Monkey IgG Fcγ ELISA Kit

Catalog Number: abs554434 Application: ELISA Reactivity: Monkey Conjugation:

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$396.83 USD

Size: 96T

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Product Details

Product Specification

Usage

Required Materials
1. Standard microplate reader (with 450nm wavelength)
2. High-speed centrifuge
3. Electric incubator
4. Mixer
5. High-precision pipettes, EP tubes, and disposable tips (5-10µL, 2-20µL, 20-200µL, 200-1000µL)
6. Double-distilled or deionized water, volumetric flasks, absorbent paper, etc.

Sample Collection and Storage
1. Serum: Whole blood samples should be incubated at room temperature for 2 hours or at 4°C overnight. Centrifuge at 1000×g for 20 minutes, and the supernatant should be collected for analysis. Blood should be collected in disposable, pyrogen-free, and endotoxin-free tubes. Store at -20°C or -80°C and avoid repeated freezing and thawing. 2. Plasma: Within 30 minutes of sample collection, centrifuge the sample at 1000 × g for 15 minutes at 2-8°C. Remove the supernatant for analysis. EDTA-Na2CO3 is recommended as an anticoagulant to avoid hemolysis or high-lipidemia samples. Store at -20°C or -80°C. Avoid repeated freezing and thawing. 3. Tissue Homogenate: Wash an appropriate amount of tissue in pre-chilled PBS (0.1M, pH 7.0-7.2) to remove blood (lysed red blood cells in the homogenate may affect the measurement results). Weigh the tissue and mince it. Add the appropriate volume of PBS (generally a 1:9 mass-to-volume ratio; the specific volume can be adjusted according to experimental needs and recorded. It is recommended to add protease inhibitors to the PBS) to the homogenizer and grind thoroughly on ice. To further lyse tissue cells, the homogenate can be sonicated or repeatedly frozen and thawed (keep the homogenate in an ice bath during sonication. Repeat the freeze-thaw cycle twice). Finally, centrifuge the prepared homogenate at 5000×g for 5-10 minutes. Collect the supernatant for testing. (Tissue homogenates should also be tested for protein concentration to obtain a more accurate test substance concentration per milligram of protein.)
4. Cell culture supernatant: Centrifuge the cell culture supernatant at 1000×g for 20 minutes to remove impurities and cell debris. Collect the supernatant for testing and store at -20°C or -80°C, avoiding repeated freezing and thawing.
5. Urine: Collect the first morning urine (midstream) or a 24-hour urine sample. Centrifuge at 2000×g for 15 minutes, collect the supernatant, and store the sample at -20°C, avoiding repeated freezing and thawing.
6. Saliva: Collect the sample using a saliva sample collection tube, then centrifuge at 1000×g for 15 minutes at 2-8°C. Remove the supernatant for testing, or aliquot and store at -20°C. Avoid repeated freezing and thawing.
7. Other biological samples: Centrifuge at 1000×g for 20 minutes, remove the supernatant, and test.

Sample Dilution Principles
If your test sample requires dilution, the following general dilution principles are provided:
1. 50-fold dilution: One-step dilution. Add 5 μL of sample to 245 μL of Standard & Sample Diluent for a 50-fold dilution.
2. 100-fold dilution: One-step dilution. Add 5 μL of sample to 495 μL of Standard & Sample Diluent for a 100-fold dilution.
3. 1000-fold dilution: Two-step dilution. Add 5 μL of sample to 95 μL of Standard & Sample Diluent for a 20-fold dilution. Then add 5 μL of the 20-fold diluted sample to 245 μL of Standard & Sample Diluent for a 50-fold dilution, for a total of 1,000-fold dilution. 4. 100,000-fold dilution: Three-step dilution. Add 5 μL of sample to 195 μL of Standard & Sample Diluent for a 40-fold dilution. Then add 5 μL of the 40-fold diluted sample to 245 μL of Standard & Sample Diluent for a 50-fold dilution. Finally, add 5 μL of the 2,000-fold diluted sample to 245 μL of Standard & Sample Diluent for a 50-fold dilution, for a total of 100,000-fold dilution. 5. The volume of liquid used in each dilution step should be no less than 3 μL, and the dilution factor should not exceed 100-fold. Too small a sample volume can easily lead to greater errors during mixing. Ensure thorough mixing at each dilution step to avoid foaming.
6. For very high dilution ratios, dilute with PBS first, and then use the standard and sample diluent provided in the kit as the final step.

Sample Dilution Recommendations

1. Normal, fresh serum/plasma samples are recommended for testing (original solution).
2. Due to individual variability, the recommended dilution ratio is for reference only. For actual testing, please estimate the sample concentration range in advance and determine the dilution ratio for the sample to be tested through preliminary experiments.

