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首页> 外文期刊>Chemicke Zvesti >Comparison between direct and indirect immunofluorescence method for determination of somatic cell count
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Comparison between direct and indirect immunofluorescence method for determination of somatic cell count

机译：直接和间接免疫荧光法测定体细胞计数的比较

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摘要

A sensitive indirect immunofluorescence (IF) assay for bovine neutrophil and somatic cell counting was developed. The obtained indirect IF was compared with direct IF. The direct IF method uses a single-fluorophore-conjugated antibody directed against the target of interest. The indirect IF method uses two antibodies. The primary antibody is unconjugated. The secondary antibody is directed against the primary and has fluorescent marker. Calibration curves for somatic cells (SC) in buffer model solutions were obtained by both methods, using fluorescence spectrophotometry. The measured linear range of somatic cells by the direct method was from 2 x 10(4) to 3 x 10(6) cells and by the indirect method was from 3 x 10(4) to 3 x 10(6) cells. The signal obtained by the indirect method was higher than the direct method at low concentrations of SC (from 30,000 to 100 000 cells/mL). That signal amplification probably is due to more than one fluorescent secondary antibody coupling to bound primary antibody. The same effect is observed by fluorescence microscopy. Consequently, the indirect IF method is more sensitive than the direct IF method. Conjugated primary antibodies are more expensive than their unconjugated counterparts, and secondary antibodies are relatively inexpensive. Therefore, using the same conjugated secondary antibody to detect different primary antibodies is cost-effective. Furthermore, a two-color staining microscopic procedure was proposed for simultaneous estimation of total SC count and neutrophil cell count.
机译：开发了对牛中性粒细胞和体细胞计数的敏感间接免疫荧光（IF）测定。如果与直接相比，所获得的间接。直接IF方法使用针对感兴趣的靶标的单荧光团缀合的抗体。间接如果方法使用两种抗体。初生抗体是未舒张的。二次抗体涉及初级并具有荧光标记物。使用荧光分光光度法通过两种方法获得缓冲模型溶液中体细胞（SC）的校准曲线。通过直接方法测量的体细胞的线性范围为2×10（4）至3×10（6）个细胞，间接方法为3×10（4）至3×10（6）个细胞。通过间接方法获得的信号高于低浓度Sc（从30,000至100000个细胞/ ml）的直接方法。该信号放大可能是由于多于一个荧光二抗偶联与结合的一抗。通过荧光显微镜观察到相同的效果。因此，间接如果方法比直接IF方法更敏感。共轭的原代抗体比其未缀合的对应物更昂贵，二次抗体相对便宜。因此，使用相同的共轭二抗来检测不同的原代抗体是具有成本效益的。此外，提出了一种双色染色微观程序，用于同时估计总Sc计数和中性粒细胞细胞计数。

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