Optimized simple and affordable procedure for differentiation of monocyte-derived dendritic cells from LRF: An accessible and valid alternative biological source.

Dendritic cells are one of the most popular immune cells, which plays a remarkable role in both immunotherapy and tolerance induction. Due to unwanted side effects of leukocyte presence in donated blood, the policy of blood service is the pre-storage reduction of leukocytes, which today, filtration is the most common method for this purpose. The filtration method has led to diminished access to Buffy coat as a generally used conventional source of biological cells. We developed a simple, affordable, and reproducible method for dendritic cell differentiation from filter-derived monocytes and, the results of the filter study were compared with differentiated DCs from the conventional buffy coat-derived monocytes. The Monocytes were recovered from leukoreduction filter using an optimized protocol with supplemented PBS buffer. Following the adhesion method, CD14+ Monocyte-enriched population with the purity of 94 % was obtained. After cytokine stimulation over a 6-day period and maturation induction by LPS, differentiated DCs were evaluated for morphology, surface markers (CD86, CD40, CD83 and, HLA-DR), antigen uptake potency and IL-12 secretion. Analysis and comparison of the results represented no significant difference between the two groups. Accordingly, we conclude that leukoreduction filters could be introduced as a reliable and research-grade source of monocyte for DC generation in biological research.

Determination of dissociation constants of anti-ALP monoclonal antibodies by an ELISA - based method.

BACKGROUND: Dissociation constant (Kd) is of major significance in immunoassay. Since affinity may be influenced by the immunoassay methodology it is important to determine this parameter under the condition of the assay used. OBJECTIVE: To employ a rapid and simple ELISA- based method for measuring dissociation constants of two Alkaline Phosphatase (ALP) specific MAbs (A1G8F7 and A1G9G3) established in our laboratory. METHODS: A simple and rapid method on enzyme- linked immunosorbent assay (ELISA) was developed for measuring the dissociation constant of antibody ­ antigen reactions. In this method the ability of increasing amounts of antigen to bind to antibody measured in the fluid phase. RESULTS: Based on the data obtained from this study, the dissociation constants of A1G8F7 and A1G9G3 MAbs were 3.8 × 10−9 and 4.3 × 10−9 M, respectively. CONCLUSIONS: A1G8F7 and A1G9G3 MAbs with reasonably high affinity are suggested for used in very immunoassay such as ELISA, immunocytochemistry and immunihistochemistry (APAAP method).