 was higher than 1:256000. The result shows that purified PE_PGRS45 can induce New Zealand rabbits to produce high-titer antibodies. In conclusion, the recombinant protein PE_PGRS45 was successfully expressed in E. coli and specific antiserum was prepared, which will be followed by further evaluation of these specific antigens to develop highly sensitive and specific diagnostic tests for tuberculosis.

Characterization of an antigenic serine protease in the Trichinella spiralis adult.

Serine proteases have been identified as important molecules that are involved in many parasitic infections, and these molecules have also been suggested to play important roles in Trichinella spiralis infections. In the present study, the antigenic serine protease gene Ts-ADSp-7, which was screened from a cDNA library of Trichinella spiralis Adults at 3 days post-infection (p.i.), was cloned and expressed in Escherichia coli. The encoded protein, Ts-ADSp-7, revealed a potential trypsin-like serine protease domain but lacked substrate banding site at position 227 and protease activity. Transcription could be detected in the Adult and muscle larval stage but not in the newborn larval stage, where no fluorescent signal was detected. Western blot analysis revealed that the 3 days p.i. Adults and muscle larvae could secrete Ts-ADSp-7. Interestingly, strong fluorescent signal of Ts-ADSp-7 could be detected in the nucleoli of the enlarged muscle cell nuclei from 12 to 16 days p.i. and in the ß-stichosomes of the muscle larvae from 16 to 35 days p.i.. The coagulation assay indicated that Ts-ADSp-7 could inhibit intrinsic coagulation pathway. Regarding the putatively important function of the serine protease in the helminth infection to hosts, a total of 81 serine proteases were found in the parasite and mainly comprised eight subfamilies. These subfamilies exhibited high similarity to transmembrane serine protease, coagulation factor XI, lipocalin, guanylin, ceropin, kallikrein, and plasminogen. Moreover, stage specificity was detected in several subfamilies. In summary, the putatively inactive serine protease-like protein Ts-ADSp-7 could inhibit blood coagulation, and the protein is located in the enlarged nuclei of nurse cells during capsule formation. Furthermore, members of the serine protease family in the parasite might be important molecules in the parasite-host interaction.

Purification of a Human Prostate Specific Antigen.

Rabbit antiserum raised against the crude extract of normal human prostatic tissue contained antibodies to a prostatic tissue-specific antigen as shown by immunoprecipitation techniques. Using this antiserum a prostate antigen was detected in normal, benign hypertrophic, and malignant prostatic tissues, but not in other human tissues. The prostate antigen was purified to homogeneity from prostatic tissues and showed a single protein band on analytical polyacrylamide gel electrophoresis and isoelectric focusing. This report thus presents the first demonstration of the purification of a prostate-specific antigen that does not represent prostatic acid phosphatase.

Generation of specific antisera directed against D-amino acids: focus on the neuroanatomical distribution of D-glutamate and other D-amino acids.

This review updates the findings about the anatomical distribution (using immunohistochemical techniques) and possible functions of D-glutamate in the central nervous system of mammals, as well as compares the distribution of D-glutamate with the distribution of the most studied D-amino acids: D-serine and D-aspartate. The protocol used to obtain highly specific antisera directed against D-amino acids is also reported. Immunoreactivity for D-glutamate was found in dendrites and cell bodies, but not in nerve fibers. Perikarya containing D-glutamate were found in the mesencephalon and thalamus. The highest density of cell bodies was found in the dorsal raphe nucleus, the mesencephalic central grey matter, the superior colliculus, and in the subparafascicular thalamic nucleus. In comparison with the distribution of immunoreactive cell bodies containing D-serine or D-aspartate, the distribution of D-glutamate-immunoreactive perikarya is less widespread. Currently, the physiological actions mediated by D-glutamate in the brain are unknown but the restricted neuroanatomical distribution of this D-amino acid suggests that D-glutamate could be involved in very specific physiological mechanisms. In this sense, the possible functional roles of D-glutamate are discussed.

A novel approach for preparation of the antisera reagent for potency determination of inactivated H7N9 influenza vaccines.