Pre-Test Preparation

1. Remove the kit from the refrigerator 30 minutes in advance and equilibrate to room temperature.
2. Dilute the 25× concentrated wash buffer to 1× working solution with double-distilled water. Return any unused portion to 4°C. 3. Standards: Add 0 mL of Universal Standard & Sample Diluent to the lyophilized standard. Tighten the cap and let stand for 10 minutes to fully dissolve. Then gently mix (concentration is 20 ng/mL). Then, serially dilute the standard to 20 ng/mL, 10 ng/mL, 5 ng/mL, 2.5 ng/mL, 1.25 ng/mL, 0.63 ng/mL, and 0.32 ng/mL. Use the standard diluent (0 ng/mL) as a blank well. Prepare the required amount of standard and set aside. It is recommended to add the prepared standard to the sample within 15 minutes; do not let it sit for an extended period. 4. Biotinylated Antibody Working Solution: Before the experiment, calculate the required amount of biotinylated antibody working solution (calculated as 100 μL/well, and 100-200 μL should be added during the actual preparation). 15 minutes before use, dilute the concentrated biotinylated antibody (1:100) with biotinylated antibody diluent to the working concentration and use it on the same day. Dilution guidelines: Add 1 μL of concentrated biotinylated antibody to 99 μL of biotinylated antibody diluent and mix thoroughly with a pipette. 5. Enzyme Conjugate Working Solution: Before the experiment, calculate the required volume for the experiment (assuming 100 μL/well; add 100-200 μL more). 15 minutes before use, dilute the concentrated HRP enzyme conjugate (1:100) with enzyme conjugate diluent to the working concentration for use that day. Dilution guidelines: Add 1 μL of concentrated enzyme conjugate to 99 μL of enzyme conjugate diluent and mix thoroughly with a pipette. 6. TMB Substrate: Pipette the required volume of solution. Do not return any remaining solution to the reagent bottle. 1. Equilibrate all materials and prepared reagents to room temperature before use. Mix all reagents thoroughly before use, taking care not to create any foam. 2. The user should calculate the number of samples likely to be used throughout the assay. Please reserve sufficient sample in advance. 3. Estimate concentrations before assay. If these values are outside the standard curve range, the user must determine the optimal sample dilution for their specific assay.

Operation Overview 1. After the kit and samples have equilibrated to room temperature, add 100 μL of standard working buffer (serial dilutions as per the instructions) or 100 μL of sample to each well and incubate at 37°C for 80 minutes. 2. Discard the liquid from the ELISA plate, add 200 μL of wash buffer to each well, and wash three times. After patting dry, add 100 μL of biotinylated antibody working solution to each well and incubate at 37°C for 50 minutes. 3. Discard the liquid from the ELISA plate, add 200 μL of wash buffer to each well, and wash three times. After patting dry, add 100 μL HRP enzyme working solution to each well and incubate at 37°C for 50 minutes.
4. Discard the liquid in the ELISA plate, add 200 μL wash buffer to each well, and wash five times. After patting dry, add 90 μL TMB to each well and incubate at 37°C for 20 minutes.
5. Add 50 μL stop solution to each well, read immediately at 450 nm, and calculate the results.

