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Antigens
Published in Constantin A. Bona, Francisco A. Bonilla, Textbook of Immunology, 2019
Constantin A. Bona, Francisco A. Bonilla
Several mechanisms may lead to alterations in antigen structure and interaction with antibody. Genetic mutations leading to amino acid substitutions may alter structure in three ways: changing a contact residue; creating or destroying a glycosylation signal sequence; or altering higher order structure (Figure 3–10). The binding of metal or organic prosthetic groups important for the function of the antigen often alter structure. For example, some antibodies binding metmyoglobin (containing heme) do not bind apomyoglobin (without heme).
Semen Analysis
Published in Botros Rizk, Ashok Agarwal, Edmund S. Sabanegh, Male Infertility in Reproductive Medicine, 2019
Meaghanne K. Caraballo, Alyssa M. Giroski, Rakesh Sharma, Ashok Agarwal
The principle of the assay is based upon the ability of all antioxidants in the seminal plasma to inhibit the oxidation of the ABTS (2,20-Azino-di-[3-ethylbenzthiazoline sulphonate]) to ABTS+ resulting in the change of the absorbance at 750 nm to a degree that is proportional to the concentration of the antioxidant in the sample [30]. The capacity of the antioxidants present in the sample to prevent ABTS oxidation is compared with that of standard 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox), a water-soluble tocopherol analogue. Ten microliters of metmyoglobin and 150 µL of chromogen are added to all standard and sample wells. The reaction is initiated by adding 40 µL of hydrogen peroxide as quickly as possible. The plate is incubated for 5 minutes at room temperature on a horizontal shaker and absorbance monitored at 750 nm using a microplate reader. Results are reported as micromoles of Trolox equivalent. Values less than 1,900 micromoles of TAC are considered as abnormal and frequently reported in men who are infertile and are indicative of OS [29].
Antioxidant assays
Published in Roger L. McMullen, Antioxidants and the Skin, 2018
When conducting the ABTS assay there are several different experimental approaches one may take. These were outlined originally by Miller and Rice-Evans, who were pioneers of the ABTS assay:20Decolorization assay: The radical, ABTS•+, is formed by one of the selected methods and it is allowed to reach a steady-state concentration. Once the radical is formed, the antioxidant to be tested may be added to the reaction assembly. As a result, ABTS•+ is converted to ABTS resulting in a change in the absorption spectrum—a color loss. Normally, the loss in absorbance at a particular wavelength is followed and this is used as a measure of the total antioxidant activity.Inhibition assay: In order to generate the radical, ABTS•+, the metmyoglobin–H2O2 system is used. First, ABTS and H2O2 are added to the reaction vessel. Metmyoglobin is later added, triggering the conversion of ABTS to ABTS•+. At a fixed point in time, key properties are measured.Inhibition assay: All compounds are added to the reaction vessel simultaneously, resulting in the onset of the conversion of ABTS to ABTS•+. Again, the metmyoglobin–H2O2 system is used to generate the radical. Various antioxidants can be compared on the basis of reaction rates.Lag time measurement assay: As earlier, all compounds are added to the vessel simultaneously resulting in the initiation of ABTS to ABTS•+ conversion. In this experiment, one measures the time required to reach a steady-state concentration of ABTS•+.
Broiler welfare is preserved by long-term low-dose oral exposure to zinc oxide nanoparticles: preliminary study
Published in Nanotoxicology, 2021
Manal A. M. Mahmoud, Doha Yahia, Doaa S. Abdel-Magiud, Madeha H. A. Darwish, Mahmoud Abd-Elkareem, Usama T. Mahmoud
Measurement of total antioxidant activity in serum samples was performed using test kits. Total antioxidant status was measured using a commercial test kit supplied by Bio-diagnostics (Dokki, Giza, Egypt) and using a UV spectrophotometer (Optizen 3220 UV; Mecasys Co. Ltd., Seoul, South Korea), in accordance with the method described by Koracevic et al. (2001). The basis of this method is a change of 2,2-azino-di(3-ethylbenzthiazoline) sulfonate (ABTS), incubated with peroxidase (metmyoglobin) and H2O2 into its cationic form. This leads to a change in color of the sample examined proportional to the concentration. After the addition of 1 ml of ABTS incubated with metmyoglobin to 20 ml of the supernatant, the sample was centrifuged and the absorbance was read at a wavelength of 600 nm. Then, 200 ml of H2O was added and another reading was obtained after 3 min. The data are presented as mmol/L.
Effect of Antioxidant Supplementation on Total Antioxidant Status in Indian Children with Type 1 Diabetes
Published in Journal of Dietary Supplements, 2019
Lavanya Parthasarathy, Vaman Khadilkar, Shashi Chiplonkar, Anuradha Khadilkar
Serum samples were assayed for TAS, measured by spectrophotometry, with a commercially available TAS kit (Randox kit, United Kingdom,) following the instructions of the manufacturer. The commercial kit by Randox is based on the method described by Miller, Rice-Evans, Davies, Gopinathan, and Milner (1993). The assay is based on the reduction of free radicals (ABTS•+ – 2,2’-azinobis(3-ehylbenzothiazoline-6-sulfonate)) measured as a decrease of absorbance at 600 nm at 3 min by antioxidants. The ABTS•+ radical cation is formed by the interaction of ABTS with ferrylmyoglobin radical species, generated by the activation of metmyoglobin with hydrogen peroxide. The suppression of the absorbance of the ABTS•+ radical cation by serum antioxidants was compared with Trolox (6-hydroxy-2, 5, 7, -tetramethylchroman-2- carboxylic acid), which is included as part of the TAS kit. The results are expressed as mmol/l of Trolox equivalents. Control of blood sugar in children with diabetes was evaluated by measuring glycosylated hemoglobin (HbA1C %), which was measured by high-performance liquid chromatography (HPLC, BIO-RAD, Germany).
Laboratory assessment of oxidative stress in semen
Published in Arab Journal of Urology, 2018
Ashok Agarwal, Emily Qiu, Rakesh Sharma
The TAC assay measures the total antioxidant available in the sample – both enzymatic and non-enzymatic, and presence of macromolecules. The TAC is measured in the clear seminal plasma after removing all the cellular components from the seminal ejaculate. TAC can be measured using a TAC assay kit. The components consist of assay buffer, standard Trolox, chromogen and metmyoglobin (Fig. 4). Hydrogen peroxide is used to initiate the reaction. TAC inhibits ABTS (2,2′-azino-di-[3-ethylbenzthiazolinesulfonate]) to ABTS+ by metmyoglobin [16]. Under the induced reaction conditions, the antioxidants in the seminal plasma suppress the absorbance at 750 nm to a degree that is proportional to the concentration of antioxidant. By comparing to standard Trolox, a water-soluble tocopherol analogue, the amount of antioxidant is measured in the sample by a colorimeter [16]. The unit of measurement of TAC is micromoles Trolox equivalent. The reference value for normal antioxidant value in a sample is >1950 micromoles of Trolox equivalent. Lower TAC values are indicative of increased OS or reduced ability of antioxidants to scavenge the formation of ROS in semen samples from infertile men (Fig. 5). Whilst this test is a rapid colorimetric method and able to measure total amount of antioxidants available in seminal plasma sample, it does not measure any enzymatic antioxidants [3,17]. Similar to other traditional diagnostic tests, this test is expensive to be incorporated in a clinical setting as a routine diagnostic laboratory test for male infertility.