Serum concentration of Follicle Stimulating Hormone (FSH) was determined by a Microplate Immunoenzymometric assay (IEMA/ELISA) using Accu-bind ELISA Microwells (Follicle Stimulating Hormone (FSH) Test System Product Code: 6425–300) from Monobind Inc. Lake Forest, CA 92630, USA.
Accubind elisa microwells
Manufactured by Monobind
Sourced in United States
The AccuBind ELISA Microwells are a laboratory product designed for enzyme-linked immunosorbent assay (ELISA) testing. The microwells are pre-coated with specific antibodies and are used to detect and quantify the presence of target analytes in samples.
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9 protocols using accubind elisa microwells
1
Serum FSH Quantification by IEMA/ELISA
2
Serum Luteinizing Hormone Quantification
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Serum concentration of Luteinizing Hormone (LH) was determined by a Microplate Ezyme Immunoassay, Colorimetric using Accu-bind ELISA Microwells (Luteinizing Hormone (LH) Test System Product Code: 625–300) from Monobind Inc. Lake Forest, CA 92630, USA.
3
Sturgeon Sex Steroid Profiling
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First, fish were anesthetized with 400 ppm clove powder extract (Abdollahpour et al., 2019) .
Blood samples (3 fish into each tank) was collected from the behind of the anal fin with 5 ml heparinized syringe at the time of first injection, 10 h after the first injection and at ovulation time. Then the samples were transferred to the tubes, centrifuged (1,600 × g for 10 min) to separate plasma and stored at -20°C for later analysis.
Sex steroid concentration (ng mL -1 ) of testosterone (T, Cat. No: 3725-300), 17β estradiol (E2, Cat. No: 4925-300) and progesterone (P, Cat. No: 4825-300) were measured using enzymelinked immunosorbent assay (ELISA) using commercial kits (AccuBind ELISA Microwells, Monobind, Inc. Lake Forest, CA, USA) based on manufacturer's instruction. The intra-and inter-assay coefficients of variation were 5.8%, 9.8% (n=10), 6.4%, 9.5% (n=10) and 5.1%, 7.5% (n=10) for E2, T and P, respectively. Testosterone and progesterone were selected based upon their potential roles during the final stages of maturation in female sturgeon (Semenkova et al. 2002; Skoblina et al. 2012; Khara et al. 2014 ) and the very low inter-individual variability of the plasma level during the reproductive season (Barannikova et al. 2002) .
Blood samples (3 fish into each tank) was collected from the behind of the anal fin with 5 ml heparinized syringe at the time of first injection, 10 h after the first injection and at ovulation time. Then the samples were transferred to the tubes, centrifuged (1,600 × g for 10 min) to separate plasma and stored at -20°C for later analysis.
Sex steroid concentration (ng mL -1 ) of testosterone (T, Cat. No: 3725-300), 17β estradiol (E2, Cat. No: 4925-300) and progesterone (P, Cat. No: 4825-300) were measured using enzymelinked immunosorbent assay (ELISA) using commercial kits (AccuBind ELISA Microwells, Monobind, Inc. Lake Forest, CA, USA) based on manufacturer's instruction. The intra-and inter-assay coefficients of variation were 5.8%, 9.8% (n=10), 6.4%, 9.5% (n=10) and 5.1%, 7.5% (n=10) for E2, T and P, respectively. Testosterone and progesterone were selected based upon their potential roles during the final stages of maturation in female sturgeon (Semenkova et al. 2002; Skoblina et al. 2012; Khara et al. 2014 ) and the very low inter-individual variability of the plasma level during the reproductive season (Barannikova et al. 2002) .
4
Sturgeon Sex Steroid Profiling
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First, fish were anesthetized with 400 ppm clove powder extract (Abdollahpour et al., 2019) .
Blood samples (3 fish into each tank) was collected from the behind of the anal fin with 5 ml heparinized syringe at the time of first injection, 10 h after the first injection and at ovulation time. Then the samples were transferred to the tubes, centrifuged (1,600 × g for 10 min) to separate plasma and stored at -20°C for later analysis.
Sex steroid concentration (ng mL -1 ) of testosterone (T, Cat. No: 3725-300), 17β estradiol (E2, Cat. No: 4925-300) and progesterone (P, Cat. No: 4825-300) were measured using enzymelinked immunosorbent assay (ELISA) using commercial kits (AccuBind ELISA Microwells, Monobind, Inc. Lake Forest, CA, USA) based on manufacturer's instruction. The intra-and inter-assay coefficients of variation were 5.8%, 9.8% (n=10), 6.4%, 9.5% (n=10) and 5.1%, 7.5% (n=10) for E2, T and P, respectively. Testosterone and progesterone were selected based upon their potential roles during the final stages of maturation in female sturgeon (Semenkova et al. 2002; Skoblina et al. 2012; Khara et al. 2014 ) and the very low inter-individual variability of the plasma level during the reproductive season (Barannikova et al. 2002) .
Blood samples (3 fish into each tank) was collected from the behind of the anal fin with 5 ml heparinized syringe at the time of first injection, 10 h after the first injection and at ovulation time. Then the samples were transferred to the tubes, centrifuged (1,600 × g for 10 min) to separate plasma and stored at -20°C for later analysis.
