Wayne Mohammed

Bacterial Proteins and Their Proposed Interactions with Fc or Fab Fragments of Immunoglobulins

The reactivity of Immunoglobulin Binding Proteins (IBP) to Fc and/or Fab fragments of immunoglobulins was summarized in this review. Staphylococcal protein A (SpA), Streptococcal protein G and Peptostreptococcal protein L (SpL) were the IBP reported. SpA reacted with IgG from skunk, coyote, raccoon, mule and donkey. SpG reacted almost with the entire panel of immunoglobulins and SpL binding was restricted to some immunoglobulins including raccoon, ostrich and duck. The various immunological techniques that have been used to test the binding capacity of IBP to Igs were double immunodiffusion, Enzyme-Linked Immunosorbent Assay (ELISA), SpA-affinity chromatography and immunoblot analysis. These protein-protein interactions are important because they can be used in the immunodiagnosis and in the purification of intact Igs or their fragments.

Preliminary Study of the Identification of Proteins in Tissue Fluids of theSea Mussel Isognomon alatus

The aim of this preliminary study was to investigate the presence of proteins in the tissue fluids of the sea mussel Isognomon alatus. Protein extraction was done by the chloroform-cold ethanol technique. Immunization for production of antibody to be used as reagents in Western blotting, assessment of the protein concentration by the Bradford method, protein characterization by native polyacrylamide gel electrophoresis (PAGE) were also performed as a part of the methodology of this study. The results showed a protein content of 65 mg/ml in tissue fluids and a protein band approximately of 220 kDa in PAGE that was further confirmed by the Western blotting. Future work should investigate the structure and function of the proteins separated from the tissue fluids and we considered it as a limitation of this investigation. The sea bivalve literature is scanty. However the limitation of this work we still can conclude that there are high molecular weight proteins in large concentrations in tissue fluids of the sea mussel Isognomom alatus.

Laying Hen's Primary Immune Response to Staphylococcal Protein A (SpA)

Our aim was to provide information about the production of Egg White Immunoglobulin (EWIg) with specificity to Staphylococcal protein-A, a surface antigen of Staphylococcus aureus and to study the inhibition of this bacterium growth in pre- and post-immunized hens. A sandwich Enzyme-Immunosorbent Assay (ELISA) showed a large concentration of anti-SpA antibodies in the eggs from hens immunized with protein A. The titer of these antibodies was at least 5 to 6-folds of that of the eggs from pre-immunized hens 10 days post-immunization. Inhibition of the growth of S. aureus by anti-SpA antibodies purified by SpA-affinity chromatography (PURE-1A) was observed in laying hens vaccinated. Growth of the bacteria in blood agar plates occurred in antibody samples from preimmunized laying hens only. Inhibition of the agglutination of SpA-bearing Staphylococcus aureus cells by purified anti-SpA antibodies was observed in vitro. The authors are not aware of previous studies of the primary immune system response developed in eggs from laying hens, so this research could set a precedent in the field of egg white immunoglobulin technology. The use of hyper-immune eggs as alternative to the use of antibiotics could be advantageous for the large amount of antibodies produced, low cost, the reduction of antigenic variation and very low toxicity.

Autoimmunity in Neurological and Psychiatric Disorders: Participation of Antibodies and Cytokines in the Immunopathogenesis of these Diseases

Angel Alberto Justiz Vaillant1*, Wayne Mohammed1, Sehlule Vuma1 and Norma Anderson2 1Department of Para-clinical Sciences, Faculty of Medical Sciences, The University of the West Indies, St. Augustine Campus, Trinidad and Tobago, West Indies 2Department of Basic Medical Sciences, The University of the West Indies, Mona campus, Jamaica, West Indies *Corresponding author: Angel Alberto Justiz Vaillant, Department of Para-clinical Sciences, Faculty of Medical Sciences, The University of the West Indies, St. Augustine Campus, Trinidad and Tobago, West Indies, E-mail: avail4883@gmail.com

Purification of Immunoglobulins and their Binding to a Bacterial ProteinLAG-HRP Conjugate

Objective: To purify IgG molecules from several species by SpA-affinity chromatography and to study the interactions of mammalian IgGs with a peroxidase-labelled SpL, SpA and SpG conjugate (SPLAG-HRP) in an enzyme-linked immunosorbent assay (ELISA). Materials and methods: The periodate method described by Nakane and Kawoi was used to prepare the SPLAG-HRP conjugate. The 10% non-denaturing sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) of sera and purified immunoglobulins was carried out to characterize molecularly the purified IgGs. The chicken IgY fraction was isolated by the chloroform-polyethylene glycol (PEG) method for its use as a negative control in the ELISA that was used to determine the affinity of different immunoglobulins to a SPLAG-HRP conjugate. Results: The SpA-affinity chromatography and the 10% non-denaturing SDS-PAGE of sera and purified immunoglobulins (IgGs) were useful separation techniques. Most purified IgGs interacted moderately with the SPLAG-HRP including IgGs from horse, dog, skunk, coyote and raccoon. The purified mammalian IgG had a molecular weight (MW) of approximately 150 kDa. Conclusion: The SPLAG-HRP was a versatile heterofunctional reagent useful for the detection of purified immunoglobulins from diverse mammalian species.

