• Monkey IgG Fcγ ELISA Kit
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Monkey IgG Fcγ ELISA Kit

Monkey IgG Fcγ ELISA Kit

Catalog Number: abs554434 Application: ELISA Reactivity: Monkey Conjugation:
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$397 USD
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Product Details

Product Specification

protein IgG Fcγ
Usage

Self-brought items required for experiments

1 , standard specification microplate reader (including wavelength 450nm ）

2 High-speed centrifuge

3 , electric thermostat box

4 Mixer,

5 , high-precision pipettes, EP Tubes and disposable tips ( 5-10μL 、 2-20μL 、 20-200μL 、 200-1000μL ）

6 , double distilled water or deionized water, volumetric flask, absorbent paper, etc.

Sample collection and storage

1 Serum: whole blood sample placed at room temperature 2 Hour or 4°C After overnight 1000×g Centrifugation 20 Minutes, take the supernatant to detect. The blood collection tubes shall be disposable non-pyrogenic, non-endotoxin tubes. deposit -20°C Or -80°C Storage, avoid repeated freezing and thawing.

2 Plasma: the sample after collection 30 Within minutes 2-8°C 、 1000×g Centrifugation 15 Minutes, take the supernatant to detect. Anticoagulants recommended EDTA-Na2 , avoid using hemolytic, hyperlipidemic samples. deposit -20°C Or -80°C Storage, avoid repeated freezing and thawing.

3 , tissue homogenate: take an appropriate amount of tissue block and put it on the pre-cooled PBS （ 01M ， pH7.0-7.2 ) to remove blood (lysed red blood cells in the homogenate will affect the measurement result), cut the tissue into pieces after weighing, and then mix it with the corresponding volume of PBS (generally according to 1:9 The mass-to-volume ratio, the specific volume can be appropriately adjusted according to the needs of the experiment, and recorded. It is recommended to PBS Adding a protease inhibitor) into a glass homogenizer and fully grinding on ice; In order to further lyse tissue cells, the homogenate can be subjected to ultrasonic disruption or repeated freeze-thaw treatment (pay attention to ice bath cooling during ultrasonic disruption, and the repeated freeze-thaw method can be repeated 2 Times). Finally, the prepared homogenate is mixed in 5000×g Centrifugation 5-10 Minutes, take the supernatant to detect. (T tissue homogenates require simultaneous detection of protein concentrations to obtain a more accurate test substance concentration per milligram of protein.)

4 Cell culture supernatant: the cell supernatant was taken from 1000×g Centrifugation 20 Minutes, impurities and cell debris were removed. Take the supernatant to detect and place it in -20°C Or -80°C Store, but repeated freezing and thawing should be avoided.

5 , urine: Please collect the first urine in the morning (middle urine), or 24 Hourly urine, 2000×g Centrifugation 15 Minutes later the supernatant was collected and the sample was stored at -20°C And repeated freezing and thawing should be avoided.

6 Saliva: collecting a sample with a saliva sample collection tube, and then at 2-8°C,1000×g Centrifugation 15 Minutes, take the supernatant to detect, or sub-package -20°C Save. Avoid repeated freezing and thawing.

7 Other biological samples: please 1000×g Centrifugation 20 Minutes, take the supernatant to detect.

Principles of sample dilution

If your test sample needs to be diluted, refer to the general dilution principles below:

1 Dilution 50 Times: One-step dilution. Take 5μL Sample to 245μL Standard & In the sample dilution, is 50 Double dilution;

2 Dilution 100 Times: One-step dilution. Take 5μL Sample to 495μL Standard & In the sample dilution, is 100 Double dilution;

3 Dilution 1000 Times: Two-step dilution. Take 5μL Sample to 95μL Standard & In the sample dilution, is 20 Dilute, and then take 5μL20 Double dilute sample to 245μL Standard & In the sample dilution, is 50 Double dilution, co-dilution 1000 Times;

4 Dilution 100000 Times: Three-step dilution. Take 5μL Sample to 195μL Standard & In the sample dilution, is 40 Dilute, and then take 5μL40 Double dilute sample to 245μL Standard & In the sample diluent, do 50 Time dilution, and finally take 5μL2000 Double dilute sample to 245μL Standard & In the sample diluent, do 50 Double dilution, total dilution 100000 Times;

5 The amount of liquid taken during each step of dilution is not less than 3μL , the dilution factor is not more than 100 Times. Too small sampling volume can easily cause greater errors in the mixing process, and each dilution step needs to be mixed evenly to avoid foaming.

6 , when the dilution factor is very high, you can use it first PBS Dilution, last step using standard in kit & Sample dilution.

