Immunofluorescence methods rely on specific target antigen labeling (somatic cell) with antibody-fluorescent dye conjugate. Anti- neutrophil antibody was chemically conjugated to fluorophore (FITC). The fluorophore allows measuring the remaining unbound conjugate (Ab-FITC) in the solution by fluorescence spectrophotometer. Two different immunofluorescence methods were used – direct and indirect methods. Direct immunofluorescence method uses a single antibody directed against the target of interest. The primary antibody is directly conjugated to a fluorophore. Indirect immunofluorescence method uses two antibodies. The primary antibody is unconjugated and a fluorophore-conjugated secondary antibody directed against the primary antibody is used for detection.
The calibration curves for somatic cells in buffer model solutions were obtained. The concentrations of somatic cells in model solutions were from 10x103 to 3 000x103 cells. The measured linear range of somatic cells in buffer samples by direct method was from 20x103 to 3 000x103 cells and by indirect method was from 40x103 to 3 000x103 cells. The signal obtained in direct methods may seem weak compared to indirect methods, where significant signal amplification is due to secondary antibodies. The indirect method was more sensitive than direct method. Conjugated primary antibodies are usually more expensive than their unconjugated counterparts. Secondary antibodies are relatively inexpensive compared to primary antibodies. Further cost savings may be made by using the same conjugated secondary antibody to detect different primary antibodies.
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