BACKGROUND: The potency of inactivated influenza vaccines is determined using a single-radial immunodiffusion (SRID) assay and requires standardized reagents consisting of a Reference Antigen and an influenza strain-specific antiserum. Timely availability of reagents is a critical step in influenza vaccine production, and the need for backup approaches for reagent preparation is an important component of pandemic preparedness. OBJECTIVES: When novel H7N9 viruses emerged in China in 2013, candidate inactivated H7N9 influenza vaccines were developed for evaluation in clinical trials, and reagents were needed to measure vaccine potency. METHODS: We previously described an alternative approach for generating strain-specific potency antisera, utilizing modified vaccinia virus Ankara vectors to produce influenza hemagglutinin (HA)-containing virus-like particles (VLPs) for immunization. Vector-produced HA antigen is not dependent upon the success of the traditional bromelain-digestion and HA purification. RESULTS: Antiserum for H7N9 vaccines, produced after immunization of sheep with preparations of bromelain-HA (br-HA), was not optimal for the SRID assay, and the supply of antiserum was limited. However, antiserum obtained from sheep boosted with VLPs containing H7 HA greatly improved the ring quality in the SRID assay. Importantly, this antiserum worked well with both egg- and cell-derived antigen and was distributed to vaccine manufacturers. CONCLUSIONS: Utilizing a previously developed approach for preparing vaccine potency antiserum, we have addressed a major bottleneck encountered in preparation of H7N9 vaccine reagents. The combination of br-HA and mammalian VLPs for sequential immunization represents the first use of an alternative approach for producing an influenza vaccine potency antiserum.

Expression and antibody generation of the cancer-testis antigen, BIOT2-S.

Biot2-S is a mouse cancer-testis antigen gene that was identified using the cross-reactive serological analysis of recombinant cDNA expression libraries (SEREX) technique in the State Key Laboratory of Biotherapy, West China Hospital, Sichuan University. To express BIOT2-S and generate its antibody for further investigation, the Biot2-S prokaryotic recombinant expression vector Biot2-S/pGEX6P-1 was constructed with Escherichia coli DH5α as a cloning vector, and BIOT2-S was expressed in E. coli Rosetta (DE3). The recombinant BIOT2-S was expressed in the form of an inclusion body and the targeted recombinant BIOT2-S was produced at the level of approximately 25% total bacterial proteins after being induced with optimum conditions (0.2 mM isopropyl-ß-D-thiogalactopyranoside for 6 h at 37°C). The target protein was purified by glutathione S-transferase (GST)-trap FF affinity chromatography and detected by western blot. The purified recombinant protein was further confirmed by electrospray ionization quadrupole time-of-flight mass spectrometry after removal of the GST-tags. Then the purified BIOT2-S was used to immunize adult rabbits to generate its antibody. The antibody was purified and its specificity determined. The titer of the antibody was shown to reach 10(4) and the antibody was demonstrated to be able recognize the corresponding protein in the testes of mouse and chicken; the tumor cell lines CT-26 and S180 also reacted with the antibody. This study provides a valuable foundation for further research on the cancer-testis antigen BIOT2-S.

[Prokaryotic expression of vp3 gene of Muscovy duck parvovirus, and its antiserum preparation for detection of virus multiplication].

New epidemic broke out in recent year which was suspected to be caused by variant Muscovy duck parvovirus (MDPV). For this reason, new MDPV detection methods are needed for the new virus strains. In this study, a pair of primers were designed according to the full-length genome of MDPV strain SAAS-SHNH, which were identified in 2012, and were used to amplify the vp3 gene of MDPV by polymerase chain reaction. After being sequenced, the vp3 gene was subcloned into the prokaryotic expression vector PET28a. The recombinant plasmid was transformed into E. coli BL21 and induced with IPTG. SDS-PAGE and Western blotting analysis showed the MDPV vp3 gene was successfully expressed. After being purified by Ni2+ affinity chromatography system, the recombinant protein was used as antigen to immunize rabbits to obtain antiserum. Western blotting analysis showed that the acquired antiserum could react specifically with VP3 protein of J3D6 strain and MDPV vaccine strain. The antiserum could also be used for detection of cultured MDPV from primary duck embryo fibroblasts by immune fluorescence assay (IFA). It could be concluded that the VP3 protein and its antibody prepared in the research could be used for detection of VP3 antiserum and antigen respectively.

Effective equine immunization protocol for production of potent poly-specific antisera against Calloselasma rhodostoma, Cryptelytrops albolabris and Daboia siamensis.

