Binding characteristics of selected commercial antibodies against recombinant HIV Subtype D P24 antigens

A research dissertation submitted to the Directorate of Graduate Research and training in partial fulfilment of the requirements for the award of the degree of Masters of Science in Immunology and Clinical microbiology of Makerere UniversityThe standard method for monitoring HIV treatment response is quantitative RT-PCR, but it is difficult to implement in low and middle income countries due to infrastructure and personnel requirements. Point of care technologies for HIV VL determination are needed, but available ones mostly rely on nucleic acid amplification with requirements that still limit their access in these areas. Earlier p24-based technologies as alternatives had low accuracy. One of the major reasons for their failure is poor binding characteristics of anti-p24 antibodies against diverse HIV subtypes, and a lack of reagents to sufficiently disrupt host p24 antigen-antibody immune complexes. Our study aimed at characterizing commercially available anti-p24 antibodies against HIV-1 p24 antigens from which the best antibodies would be used in the development of the innovative rapid diagnostic assay. From April to November, 2022, we conducted a laboratory based innovative study from Makerere University at the Department of Immunology and Molecular Biology. HIV-1 recombinant p24 protein were obtained from MyBioSource & Two anti-P24 antibodies from; Genscript and RayBioTec. We planned to design and optimize an antigen capture ELISA an use it determine the binding avidity of the commercial antibodies by the chaotropic method using Potassium Thiocyanate as the chaotropic agent. The antibody with best binding avidity would be chosen for use in the development of semi-quantitative locally relevant HIV-1 viral load assay. Controls of the antigen capture ELISA failed with ODs ≥1. Reproducibility of the experiments was very poor. Troubleshooting by variation of blocking buffers including use of 5% skimmed milk as an alternative, use of 1:1000 dilution instead of 1:500 dilution of HRP and even change of the secondary antibody from the Goat Anti-HIV-1 p24 polyclonal Antibody, HRP to Goat anti-Mouse to Goat anti-Mouse IgG Antibody, HRP did not improve the antigen capture ELISA. With the above challenges coupled with time constraints and the lack of other relevant resources like precoated plates, we failed fully trouble shoot, redesign and optimize the antigen capture ELISA and later on characterize the primary antibodies for which the study was intended. However, we designed an indirect antigen down ELISA which can be deployed for other purposes. Precoated ELISA plates, characterization of all reagents before use can be deployed in later experiments to improve the antigen capture ELISA design and optimization

Contamination of microbial pathogens and their antimicrobial pattern in operating theatres of peri-urban eastern Uganda: a cross-sectional study

Abstract Background Microbial contamination of hospital environment, especially in operating theatres (OT) and other specialized units has greatly contributed to continuous and multiple exposure to nosocomial infections by patients and the public. We purposed to assess microbial contamination of operating theatres and antibacterial sensitivity pattern of bacteria isolated from theatres of Mbale Regional Referral Hospital, Eastern Uganda. Methods We employed a laboratory based cross-sectional study design. Swabbing of different surfaces and settle plate establishment in 4 various operating theatres was carried out. A total of 109 samples were collected, 31 air samples and 78 swabs from four operating theatres. Samples were collected in the mornings after disinfection prior to start of daily operations. Antibacterial sensitivity testing of isolated bacterial pathogens was performed by Kirby Bauer disc diffusion method following standard operating procedure. Colony counts for the settle plates were carried out using a colony counter. Results All the four theatres had their mean colony counts exceeding the acceptable limit of 5 cfu/dm2/h. Gynaecology theatre had up to 261 cfu/dm2/h and Ophthalmology operating theatre had approximately 43 cfu/dm2/h. A total of 14 different organisms were isolated with Pseudomonas spp. [23.9%]; Bacillus spp. [17.5%] and Aspergillus spp. [15.8%] being the most common contaminants respectively. Other isolates included Enterococcus spp., Rhizopus spp. and Coagulate Negative Staphylococcus isolates especially from settle plates. Most bacterial isolates showed considerable resistance to antibacterial agents. Pseudomonas spp. was resistant to chloramphenicol (53.6%) and cotrimoxazole (57.1%). Most of the bacterial pathogens were sensitive to imipenem [83.3%]. Conclusions There is moderate contamination of operating theatres of Mbale Regional Referral Hospital. Common organisms were Pseudomonas, Bacillus, and Aspergillus spps. Resistance was observed against chloramphenicol and cotrimoxazole. More caution is necessary to carefully disinfect the operating theatres at Regional referral settings and similar tertiary health care centres with more emphasis on obstetrics and gynecology theatres. Diagnosis and care of patients at such clinical settings should consider the possibility of antibiotic resistance

Additional file 3: of Contamination of microbial pathogens and their antimicrobial pattern in operating theatres of peri-urban eastern Uganda: a cross-sectional study

Data description (DOCX 13 kb

Additional file 2: of Contamination of microbial pathogens and their antimicrobial pattern in operating theatres of peri-urban eastern Uganda: a cross-sectional study

Data S2. Susceptibility pattern of microbial pathogens isolated from operating theatres of Mbale Regional Referral Hospital, Eastern Uganda. Indicating value labels for data 1 and data 2. (CSV 24 kb

Additional file 1: of Contamination of microbial pathogens and their antimicrobial pattern in operating theatres of peri-urban eastern Uganda: a cross-sectional study

Data S1. Microbial pathogen contamination of operating theatres of Mbale Regional Referral Hospital, Eastern Uganda. (CSV 3 kb

CBRN Centres of Excellence Newsletter, Volume 20 – Women in CBRN

CBRN Centres of Excellence Newsletter, Volume 20 – Women in CBRN, November 2024JRC.02 - Euratom Coordinatio