To note that the anti-PCP IgG2/anti-PCP IgG ratio was similar in HEU and HUU infants (0.282 vs 0.286), suggesting that the transplacental passage of anti-pneumococcus IgG was globally lower in HEU infants, without a selective reduction of the specific anti-PCP IgG2. Clindamycin palmitate HCl Generally, higher total IgG correlates with higher pathogen-specific antibody levels [33]. out of the total IgG are used to describe Clindamycin palmitate HCl the subclasses profileinfection at an age when protection is mostly depending on maternal IgG. total IgG and IgG2 subclass (generally considered associated with protection). Materials and methods Population characteristics This Clindamycin palmitate HCl study is part of a larger study (conducted between January 2019 and June 2021) aimed to assess the factors that determine maternal retention in programs for the prevention of vertical HIV transmission and to compare the Clindamycin palmitate HCl health of HIV-exposed infants under Option B?+?with that of HIV-unexposed infants (including assessment of growth, evaluation of the immune response to vaccines, and of the incidence of infectious and non-infectious events up to 1 1?year of age). The main study enrolled 163 HEU and 72 HUU infants. For the present study we included all HIV-unexposed infants who had available samples collected at 6?weeks and an equal number of HIV-exposed infants (40 HUU and 40 HEU). The mothers were enrolled at week 36 of pregnancy when demographic characteristics were recorded and clinical visits were scheduled. At delivery, and at monthly subsequent visits mother/child pairs data were collected including information about regular ART intake. The study was conducted within the structures of the DREAM (Drug Resource Enhancement against AIDS and Malnutrition) Program of the Community of S. Egidio, an Italian faith-based non-governmental organization. Three clinical sites were involved: the urban DREAM Center, in Mandala, Blantyre, and the semi-urban sites of Chileka and Machinjiri. Mother/child pairs of both groups were followed until 12?months from delivery. Blood samples from 6-week old infants were collected from the plantar surface of the infants’ heel and Dry Blood Spots (DBS) prepared by locally trained people. Using sterile lancets the drops of blood were absorbed onto each circle of Whatman 903 filter paper card. DBS were dried at room temperature for 4?h and then stored at ??20?C, in individual ziplock bags containing a desiccant until shipment to the laboratories at the Istituto Superiore di Sanit in Rome, Italy, where the DBS were stored at? ??20?C, until processing. Dried blood spot processing Two spots from each card were punched out to obtain 20 micro-disks (diameter: 3.2?mm) using a pneumatic DBS Card Punch (Analytical Sales and Services Inc., Flanders, NJ). For elution, we used a methodology already described [21] with a modification, consisting of two steps of extraction. In the first step, the final 20 micro-disks were placed together into Rabbit Polyclonal to MOBKL2A/B a low binding flat-bottom 24-well plate covered with a lid and incubated overnight at?+?4?C in 400?l of elution buffer [Phosphate Buffered Saline (PBS 1??Sigma Aldrich, Milan, Italy)?+?0.05% Tween 20 (Sigma Aldrich, Milan, Aldrich)?+?0.1% BSA (Sigma Aldrich, Milan, Italy) gently shaken with a bench-top shaker; after the first incubation, the soaked punches and elution buffer were transferred into the corresponding centrifuging system, consisting of a 15?mL centrifuge tube (Falcon Polypropylene Conical Tubes, Corning Science) that held a microtube (1.2?ml Corning Cluster Tubes, Salt Lake City, UT), and supported an uncapped 2.5?mL syringe barrel at the open end [22]. Samples were centrifuged at room temperature (RT) for 7?min 1,800 RPM. Eluate was transferred in 1.5 low-binding vials (Protein LoBind Tube, Eppendorf) and centrifuged (14,000 RPM, 15?min RT) to remove debris. In the second step, the remaining soaked punches were re-incubated with 200?l of elution buffer overnight, to remove the remaining blood. The second eluate was processed as the first one, then added to the first elution product, to obtain a final volume of about 500?l. Based on previous reports [23], a 3.2?mm punch was considered to contain 3.275?l of blood; considering a hematocrit value of 50% as acceptable for infants we calculated 1.6375?l of plasma for each 3.2?mm punch. The final dilution was therefore of 1 1:18.3 or 32.75?l (1.6375?l??20 spots) in 600?l of elution buffer. Quantification of IgG and subclasses An automatized nephelometry.