Glycol Precipitation Method Research Articles

A novel and efficient immunoglobulin Y extraction method using poloxamer-polyethylene glycol

ABSTRACTAlthough many IgY extraction methods (such as polyethylene glycol (PEG) precipitation method, octanoic acid method, water dilution method, etc.) have been established, there is still industrial drive and real need in developing scale-up IgY production methods. Some previous studies have reported that poloxamer degreasing method shows very good result in IgY extraction from egg yolk with high degreasing speed, harmlessness, simpleness in operation and minimal effect on antibody titer. In this study, we developed a new method, poloxamer-PEG method, to obtain functional IgY with high purity and yield. First, the delipidation solution was added into the diluted yolk samples, and then the filtrates were collected from the diluted yolk samples after 3 hr in room temperature. PEG-6000 was added into the collected filtrates and the mixture was centrifuged after shaking on the roller mixer for 45 min at room temperature. Last, the precipitates were resuspended in 1 mL phosphate buffered solution (PBS) buffer and dialyzed overnight. The results showed that the total protein concentrate of extractive could reach at 30 mg/mL and the purity of the IgY could reach at 92.71% with the novel method, which was superior to the PEG precipitation method and water dilution method.

Coexistence of macroprolactinaemia and hyperprolactinaemia in women with oligo-/amenorrhoea is associated with high risk of pituitary adenomas

Macroprolactin may cause elevation of prolactin (PRL) concentrations measured by standard assays. In our study, we assessed the prevalence of pituitary lesions in women with macroprolactinaemia and either oligomenorrhoea or secondary amenorrhoea. Pituitary MRI scans were performed in 61 women aged 31.0 ± 6.7 years (mean ± SD), with raised PRL concentrations due to macroprolactinaemia, detected by 25% polyethylene glycol (PEG) precipitation method (PRL recovery <40%). After PEG precipitation of macroprolactin, free PRL concentrations were still raised in 36 (59%) women. Microadenomas were detected in 10 patients out of 61 (16.4%), with raised free PRL in 9 of these cases, while macroadenomas were detected in 4 out of 61 (6.6%) women, all of whom also had raised free PRL. In case of coexistence of macroprolactinaemia and raised free PRL after PEG precipitation of macroprolactin, the chance of finding of either a micro- or a macroadenoma was 36% (13 cases out of 36). We conclude that hyperprolactinaemia and macroprolactinaemia may coexist in the same patient. Furthermore, if free PRL is raised after PEG precipitation of macroprolactin, then the chance of finding either a pituitary micro- or macroadenoma in women with oligo-/amenorrhoea is over 30%. Therefore pituitary magnetic resonance imaging is mandatory in all such cases.

Prevalence of macroprolactinaemia in regularly menstruating women with non-toxic goitre or autoimmune thyroid disease

BackgroundThe so called “big-big” prolactin (Prl), also known as macroprolactin is formed by Prl-immunoglobulin (Prl-IgG) complexes and may cause elevation of serum Prl concentrations measured by standard assays, potentially leading to unnecessary investigations and/or treatment. In our study, we have endeavoured to assess the prevalence of macroprolactinaemia in euthyroid, regularly menstruating women with thyroid disease, as well as to assess whether autoimmune thyroid disease may result in an increased prevalence of macroprolactinaemia.Material and methodsWe measured serum Prl in 182 regularly menstruating women aged 32.7 ± 7.5 years (mean ± SD, range 17–46 years) who attended endocrine clinic either for investigation of non-toxic goitre (n = 86, age 33.2 ± 7.8 years) or with autoimmune thyroid disease (n = 96, age 32.3 ± 7.2 years). Autoimmune thyroid disease was defined as raised titre of at least one anti-thyroid antibody [anti-thyroid peroxidase (anti-TPO), anti-thyroglobulin (anti-Tg) and/or anti-TSH-receptor (anti-TSH-R) antibodies]. All women were clinically and biochemically euthyroid, either without or on treatment with L-thyroxine. In those with raised Prl (i.e., above 530 mIU/l) we ruled out the presence of macroprolactinaemia by polyethylene glycol (PEG) precipitation method.ResultsThere was no significant age difference between women with and without autoimmune thyroid disease (p = 0.84). Raised Prl concentrations were found in 10 women with thyroid disease (5.5%), and of those a significant macroprolactinaemia (i.e., reduction of Prl concentrations of more than 60% after PEG precipitation) was found in 9 subjects (4.94%). There were no differences in the prevalence of macroprolactinaemia between women with autoimmune thyroid disease (4 out of 96), and without autoimmune thyroid disease (5 out of 86, p = 0.75).ConclusionsApproximately one out of twenty women with regular menses is likely to have raised serum Prl that is usually caused by the presence of macroprolactinaemia. Though structure of macroprolactin involves Prl-IgG complexes, there is no evidence that autoimmune thyroid disease is associated with raised prevalence of macroprolactinaemia.

EFFECT OF IMMUNOGLOBULIN Y PURIFIED FROM IMMUNIZED HEN EGGS ON THE GROWTH OF STAPHYLOCOCCUS AUREUS

A novel purification method of egg yolk immunoglobulin (IgY) based on precipitation using agarPEG was developed. This me thod was compared with chloroform extraction and polyethylene glycol (PEG) precipitation methods. The results showed the protein contents were high with chloroform method followed by agarPEG then PEG method. The purity of resultant I gY was homogeneous with agar� PEG method followed by PEG method then chloroform extraction method. The IgY purified by agar -PEG method, obtained from hens immunized by formalintreated S. aureus , showed a significant reduction in bacterial growth and the growth inhibition was dependent on specificIgY concentration.