Snake envenomation has been estimated to affect 1.8 million people annually with about 94,000 deaths mostly in poor tropical countries. Specific antivenoms are the only rational and effective therapy for these cases. Efforts are being made to produce effective, affordable and sufficient antivenoms for these victims. The immunization process, which has rarely been described in detail, is one step that needs to be rigorously studied and improved especially with regard to the production of polyspecific antisera. The polyspecific nature of therapeutic antivenom could obviate the need to identify the culprit snake species. The aim of this study was to produce potent polyspecific antisera against 3 medically important vipers of Thailand and its neighboring countries, namely Cryptelytrops albolabris "White lipped pit viper" (CA), Calleoselasma rhodostoma "Malayan pit viper" (CR), and Daboia siamensis "Russell's viper" (DS). Four horses were immunized with a mixture of the 3 viper venoms using the 'low dose, low volume multi-site' immunization protocol. The antisera showed rapid rise in ELISA titers against the 3 venoms and reached plateau at about the 8th week post-immunization. The in vivo neutralization potency (P) of the antisera against CA, CR and DS venoms was 10.40, 2.42 and 0.76 mg/ml, respectively and was much higher than the minimal potency limits set by Queen Soavabha Memorial Institute (QSMI). The corresponding potency values for the QSMI monospecific antisera against CA, CR and DS venoms were 7.28, 3.12 and 1.50 mg/ml, respectively. The polyspecific antisera also effectively neutralized the procoagulant, hemorrhagic, necrotic and nephrotoxic activities of the viper venoms. This effective immunization protocol should be useful in the production of potent polyspecific antisera against snake venoms, and equine antisera against tetanus, diphtheria or rabies.

[Expression and purification of HPV16 E2 protein and preparation of its antiserum].

OBJECTIVE: To express and purify the human papillomavirus type 16 (HPV16) E2 protein in prokaryotic bacteria and prepare the antiserum of HPV16 E2. METHODS: After amplified by PCR, HPV16 E2 was inserted into pET21b vector. The recombinant pET21b-HPV16E2 vector was transfected into E.coli BL21 (DE3). Expression product was identified after induction. Through purification, denaturation and renaturation, soluble protein was obtained. With the HPV16 E2 protein, we immunized BALB/c mice and examined mouse IFN-γ, CD4(+); T cells, CD8(+); T cells, CD4/CD8 ratio and antiserum titer. RESULTS: Restriction digestion and DNA sequencing showed pET21b-HPV16E2 was constructed successfully. Relative molecular mass (Mr;) of HPV16 E2 was 42 000 in SDS-PAGE and the specificity of the protein was confirmed with Western blotting. The antiserum could specifically bind with HPV16 E2 protein. In the immunized BALB/c mice, antiserum titre, CD4(+); T cell count and CD4/CD8 ratio increased, while mouse IFN-γ did not change obviously. CONCLUSION: Soluble HPV16 E2 protein was obtained successfully. The antiserum of high titer against HPV16 E2 was prepared in mice.

Indirect homologous competitive enzyme-linked immunosorbent assay for the detection of a class of glycosylated dihydrochalcones.

Hesperetin dihydrochalcone 4'-glucoside, 1, and phloretin 4'-glucoside, 2, belong to a family of dihydrochalcone glycosides that exhibit flavorant properties. In this study was developed a competitive, indirect homologous ELISA for the detection of targets 1 and 2 in fermentation media. Immunogen and coating antigen were prepared by conjugating hapten, 4-(3-oxo-3-(2,6-dihydroxy-4-glucoside phenyl)propyl) benzoic acid, to thyroglobulin and bovine serum albumin, respectively. Antibodies raised in rabbits M6122, M6123, and M6124 and the coating antigen were screened and characterized to determine their optimum concentrations. The optimized ELISA, developed with antibody M6122, gave IC50 values of 27.8 and 21.8 ng/mL for 1 and 2, respectively. Selectivity of the assay was assessed by measuring cross-reactivity of antibody M6122 to related congeners such as aglycones and the 2'-glycosides of hesperetin dihydrochalcone, 5 and phloretin, 6. Antibody M6122 showed very low recognition of 5 and virtually no recognition of the aglycones and 6.

An investigation into the potential use of nanoparticles as adjuvants for the production of polyclonal antibodies to low molecular weight compounds.

Two nanoparticle based adjuvants were assessed for their ability to produce polyclonal antibodies in rabbits to low molecular weight target analytes, i.e. veterinary drugs banned from use in food producing animals. The nanoparticles, Montanide IMS 251 and amphiphilic poly (γ-glutamic acid) were compared against a mineral oil adjuvant, Montanide ISA 50, which had previously been shown to be successful in producing antibodies to haptens whilst being safe to use with respect to the welfare of the host animals. The adjuvants were assessed for their tendency to cause adverse effects to the host animals and by the quality of the antibodies generated in terms of assay sensitivity. None of the three adjuvants employed in the trial generated any measurable adverse effects in the host animals. While the mineral oil adjuvant produced higher titres of antibodies the nanoparticle adjuvants were found to produce antibodies of statistically comparable sensitivity. Based on IC(50) values, six antisera displayed potential to detect the required level of the target compounds; five of these were produced by rabbits immunised with the two different nanoparticle adjuvants. As antibody sensitivity is the main performance criteria of an analytical immunoassay, it can be concluded that the nanoparticle adjuvants under evaluation are fit for the purpose described in this study.