Sample dilution recommendations

1 Normal fresh serum, / Plasma Sample Recommendation (Original solution) Testing.

2 Due to individual differences, the recommended dilution factor is for reference only. For actual testing, please estimate the concentration range of the sample in advance, and determine the dilution factor of the sample to be tested through pre-experiments.

Preparation for testing

1 , please advance 30 Minutes remove the kit from the refrigerator and equilibrate to room temperature.

2 , using double-distilled water 25× The concentrated wash liquid is diluted to 1× Working fluid, put back unused 4°C 。

3 Standard: Add standard & Sample Universal Diluent 0mL Into the lyophilized standard, screw the tube cap tightly and let stand 10 Minutes, and after it is fully dissolved, gently mix (concentration of 20ng/mL ）。 Thereafter, double dilution is carried out to 20ng/mL ， 10ng/mL ， 5ng/mL ， 2.5ng/mL ， 1.25ng/mL ， 0.63ng/mL ， 0.32ng/mL Standard dilution ( 0ng/mL ) is a blank hole. Configure the standard according to the amount you need for later use. The configured standards are recommended in 15 Add the sample within minutes, and it is not recommended to leave it for too long.


4 , biotinylated antibody working solution: calculate the dosage required for the current experiment before the experiment (according to 100μL/ Hole meter, should be configured more in actual configuration 100-200μL ), before use 15 Min, concentrated biotinylated antibody was diluted with biotinylated antibody diluent ( 1:100 ) into working concentration, use on the same day. Dilution principle 1μL Concentrated biotinylated antibody is added to 99μL In the biotinylated antibody dilution, mix well with a pipette.

5 , enzyme conjugate working solution: calculate the dosage required for the current experiment before the experiment (according to 100μL/ Hole meter, should be configured more in actual configuration 100-200μL ）。 Before use 15 Minutes, dilute and concentrate with enzyme conjugate diluent HRP Enzyme conjugate ( 1:100 ) into working concentration, use on the same day. Dilution principle 1μL The concentrated enzyme conjugate is added to 99μL The enzyme conjugate dilution was mixed with a pipette.

6 、 TMB Substrate —— Pipette the desired dose of solution and do not pour the residual solution back into the reagent vial again.

Preparation before the experiment

1 All materials and prepared reagents were equilibrated to room temperature prior to use. Before use, mix all reagents thoroughly, taking care not to create any foam.

2 The user should calculate the number of samples that may be used throughout the test. Please reserve enough samples in advance.

3 Please estimate the concentration before measurement. If these values are not within the standard curve range, the user must determine the optimal sample dilution for their particular experiment.


OPERATION

1 After the kit and sample are equilibrated at room temperature, standard working buffer is added to each well 100μL( Gradient dilution according to instructions ) Or sample 100μL,37°C incubation 80 Minutes.

2 Discard the liquid in the enzyme plate and add it to each well 200μL Wash buffer, wash 3 Times. After patting dry, each well was added 100μL Biotinylated antibody working solution, 37°C incubation 50 Minutes.

3 Discard the liquid in the enzyme plate and add it to each well 200μL Wash buffer, wash 3 Times. After patting dry, each well was added 100μLHRP Enzyme working solution, 37°C incubation 50 Minutes.

4 Discard the liquid in the enzyme label plate and add it to each well 200μL Wash buffer, wash 5 Times. After patting dry, each well was added 90μLTMB,37°C incubation 20min 。

5 , adding per well 50μL Stop liquid, 450nm Take immediate readings and calculate results.

Operation steps

1 Before the experiment starts, all reagents should be balanced to room temperature, and all reagents should be configured in advance. When reagents or samples are diluted, they should be mixed evenly, and foaming should be avoided as much as possible when mixing evenly. If the sample concentration is too high, dilute with a sample diluent to bring the sample within the range of the kit.

2 Add standard substance or sample to be tested 100μL (If the sample needs to be diluted, please refer to the sample dilution principle for the dilution method). Be careful not to have bubbles. When adding the sample, add the sample to the bottom of the well of the enzyme label plate, try not to touch the well wall, gently shake and mix well, and add the enzyme label plate. Cover or coating, 37°C incubation 80 Minutes. To ensure the validity of the experimental results, please use a new standard solution for each experiment.

3 Discard the liquid in the hole, spin dry, and wash the plate 3 Times. For each well 200μL Washing with washing solution, soaking 1-2 Minutes, shake off the liquid in the labeled plate (or wash the plate with a plate washer). After the last wash was complete, the plate was pat dry on absorbent paper.

4 Add biotin antibody working solution to each well 100μL (can be advanced 15 Preparation in minutes), the enzyme labeled plate is coated, 37°C incubation 50 Minutes.

