Rastawicki Waldemar, Smietańska Karolina, Rokosz Natalia, Jagielski Marek
Zakład Bakteriologii Narodowego Instytutu Zdrowia Publicznego-Pańistwowego Zakładu Higieny w Warszawie.
Med Dosw Mikrobiol. 2012;64(1):79-85.
In presented study we investigated the effect of multiple freeze-thaw cycles of human sera on the determination of IgA, IgG and IgM antibodies to selected bacterial antigens.
A panel of 15 serum samples with elevated levels of antibodies to Mycoplasma peumoniae, Yersinia enterocolitica and Salmonella spp. were used (5 positive sera for each pathogen). One set of aliquots designed as the baseline, was taken and stored at 4-8o C for the remainder for the study. The remaining seven sets of aliquots were divided into two parts and repeatedly frozen respectively at two different temperatures: -65 degrees C and -25 degrees C. Once a day the aliquot sets were removed from the freezer and allowed to stand at room temperature for approximately 1 h until completely thawed. For the determination of the level of antibodies the sera after: 2, 5, 10, 15, 20, 25 and 30 freeze/cycle were used. The measurement of IgA, IgG and IgM antibodies was done using a home-made ELISA with four different antigens: whole-cell antigen of M. pneumoniae FH strain, LPS and Yop antigens of Y. enterocolitica serotype O:3 and LPS extracted by Westphal method from Salmonella serogroup B +D. The results were presented as the arithmetic mean of the antibody titre in five sera which were treated by the same number of freeze-thaw cycles.
There was no significant statistic difference between levels of antibodies in unfrozen and frozen sera even after 30 freeze-thaw cycles. Depending of the antigen used in ELISA a slight varations in the level of antibodies were observed but the changes were small and not clinically significant. Examination of the ELISA values does not suggest any consistent nonlinear trend in levels of IgA, IgG and IgM antibodies in sera frozen at -65 degrees C as well at -25 degrees C.
Our study demonstrates that the IgA, IgG and IgM antibody activity levels measured for M. pneumoniae, Y enterocolitica and Salmonella antigens are stable even after 30 freeze-thaw cycles.
在本研究中，我们调查了人血清多次冻融循环对选定细菌抗原的IgA、IgG和IgM抗体测定的影响。
使用一组15份血清样本，这些样本针对肺炎支原体、小肠结肠炎耶尔森菌和沙门氏菌属的抗体水平升高（每种病原体5份阳性血清）。取一组作为基线的等分试样，并在4-8℃下储存，用于研究的其余部分。其余七组等分试样分为两部分，分别在-65℃和-25℃两个不同温度下反复冷冻。每天从冰箱中取出等分试样组，使其在室温下放置约1小时，直到完全解冻。为了测定抗体水平，使用经过2、5、10、15、20、25和30次冻融循环后的血清。使用自制的酶联免疫吸附测定法（ELISA），采用四种不同抗原测定IgA、IgG和IgM抗体：肺炎支原体FH菌株的全细胞抗原、小肠结肠炎耶尔森菌O:3血清型的脂多糖（LPS）和Yop抗原，以及用韦斯特法从B+D血清群沙门氏菌中提取的LPS。结果以经过相同次数冻融循环处理的五份血清中抗体滴度的算术平均值表示。
即使经过30次冻融循环，未冷冻血清和冷冻血清中的抗体水平之间也没有显著的统计学差异。根据ELISA中使用的抗原，观察到抗体水平有轻微变化，但变化很小且无临床意义。对ELISA值的检查未表明在-65℃和-25℃下冷冻的血清中IgA、IgG和IgM抗体水平存在任何一致的非线性趋势。
我们的研究表明，针对肺炎支原体、小肠结肠炎耶尔森菌和沙门氏菌抗原测定的IgA、IgG和IgM抗体活性水平即使在30次冻融循环后仍保持稳定。