Detection of feline calicivirus as norovirus surrogate in food and water sources using filtration and real-time RT-PCR

Norovirus (NoV) is a major cause of acute non-bacterial gastroenteritis in all age groups worldwide. To detect NoV from foods, polyethylene glycol (PEG) precipitation or ultracentrifugation methods are generally used with reverse transcription (RT)-PCR. These methods need to use complicated procedures and varied buffers depending on the kinds of food matrices. In this study, we suggested a universal method to recover NoV in food and water samples as a prior step to real-time RT-PCR. As a NoV surrogate model, feline calicivirus (FCV) was used. FCV was artificially inoculated to samples, and then concentrated by the adsorption-elution method using negatively charged membrane filters. The detection limit was 4.3×101 PFU/250 mL for distilled water, 4.3×102 PFU/250 mL for environmental waters, and 4.3×102 PFU/15 g for lettuce and oyster. We were able to identify the possibility of one universal and time-saving method to detect NoV in food and water samples without modifications.

Immune complexes in blood serum of calves with clinical symptoms of bronchopneumonia

Pneumonia in preruminant calves is a multifactorial disease. Infectious agents, the environment, management and the immune status of the calves are all important factors in determining the outcome of an infection. Until today, the level and composition of circulating immune complexes in preruminant calves with pneumonia have not been studied in detail. We performed this work with the aim to determine whether pneumonia in three-month-old calves is followed by changes in the immune complex level and changes in the ?-globulin level as their possible constituents. Immune complexes from the calves? sera were isolated by polyethylene glycol (PEG) precipitation methods. Optical density at 350 nm (OD350) of redissolved precipitates was measured to determine the circulating immune complexes level. The OD350 level of PEG precipitates of calves with pneumonia at the time of diagnosis was 0.577?0.206 and it was statistically significantly higher (p&lt;0.001) than OD350 the level of PEG precipitates of healthy calves (0.286?0.080). Electrophoretic analysis of sera and PEG precipitates showed that both slow and fast ?-globulins are found among serum and immune-complexes' ?-globulins, but the concentration of fast ?-globulins was significantly lower in sera of diseased calves. The level of PEG precipitable immune complexes was not correlated with the concentration of serum and PEG precipitable g-globulins. The results of this study have shown that by relatively simple PEG precipitation assay it is possible to detect an increased level of circulating immune complexes in calves with pneumonia. This can be used as an additional diagnostic parameter for the detection and follow up of the disease.

Hypoglycaemia caused by atypical insulin antibodies in a patient with benign monoclonal gammopathy

We describe a 48-year-old woman with recurrent severe hypoglycaemia apparently caused by a paraprotein with insulin-binding capacity. Very high fasting values were found for serum insulin (170 and > 250 mU l-1) as well as for proinsulin 125 pmol l-1 and an insulinoma was suspected. Hypoglycaemia developed after an oral glucose tolerance (OGTT) test but not during fasting for 48 h. Free insulin and C-peptide were normal during OGTT whereas serum insulin was very high. 125I-insulin binding to serum, determined with a polyethylene glycol (PEG) precipitation method was high (40%), and equally high after addition of 1.7 x 10(-5) mol l-1 cold insulin to estimate non-specific binding. By adding very high concentrations of cold insulin, displacement of 125I-insulin bound to serum was found (50% displacement at 4 x 10(-5) mol l-1). No immunoglobulin G (IgG) insulin antibodies were detected by radioimmunoelectrophoresis. On agarose electrophoresis a small paraprotein (4 g l-1) in the gamma-globulin fraction was detected. 125I-insulin binding to this paraprotein was demonstrated. We conclude that if insulin autoantibodies are suspected as a cause of hypoglycaemia screening for insulin antibodies should always be done with a PEG-precipitation method.

Study of circulating immune complexes in three hematologic diseases: Acute myeloid leukemia, acute lymphatic leukemia, and hematosarcoma

Circulating immune complexes (CIC) were detected by means of the polyethylen glycol (PEG) precipitation method, in the serum of untreated patients with hematologic diseases including acute myeloid leukemia (AML), acute lymphatic leukemia (ALL) and hematosarcoma (HS). Immunoglobulins (Ig) and the C 4 fraction of complement were quantitated in the precipitate dissociated by potassium cyanate (KCNO) and in the serum. When compared to control sera, the results showed a simultaneous increase of both precipitate and Ig component. The proportion of each Ig in the precipitate was stable for the controls but variable for the patients. On the whole precipitated proteins, 30% were systematically quantitated in patients and controls. In the remaining portion were noticed Clq and C 3 fractions of complement as well as haptoglobin and albumin. The nature of the antigen was discussed.

An alternative method of large scale plasma fractionation for the isolation of serum albumin.

Human plasma may be separated into five fractions using the method described by Cohn in 1946. Although there are several drawbacks to alcohol precipitation, especially in albumin isolation, it is still used throughout the world. This paper describes an alternative procedure for albumin isolation from plasma or albumin-containing plasma fractions using a combined heat fraction/polyethylene glycol precipitation method. No polyethylene glycol is detected in the final product which is immunoelectrophoretically 100% pure, salt poor, heat resistant during pasteurization, and stable during long-term room temperature storage. The yield is at least 90% of the original plasma albumin. In comparison with the Cohn method, fractionation time and expense are significantly reduced.