Procedure
1. Before beginning the experiment, all reagents should be equilibrated to room temperature and prepared in advance. When diluting reagents or samples, mix thoroughly and avoid foaming. If the sample concentration is too high, dilute it with sample diluent to bring the sample within the detection range of the kit. 2. Add 100 μL of the standard or sample to be tested (if the sample needs to be diluted, refer to the sample dilution guidelines for dilution methods). Be careful not to create bubbles. Add the sample to the bottom of the ELISA plate well, avoiding contact with the well walls. Gently shake to mix. Cover the plate with a film or membrane and incubate at 37°C for 80 minutes. To ensure the validity of the experimental results, use a fresh standard solution for each experiment. 3. Discard the liquid in the wells, spin dry, and wash the plate three times. Wash each well with 200 μL of wash buffer, soaking for 1-2 minutes. Spin off the liquid in the plate (or wash the plate using a microplate washer). After the final wash, pat the plate dry on absorbent paper. 4. Add 100 μL of biotin antibody working solution to each well (can be prepared 15 minutes in advance). Cover the plate with a film and incubate at 37°C for 50 minutes. 5. Discard the liquid in the wells and wash the plate three times. Wash each well with 200 μL of wash buffer, soak for 1-2 minutes, and discard the liquid from the plate (or wash the plate using a plate washer). After the final wash, pat the plate dry on absorbent paper. 6. Add 100 μL of enzyme conjugate working solution to each well (can be prepared 15 minutes in advance) and incubate at 37°C for 50 minutes. 7. Discard the liquid from the wells and wash the plate five times. Wash each well with 200 μL of wash buffer, soak for 1-2 minutes, and discard the liquid from the plate (or wash the plate using a plate washer). After the final wash, pat the plate dry on absorbent paper. 8. Add 90 μL of TMB chromogenic substrate solution to each well and incubate at 37°C in the dark for 20 minutes (this period may be shortened or extended depending on the actual color development, but should not exceed 30 minutes). 9. Add 50 μL of stop solution to each well to stop the reaction (the blue color will immediately turn yellow). The order of adding the stop solution should be consistent with the order of adding the colorimetric reagent. To ensure accurate experimental results, add the stop solution as soon as possible after the substrate reaction time expires. 10. Immediately measure the optical density (OD) of each well using a microplate reader at a wavelength of 450 nm. The instrument should be preheated and the assay program set before use. Calculation of Results 1. Subtract the OD value of the blank well from the OD value of each standard and sample. If replicates are used, the average value should be used for calculation. 2. For ease of calculation, although concentration is the independent variable and OD value is the dependent variable, the OD value of the standard is used as the horizontal axis (X-axis) and the concentration of the standard is used as the vertical axis (Y-axis). For intuitive visualization of the experimental results, the graphs show raw data rather than logarithmic values. The OD values of the standard curve will vary due to differences in experimental operating conditions (such as operator, pipetting technique, plate washing technique, and temperature). The standard curve provided is for reference only. Experimenters need to establish a standard curve based on their own experiments. The OD value of the sample used can be used to calculate the sample concentration on the standard curve, and then multiplied by the dilution factor to obtain the actual concentration of the sample. It is recommended to use professional curve drawing software, such as CurveExpert.

Concentration(ng/mL)	OD	CorrectedOD
20	2.135	2.053
10	1.612	1.53
5	1.162	1.08
2.5	0.875	0.793
1.25	0.557	0.475
0.63	0.377	0.295
0.32	0.236	0.154
0	0.082	0.000

1. Precision
Intra-plate precision (precision within the assay): CV% <8%
Three samples of known concentrations were tested 20 times on one ELISA plate to assess intra-plate precision.
Inter-plate precision (precision between assay plates): CV% <10%
Three samples of known concentrations were tested 40 times on three different ELISA plates to assess the precision of the assay.

2. Recovery
Recovery experiments were performed by adding monkey Fcγ to different samples at known concentrations to obtain the range and average recovery.

Sample Type	Recovery Range	Average Recovery
Serum (n=5)	92-107%	99%
EDTA plasma (n=5)	83-96%	89%
heparin plasma (n=5)
100%

3. Linearity
The sample spiked with monkey Fcγ was diluted 2-fold, 4-fold, 8-fold, and 16-fold to perform a recovery experiment.

Sample type	1:2	1:4	1:8	1:16
Serum (n=5)	81-97%	82-96%	95-102%	85-92%
EDTA plasma (n=5)	90-96%	87-102%	85-97%	96-96%
heparin plasma (n=5)	87-96%	93-108%	92-102%	91-99%

Sensitivity

0.2 ng/mL

Theory

This kit utilizes a sandwich assay. A specific anti-monkey Fcγ antibody is coated onto a 96-well microplate. A monkey Fcγ standard or sample is added to the microwells, allowing the monkey Fcγ protein in the standard or sample to bind to the anti-monkey Fcγ antibody immobilized on the microplate. Biotinylated anti-monkey Fcγ antibody is then added. Unbound biotinylated antibody is washed away, and HRP-labeled streptavidin is added. After further thorough washing, TMB substrate is added for color development. TMB is converted to blue by peroxidase catalysis and to the final yellow by acid. The intensity of the color is positively correlated with the amount of monkey Fcγ protein in the sample. Absorbance (OD) is measured at 450 nm using a microplate reader, and sample concentration is calculated by plotting a standard curve.