[Human CD96 gene cloning, expression and identification].

OBJECTIVE: To construct and express human CD96 gene outer membrane domain (hCD96om) in prokaryotic cells and prepare rabbit polyclonal antibody of hCD96om. METHODS: hCD96om was amplified by RT-PCR from the peripheral blood of patients with acute myeloid leukemia and inserted into prokaryotic expression vector pET32a(+) to construct the recombinant plasmid pET32-CD96. The expression of hCD96om was induced by IPTG in BL21(DE3) cells, and the expression product was identified by Western blotting. The anti-hCD96 polyclonal antibody was prepared by immunization of rabbits with the fusion protein. The specificity of anti-hCD96 antibody was determined by Western blotting. RESULTS: hCD96om protein was expressed in E.coli BL21(DE3) cells in the form of inclusion body, with a relative molecular mass around 37 kD. Western blotting showed a specific reaction of the prepared antiserum with the 70 kD protein extracted from human leukemia cell line HL-60 cells and with the 37 kD hCD96om fusion protein. CONCLUSION: The CD96 gene of human has been successfully cloned and expressed in BL21(DE3) cells, and its rabbit polyclonal antibody has been obtained.

[Evaluation of the efficiency and reactogenicity of emulsion-based adjuvant systems in the manufacture of polyclonal enteroviral diagnostic sera].

Whether various adjuvants might be used in the manufacture of commercial enteroviral diagnostic sera (EDS) was studied. The following adjuvants: Ribi, SAF-1, and TiterMax were compared; vaseline-lanoline emulsion used to prepare EDSs, as well as modified Freund's complete adjuvant served as controls. Chinchilla rabbits were intramuscularly injected enterovirus antigens (enterovirus 70 and ECHO 2) together with the adjuvant emulsions. TiterMax showed the highest efficiency comparable with the activity of Freund's adjuvant. The activities of Ribi, SAF-1, and vaseline-lanoline emulsion were 3-4 times lower. The neutralizing activity of the sera obtained after 2-3 (TiterMax) or 4-5 (Ribi, SAF-1) immunizations was maximal. Further immunizations resulted in a reduction in the titers of neutralizing antibodies. TiterMax and vaseline-lanoline emulsion caused minimal complications at the site of inoculation whereas SAF-1 and Ribi gave rise to severer inflammatory responses.

Shared antigenicity between the polar filaments of myxosporeans and other Cnidaria.

Nematocysts containing coiled polar filaments are a distinguishing feature of members of the phylum Cnidaria. As a first step to characterizing the molecular structure of polar filaments, a polyclonal antiserum was raised in rabbits against a cyanogen bromide-resistant protein extract of mature cysts containing spores of Myxobolus pendula. The antiserum reacted only with proteins associated with extruded polar filaments. Western blot and whole-mount immunohistochemical analyses indicated a conservation of polar filament epitopes between M. pendula and 2 related cnidarians, i.e., the anthozoan, Nematostella vectensis, and the hydrozoan, Hydra vulgaris. This conservation of polar filament epitopes lends further support to a shared affinity between Myxozoa and cnidarians.

Sandwich enzyme-linked immunosorbent assay (ELISA) for detection of cashew nut in foods.

The presence of undeclared cashew can pose a health risk to cashew-allergic consumers. The food industry has the responsibility to declare the presence of cashews on packaged foods even when trace residues are or might be present. The objective of this study was to develop a rapid, sensitive, and specific enzyme-linked immunosorbent assay (ELISA) for the detection of cashew residues. Raw and roasted cashews were defatted and used separately to immunize sheep, goats, and rabbits. The cashew ELISA was developed using sheep and rabbit polyclonal anti-roasted cashew sera as capture and detector reagents, respectively, with visualization through an alkaline phosphatase-mediated substrate reaction. The cashew ELISA was shown to have a limit of quantification of 1 ppm (1 µg cashew/g). The ELISA was highly specific except that substantial cross-reactivity was noted with pistachio and a lesser degree of cross-reactivity was noted with hazelnut. The performance of the ELISA was assessed by manufacturing cookies, ice cream, and milk chocolate with added known amounts (0 to 1000 ppm) of cashew. The mean percent recoveries for ice cream, cookies, and milk chocolate were 118%± 2.9%, 84.3%± 4.0%, and 104%± 3.0%, respectively. In a limited retail survey, 4/5 retail samples with cashew declared on ingredient labels tested positive for cashew compared to 5/36 samples of foods with precautionary labels indicating the possible presence of one or more tree nuts and 0/18 samples without cashew declared on the label in any manner. The cashew ELISA can be used to detect undeclared cashew residue in foods and as a potential tool for the food industry to assess the effectiveness of allergen control strategies and to guarantee compliance with food labeling regulatory requirements.

