Analysis of serum IgG antibody levels was performed by ELISA as described previously by Mpagi et al. [55 (link)] with modifications to fit analysis with cattle sera. ELISA microtiter plates (Nunc, Roskilde, Denmark) were coated with 100 μl of 200 ng/well O. ochengi or O. volvulus galectins, and O. ochengi and O. volvulus somatic extracts were used as positive controls. After overnight incubation, plates were washed four-times with PBS 0.05% Tween 20 (pH 7.2). Excess reactive sites were blocked for 2 h at room temperature with 200 μl of PBS/5% BSA for human sera and with PBS/5% (w/v) skimmed milk for cattle plasma analyses. Furthermore, individual human sera (n = 44: 35 males and 9 females) from over 10 years O. volvulus-infected individuals diluted at 1:1000, 1:3000, and 1:9000 or sera from 2 years old O. ochengi infected cattle (n = 9: four males and five females) diluted at 1:50, 1:100, and 1:200 were used. Sera from two European cattle not exposed to Onchocerca spp. (EC) from the University of Veterinary Medicine (Hannover, Germany) and healthy Europeans (naïve, n = 12: four males and eight females) individuals were included as negative controls. The secondary antibodies used were, horseradish peroxidase-conjugated goat anti-human IgG and goat anti-bovine IgG (Sigma, St. Louis, USA), respectively, each diluted at 1:5000. Plates were processed as described above.
Elisa microtiter plate
Manufactured by Thermo Fisher Scientific
Sourced in Denmark, United States
ELISA microtiter plates are a type of laboratory equipment used in enzyme-linked immunosorbent assay (ELISA) testing. These plates are designed with wells that can hold small volumes of liquid samples, allowing for the simultaneous analysis of multiple samples. The plates provide a platform for the various steps involved in the ELISA process, including the immobilization of antibodies or antigens, the addition of samples and reagents, and the measurement of the resulting color change or fluorescence.
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4 protocols using elisa microtiter plate
1
Onchocerciasis Antibody Detection by ELISA
2
ELISA for α-Taxilin Quantification
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ELISA was performed using RA (n = 100) and OA (n = 100) plasma samples and healthy (n = 62) individual plasma samples along with RASF (n = 16) and OASF (n = 16) samples. ELISA microtiter plates (Nunc, USA) were coated with 100 μl diluted sample (plasma/SF; dilution 1 : 200) and incubated at 4°C overnight. After washing, wells were blocked with 1% BSA for 1 h at RT followed by 2 h incubation with 100 μl diluted (1 : 2000) anti-α-Taxilin (Santacruz, USA) at RT. Plates were then washed and incubated with 100 μl HRP-conjugated secondary anti-mouse antibody (dilution 1 : 1000) (Jackson, USA) for 1 h at RT. The reactions were then developed with orthophenylene diamine (1 mg/ml) substrate for 15 min, terminated by adding 50 μl of 3 N·H2SO4. The absorbance was measured at 492 nm (SpectraMax Plus 384) [25 (link)].
3
Quantification of Heparin-Bound Proteins
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20 µl aliquots of acetonitrile-containing fractions from a preparative RP8-HPLC-run of heparin-bound proteins off a stratum corneum extract11 ; for a RP-HPLC run, see Fig. 1 ) were lyophilized and the residues dissolved in 5 µl distilled water. 4 µl of each sample were diluted with 45 µl 0.1 M Na-phosphate buffer (NaP), pH 7.4, and then added to an ELISA-microtiter plate (Nunc). The HRNR-ELISA-microtiter plate has been prepared by coating over night with pooled affinity-purified goat polyclonal HRNR antibodies (antigens: HRNR1075–1172, HRNR2591–2662, HRNR2726–2850)12 (link), stored at 1 mg/ml as pool below −78 °C and used at 1:200 dilution in coating buffer, blocking with 1% (w/v) freshly prepared, biotin-free BSA for 90 min, and three times washing with PBS-Tween (PBS-T, 0.05%, v/v). Samples and standards (rHRNR1075–1172 (0–500 ng/ml 0.1 M Na-P buffer, pH 7.4) were added to the ELISA plate, incubated for 45 min at 37 °C, followed by adding biotinylated pooled polyclonal HRNR antibodies (see above, from a 1 mg/ml pool), at 1:200 dilution in PBS-T (0.05%, v/v) and a 45 min at 37 °C incubation. After three-fold washing in PBS-T, streptavidin-peroxidase conjugate (1:10,000) was added, incubated for 30 min and after 6-fold washing with PBS, peroxidase substrate (T-ABTS- solution) was added and monitored at 405 nm and 492 nm.
4
Chimeric Lectin Binding Assay
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The embryonic alpha-1-acid glycoprotein (0.1 µg/mL) was dissolved in 0.1 M carbonate buffer, pH 9.5, containing 0.15 M NaCl, and 150 µl was added to the first well and was two-fold serially diluted in the same buffer in each well of a polystyrene 96-well ELISA microtiter plate (Maxisorp, Nunc), and then incubated at 4°C overnight. After incubation, the plate was washed three times with the buffer containing 0.01 M Tris-HCl, pH 7.5, 0.15 M NaCl, 0.05% Triton X-100 (TBS-T) and then three times with water. After that, 160 µL of bovine serum albumin (1 µg/mL) in TBS-T was added to each well to block non-specific binding sites. The plate was then incubated for 1 hour at room temperature and washed as described above. 150-µL aliquots of the samples containing chimeric lectin CmAP/MBL-AJ with concentration 100 µg/mL were added to each well. TBS-T was used as negative control, and interaction of MBL-AJ with adsorbed antibodies under the same conditions served as a positive control. After that, the plate was kept for 1 h at room temperature and washed again. Then 150 µL of pNPP in the buffer for AP assay was added to each well. After 2–5 min of incubation, 100 µL of 2 M sodium hydroxide was added to stop the reaction. Absorbance of the resultant solution was measured at 400 nm with a plate spectrophotometer (Bio-Tek Instruments, USA).
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