Detection Type

serum, plasma, tissue homogenates, cell lysates, cell culture supernates and other biological fluids

Composition

Name	96TConfigurationConfiguration	Save conditions
ELISA plate	8 holes×12 strips	4℃/-20℃
Freeze-dried standard	2	4℃/-20℃
Standard & sample dilution	20mL	4℃/-20℃
Concentrated biotinylated antibody (100×)	120uL	Biotinylated Antibody Dilution Buffer	12mL	4℃/-20℃
Concentrated HRP Enzyme Conjugate (100x)	center;"> 120uL	4℃/-20℃
Enzyme conjugate diluent	12mL	4℃/-20℃
Concentrated detergent 25×	20mL	4℃/-20℃
Chromogenic substrate solution (TMB)	10mL	4℃/-20℃ (avoid light)
Reaction stop solution	6mL	4℃/-20℃
Sealing board lamination	2	Normal temperature
Product Instructions	1	Room Temperature

1. After opening the package, please promptly check that all items are complete. The batch numbers of all reagents are shown on the labels.
2. Concentrated wash solution may precipitate salts when stored at low temperatures. Warm it in a water bath to aid dissolution during dilution.
3. A small amount of water-like substance may appear in the wells of a newly opened ELISA plate. This is normal and will not affect the experimental results. 4. All kit components have been formulated and quality controlled to function successfully as one kit. Do not mix or substitute reagents or materials from other kits. Performance cannot be guaranteed if used alone or substituted.

General Notes

1. This kit is for laboratory research and development use only and is not intended for use on humans or animals. 2. Reagents should be handled as hazardous materials and should be handled with care and disposed of properly. 3. Always wear gloves, a lab coat, and protective eyewear to avoid contact between skin and eyes with the stop solution and TMB. In case of accidental contact, rinse thoroughly with water. 4. Samples should be clear and transparent, and suspended matter should be removed by centrifugation. Hemolysis of the sample can affect the results, so hemolyzed samples should not be used. 5. Samples collected for testing within one week can be stored at 4°C. If testing cannot be performed immediately, aliquot the sample into single-use amounts and freeze at -20°C (for testing within one month) or -80°C (for testing within three to six months). Avoid repeated freeze-thaw cycles. Bring the sample to room temperature before use. 6. Ensure all components of the kit are dissolved and mixed thoroughly before use. Discard any unused standard after reconstitution. 7. Concentrated biotinylated antibody and enzyme conjugate solutions are relatively small and may disperse throughout the tube during transportation. Before use, centrifuge at 1000 × g for 1 minute to allow any liquid on the tube walls or cap to settle to the bottom. Mix the solution by carefully pipetting 4-5 times before use. Prepare the standard, biotinylated antibody working solution, and enzyme conjugate working solution according to the required volume and use the corresponding diluents. Do not mix them. 8. Concentrated wash buffer may crystallize after removal from the refrigerator. This is normal. Dissolve the crystals completely in a water bath or incubator before preparing the wash buffer (do not heat above 40°C). The wash buffer should be at room temperature before use. 9. Samples should be added quickly, preferably within 10 minutes for each addition. To ensure accuracy, replicate wells are recommended. When pipetting reagents, maintain a consistent order of addition from well to well. This will ensure consistent incubation times for all wells. 10. During the wash process, any remaining wash solution in the reaction wells should be patted dry on absorbent paper. Do not place filter paper directly into the reaction wells to absorb water. Before reading, be sure to remove any remaining liquid and fingerprints from the bottom of the wells to avoid affecting the microplate reader reading. 11. The color developer, TMB, should be protected from direct sunlight during storage and use. After adding the substrate, carefully observe the color change in the reaction wells. If a gradient is already evident, terminate the reaction early to prevent excessive color from darkening and affecting the microplate reader reading. 12. All test tubes and reagents used in the experiment are disposable. Reuse is strictly prohibited, as this will affect the experimental results. 13. Please wear a lab coat and latex gloves for proper protection during the experiment, especially when testing blood or other body fluid samples. Please follow the national biological laboratory safety regulations. 14. Components from different batches of the kit should not be mixed (except for the wash solution and the reaction stop solution). 15. The enzyme labeling strips in the kit are removable. Please use them in batches according to experimental needs. 16. This kit may not be suitable for testing samples in certain experimental settings where the validity of the assay itself is uncertain, such as gene knockout experiments. Certain natural or recombinant proteins, including both prokaryotic and eukaryotic recombinant proteins, may not be detected due to incompatibility with the detection and capture antibodies used in this product. 17. This kit has not been compared with similar kits from other manufacturers or products using different methods to detect the same target, so inconsistent test results cannot be ruled out. 18. Due to current conditions and technological limitations, it is not possible to fully identify and analyze all raw materials. This product may present certain quality and technical risks. 19. Final experimental results are closely related to the validity of the reagents, the experimenter's instructions, and the experimental environment. Please ensure that sufficient sample preparation is available. 20. To ensure the accuracy of experimental results, please strictly follow the instructions and do not mix reagents from other companies.

Storage Temp.

Unopened test kit, sealed and stored at 2-8℃, valid for 6 months

Test Range

0.31-20 ng/mL

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Monkey IgG Fcγ ELISA Kit