Sex steroid concentration (ng mL -1 ) of testosterone (T, Cat. No: 3725-300), 17β estradiol (E2, Cat. No: 4925-300) and progesterone (P, Cat. No: 4825-300) were measured using enzymelinked immunosorbent assay (ELISA) using commercial kits (AccuBind ELISA Microwells, Monobind, Inc. Lake Forest, CA, USA) based on manufacturer's instruction. The intra-and inter-assay coefficients of variation were 5.8%, 9.8% (n=10), 6.4%, 9.5% (n=10) and 5.1%, 7.5% (n=10) for E2, T and P, respectively. Testosterone and progesterone were selected based upon their potential roles during the final stages of maturation in female sturgeon (Semenkova et al. 2002; Skoblina et al. 2012; Khara et al. 2014 ) and the very low inter-individual variability of the plasma level during the reproductive season (Barannikova et al. 2002) .
5
Blood Serum Analysis Protocol
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In order to separate the serum from the cells, clotted blood samples obtained by venipuncture from all subjects were centrifuged. Samples were stored at temperature of -70°C until assayed. Serum samples were evaluated for quantitative measurement of TSH and Total/Free T4 concentration by an immunoenzymometric assay and competitive enzyme immunoassay, respectively. All parameters were measured according to manufacturer's instruction by commercial kits (AccuBind ELISA Microwells, Monobind, Inc. Lake Forest, CA, USA).
6
Assessment of Metabolic Health Markers
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Fasting blood samples were collected and immediately analysed in an A15 analyzer (Biosystems) for glucose and lipid profile measures (Biosystems). Serum aliquots were obtained and maintained at -20°C for insulin measurement by ELISA (AccuBind ELISA Microwells ISO 13485 & 9001; Monobind Inc.). The homoeostasis model assessment (HOMA-2) was used to evaluate β-cell function (HOMA-B index), insulin sensitivity (HOMA-S index) and insulin resistance (HOMA-IR index). Insulin resistance was considered as a value ≥95th percentile of HOMA-IR index, adjusting for sex and age, using regional reference values for non-obese children (13) . Dyslipidaemias were considered as follows (14) : hypercholesterolaemia ≥ 5•2 mmol/l, elevated LDLcholesterol ≥ 3•3 mmol/l, low HDL-cholesterol ≤ 1 mmol/l, and hypertriacylglycerolaemia ≥ 1•1 mmol/l (for ≤9•9 years old) and ≥1•4 mmol/l (for ≥10 years old).
7
Hormone Quantification by ELISA
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The sex hormones (luteinizing hormone, follicle stimulating hormone, prolactin, testosterone, estradiol and progesterone) were assayed by ELISA technique (Monobind Inc. Lake Forest, CA, USA (Accu-Bind ELISA Microwells)).
8
Neonatal Vitamin D Assessment Protocol
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For all neonates, two milliliters of venous blood were withdrawn under complete aseptic conditions for complete blood count (CBC), blood culture and C-reactive protein (CRP) within the first 72 h after birth. 25-OH vitamin D serum level was measured shortly after birth (within 24 h) by ELISA technique using Accubind ELISA micro wells (Monobind, Inc., Lakeforest, CA, USA) [9 (link)].
Another two milliliters of maternal blood were withdrawn within 24 h after birth for (25-OH vitamin D) assessment using a radioimmunoassay method in which we used Gamma-B 25-(OH)D RIA (IDS, Boldon, UK). The coefficient of variation (CV) for inter-assay analysis was 7.6% and sensitivity less than 3 nmol/L. All samples were analyzed in duplicate and all duplicates with a coefficient of variation more than 10% were reanalyzed [10 (link)].
25-OH vitamin D serum level values were categorized in descriptive analyses as sufficient with serum levels more than 50 nmol/L, insufficient between 30–50 nmol/L and deficient when <30 nmol/L according to institute of medicine (IOM) report and the consensus report on nutritional rickets [11 (link),12 (link),13 (link)].
The study was approved by the local research ethics committee of the El-Minya university hospital for children and written informed consents were obtained from the parents of all neonates to share in the study.
Another two milliliters of maternal blood were withdrawn within 24 h after birth for (25-OH vitamin D) assessment using a radioimmunoassay method in which we used Gamma-B 25-(OH)D RIA (IDS, Boldon, UK). The coefficient of variation (CV) for inter-assay analysis was 7.6% and sensitivity less than 3 nmol/L. All samples were analyzed in duplicate and all duplicates with a coefficient of variation more than 10% were reanalyzed [10 (link)].
25-OH vitamin D serum level values were categorized in descriptive analyses as sufficient with serum levels more than 50 nmol/L, insufficient between 30–50 nmol/L and deficient when <30 nmol/L according to institute of medicine (IOM) report and the consensus report on nutritional rickets [11 (link),12 (link),13 (link)].
The study was approved by the local research ethics committee of the El-Minya university hospital for children and written informed consents were obtained from the parents of all neonates to share in the study.
9
Enzyme-Linked Immunosorbent Assay for Fasting Serum Insulin
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Fasting serum Insulin was assayed using a solid phase enzyme-linked immunosorbent assay. The assay was done using the Accu-Bind ELISA Microwells manufactured by Monobind Inc, USA.
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