Trypanosoma evansi: identification and characterization of a variant surface glycoprotein lacking cysteine residues in its C-terminal domain.

African trypanosomes are flagellated unicellular parasites which proliferate extracellularly in the mammalian host blood-stream and tissue spaces. They evade the hosts' antibody-mediated lyses by sequentially changing their variant surface glycoprotein (VSG). VSG tightly coats the entire parasite body, serving as a physical barrier. In Trypanosoma brucei and the closely related species Trypanosoma evansi, Trypanosoma equiperdum, each VSG polypeptide can be divided into N- and C-terminal domains, based on cysteine distribution and sequence homology. N-terminal domain, the basis of antigenic variation, is hypervariable and contains all the exposed epitopes; C-terminal domain is relatively conserved and a full set of four or eight cysteines were generally observed. We cloned two genes from two distinct variants of T. evansi, utilizing RT-PCR with VSG-specific primers. One contained a VSG type A N-terminal domain followed a C-terminal domain lacking cysteine residues. To confirm that this gene is expressed as a functional VSG, the expression and localization of the corresponding gene product were characterized using Western blotting and immunofluorescent staining of living trypanosomes. Expression analysis showed that this protein was highly expressed, variant-specific, and had a ubiquitous cellular surface localization. All these results indicated that it was expressed as a functional VSG. Our finding showed that cysteine residues in VSG C-terminal domain were not essential; the conserved C-terminal domain generally in T. brucei like VSGs would possibly evolve for regulating the VSG expression.

Characterization of an antiserum against Atlantic cod (Gadus morhua L.) g-type lysozyme.

In this study we describe the production and characterization of an antiserum against recombinant g-type lysozyme derived from Atlantic cod. This is also the first initial analyses of g-type lysozyme protein expression in tissues of Atlantic cod. Recombinant expression and purification of cod g-type lysozyme was used for immunization to rabbit and the rabbit sera were analysed for anti g-type lysozyme antibodies using enzyme-linked immunosorbent assay (ELISA), Western blot and immunohistochemistry. ELISA results showed that antibody titres were mounted between 12,800 and 25,600 as measured at an optical density corresponding to 50% of the maximal level. By Western blot analysis the rabbit immune serum detected a single ∼23 kDa band representing the size of the injected antigen, in both spleen and head kidney homogenates from the Atlantic cod. Immunohistochemisrty detected the native folded g-type lysozyme in tissues and revealed that g-type lysozyme positive cells were observed in haematopoietic tissue of the head kidney and in red pulp of spleen. In conclusion, the rabbit anti g-type lysozyme immune sera was developed and is effectively utilized for ELISA, Western analysis as well as for immunohistochemistry. This has allowed us to obtain new knowledge about this protein regarding localization and distribution in cod tissue.

Production and purification of polyclonal antibodies.

Polyclonal antibodies are derived from multiple B-cell clones that have differentiated into antibody-producing plasma cells in response to an immunogen. Polyclonal antibodies raised against a single molecular species of antigen recognize multiple epitopes on a target molecule resulting in signal amplification in indirect immunoassays, including immuno-electron microscopy. In this chapter, we present a basic procedure to generate polyclonal antibodies in rabbits.

Giardia lamblia: intracellular localization of alpha8-giardin.

Alpha8-giardin (α8-giardin) is a member of the multi-gene α-giardin family in the intestinal parasitic protozoan, Giardia lamblia. This gene family shares an ancestry with the annexin super family, whose common characteristic is calcium dependent binding to membranes that contain acidic phospholipids. In the present study, the antigenicity, hydrophilicity, flexibility, surface probability, and secondary structure of α8-giardin amino acids were predicted by bioinformatics applications. A specific anti-peptide antiserum, anti-P3, was used to determine the intracellular location of α8-giardin with confocal immunofluorescence microscopy and immunoelectron microscopy. The results indicated that α8-giardin was located on the plasma membrane and flagella, but not on the ventral disk. Reduction of α8-giardin transcript levels by ribozyme-mediated cleavage decreased trophozoite motility and growth rate, indicating the functional importance of α8-giardin to Giardia trophozoite biology.