Purification of the Mule (Equus mulus) IgG by Protein A - AffinityChromatography

A mule is the off-spring (F1 hybrid) of two equine species: a male donkey and a female horse. Little is known about the immune system of the mule. In this investigation we isolated and characterized molecularly the mule IgG that was purified by Staphylococcal protein A (SpA) affinity chromatography, and then the purified IgG was further study by SDS-PAGE and enzyme-linked immunosorbent assay that concluded that the intact mule IgG has a molecular weight of 150 kDa.

Immunoglobulin-binding Bacterial Proteins (IBP) Conjugates and their Reactivity with Immunoglobulin in Enzyme-Linked Immunosorbent Assays (ELISA)

The aim of this study was to create universal chimeric conjugates able to react with both avian and mammalian immunoglobulins to be used as a reagent in Enzyme-linked Immunosorbent Assays (ELISAs). The periodate method was used in the conjugation process of linking horseradish peroxidase to immunoglobulin-binding bacterial proteins. ELISAs were used to prove the efficacy of the conjugates, namely newly synthesize conjugates (NSC) in the detection of immunoglobulins. All NSC bound to mammalian immunoglobulins, but failed to bind avian immunoglobulin Y (IgY), with the exception of the SpLAG-anti-IgY-HRP that was the most versatile binding to the all panel of purified immunoglobulin and sera. In conclusion, the results of these experiments show that SpLAG-anti-IgY-HRP conjugate, a novel reagent, was the most versatile product among NSC and commercially-available conjugates.

Development of Anti HIV Gp120 and HIV Gp41 Peptide Vaccines

The aim of this preliminary study was to produce anti HIV antibodies against the fragment 579 601 of the HIV gp41 and fragments 308 331 and 421 438 of the HIV gp120 from the human immunodeficiency virus (HIV) in layer hens. Keyhole limpet hemocynin (KLH) was conjugated to HIV synthetic peptides by the glutaraldehyde method after they were dimerized by cysteine oxidation with dimethyl sulfide. Two healthy brown Leghorn layer hens (per immunogen) were injected intramuscularly on the breasts with KLH peptide conjugated vaccines. They were immunized on days 0,21,45 and 60. Enzyme linked immunosorbent assays (ELISA) were used to test for anti HIV antibodies and there was a statistical significance in the mean of optical density readings between pre immunized and post immunized animals, proving the formation of specific antibodies. These molecules can potentially be used as therapeutic agents or diagnostic reagents. The limitations of this investigation were the small number of cases, viral neutralization produced by anti HIV antibodies was not tested in cell cultures neither their capacity to inhibit the HIV entry into the CD4+ lymphocyte. We conclude that KLH peptide conjugated vaccines against regions of the gp120 and gp41 effectively produced a strong immune response in egg yolks from layer hens. Despite of limitations of this investigation we report an outcome that encourages us to design and perform a larger study, which can be done in future.

Purification of Avian IgY with Trichloroacetic Acid (TCA)

By using trichloroacetic acid (TCA) precipitation to separate egg yolk proteins, mainly IgY was demonstrated that this procedure yield twice more IgY that its counterpart polyethylene glycol (PEG). An ELISA demonstrated that it does not affect the functionality of the IgY molecule. The TCA precipitation procedure provides an efficient and rapid way to purify IgY antibody. It can be used for large scale production of high quality IgY.

Inhibition of the Reactivity of Coombs Sera with Igg-Sensitized Human Erythrocytes by Streptococcal Protein-G (Spg)

Streptococcal protein-G (SpG), type III bacterial Fc receptor, is a small globular protein produced by several Streptococcal species and it is composed of two or three nearly identical domains, each of 55 amino acids. Streptococcal protein G has been shown to have high binding affinity to sera from various mammalian species including rabbit, human, pig, goat, sheep, cow and many other animal species. Of concern are patients with invasive infections by Streptococcus spp, where large amount of secreted SpG could interfere with the outcome of the gel technique by getting false negative tests. It has been shown and reported that the bacterial protein SpA was already found to inhibit the Coombs test. We hypothesize that SpG as well as many other immunoglobulin-binding bacterial proteins with binding affinity to human IgG could cause false negative results in patients with bacteraemia. With the intention of proving this hypothesis we conducted two sets of experiments, which proved that SpG has the potential of inhibiting the gel test for the detection of sensitized erythrocytes in vitro. We concluded that is important to exercise caution, when evaluating a result of gel technique in patients with septicemia caused by IgG-producer Streptococci. The experiments used in this research were novel modifications of existent techniques and they proved reliable in demonstrating our hypothesis.