Back ground absorbance values were close to zero throughout all the experiments performed and were arbitrarily assigned a titer of 100 for every antigen tested by ELISA. The binding of antibodies with their cognate antigens was found by applying alkaline order Ivacaftor phosphataseconjugated goat anti mouse immunoglobulins, followed by incubation in p nitrophenyl phosphate. Antibody titers were determined since the greatest dilution of serum giving a noticeable absorbance reading above back ground. History in every one of the ELISAs was thought as the mean absorbance values for sera obtained from mice immunized with mouse serum albumin diluted 1 to 100 in PBS. Antibody titers unique for type 3 PS were determined in the same fashion through the use of Polysorp plates coated with type 3 PS overnight at 4 C, as previously described. Serial dilutions of sera were tested in duplicate. Our observation that MSA immunized mice demonstrated low background absorbances to each of the antigens tested by ELISA provided additional evidence Skin infection that the cohorts of mice evaluated in these experiments hadn’t previously been subjected to S. pneumoniae. Recombinant PsaA, PpmA, PspA, and whole cell lysates of S. pneumoniae strains were subjected to sodium dodecyl sulfate 120-volts polyacrylamide gel electrophoresis and electrophoretically transferred to polyvinylidene difluoride membranes for Western blot analysis. Specific blots were reacted with hyperimmune serum specific for either PsaA, PpmA, or PspA. The walls were subsequently incubated in alkaline phosphatase conjugated goat antimouse immunoglobulin G and manufactured by incubation in BCIP nitroblue tetrazolium chromogenic phosphatase substrate. Indirect immunofluorescence was performed to determine the ability of antibodies raised against recombinant pneumococcal antigens to bind for the surface of intact S. pneumoniae, as previously described. Cryopreserved bacteria comparable to 12 pneumococcal isolates were streaked individually onto blood agar plates incubated for 12 h at 37 C. Germs were re-suspended in staining buffer, washed in sterile PBS, and harvested from the dishes. Around 2 107 microorganisms were incubated with one hundred thousand serum from mice inoculated with MSA as negative controls or specific antigens. After incubation at 4 C, microorganisms were washed in staining buffer and incubated with a 1:50 dilution in staining buffer of the F 2 fragment of goat anti mouse IgG conjugated to Alexa 488 fluorescent dye. Bacteria were then washed in PBS and put through flow cytometry utilizing a Becton Dickinson benchtop flow cytometer. The information were collected and analyzed by using CellQuest software. Currently available data indicate that PspAs among traces could be divided into three families.