5 Discard the liquid in the hole and wash the plate 3 Times. For each well 200μL Washing with washing solution, soaking 1-2 Minutes, shake off the liquid in the labeled plate (or wash the plate with a plate washer). After the last wash was complete, the plate was pat dry on absorbent paper.

6 Enzyme conjugate working solution per well 100μL (can be advanced 15 Minute preparation), 37°C incubation 50 Minutes.

7 Discard the liquid in the hole and wash the plate 5 Times. For each well 200μL Washing with washing solution, soaking 1-2 Minutes, shake off the liquid in the labeled plate (or wash the plate with a plate washer). After the last wash was complete, the plate was pat dry on absorbent paper.

8 , adding per well TMB Chromogenic substrate solution 90μL ， 37°C Incubate in the dark 20 Minutes (shortened or extended as appropriate according to the actual color development, but not exceeding 30 Minutes).

9 , adding stop solution to each well 50μL , terminate the reaction (blue immediately turns yellow at this time). The sequence of addition of the terminating solution should be the same as that of the developer as possible. In order to ensure the accuracy of the experimental results, the termination solution should be added as soon as possible after the substrate reaction time expires.

10 , immediately use a microplate reader in 450nm The optical density values of each well were measured at the wavelength ( OD Value). The instrument should be preheated before use, and the testing program should be set up.

Results Calculation

1 , each standard and sample OD Value should be subtracted from the blank hole OD Value. If a double hole is set, the average value should be taken for calculation.

2 For ease of calculation, although the concentration is an independent variable and OD The value is the dependent variable, and we still use the standard when drawing OD Values as abscissa ( X Axis), the concentration of the standard is the ordinate ( Y Axis). At the same time, for the intuition of the test results, the figure provides raw data instead of logarithmic values. Due to the different experimental operating conditions ( such as operator, pipetting technology, plate washing technology and temperature conditions, etc.), the standard curve OD Values will vary. The standard curve provided is for reference only, and the experimenter needs to establish the standard curve according to his own experiment. Spent sample OD The value can be calculated on the standard curve to calculate the sample concentration and multiply it by the dilution factor, which is 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


Kit parameters
1 Precision

Intraplate precision ( Precision within the assay ):CV%<8%

Three samples with known concentrations were respectively in 1 Test on enzyme label plates 20 Times to evaluate the precision in the assay plate.

Inter-plate precision ( Measure inter-plate precision ):CV%<10%

Three samples with known concentrations were respectively in 3 Tested on different enzyme plates 40 Times to evaluate the precision of the analytical plate.
2 Recovery rate

Add monkeys at known concentrations to different samples Fcγ , do the recovery experiment, get the recovery range and average recovery rate
Sample Type
Recovery Range
Average recovery
Serum ( n=5 ）
92-107%
99%
EDTA Plasma ( n=5 ）
83-96% 89%
heparin Plasma ( n=5 ）
93-107%
100%
3 Linearity

There will be monkeys added Fcγ The samples were diluted separately 2 Times, 4 Times, 8 Times, 16 Double the recovery experiment to obtain the recovery rate range
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 adopts the principle of sandwich method. The specific anti-monkey Fcγ antibody was coated in a 96-well microplate, and the monkey Fcγ standard or sample was added to the microwells respectively, so that the monkey Fcγ protein in the standard or the monkey Fcγ protein in the sample was bound to the anti-monkey Fcγ antibody solid on the microplate, then biotinylated anti-monkey Fcγ antibody was added, the unbound biotinylated antibody was washed, HRP-labeled streptavidin was added, and then TMB substrate was added to develop color. TMB is converted to blue under peroxidase catalysis and to final yellow under the action of acid. There was a positive correlation between the depth of color and the monkey Fcγ protein in the sample. The absorbance (OD value) was measured with a microplate reader at a wavelength of 450 nm, and the sample concentration was calculated by drawing a standard curve.
Source Monkey
Synonym Monkey immunoglobulin GFc fragment (Fcγ) enzyme-linked immunosorbent assay kit
Detection Type serum, plasma, tissue homogenates, cell lysates, cell culture supernates and other biological fluids
Composition
Name 96T match set Preservation conditions
Enzyme plate 8 Hole ×12 Strip 4℃/-20℃
Lyophilized Standard 2
4℃/-20℃
Standard & Sample dilution
20mL
4℃/-20℃
Concentrated biotinylated antibodies ( 100× ）
120uL
4℃/-20℃
Biotinylated antibody dilution
12mL
4℃/-20℃
concentrate HRP Enzyme conjugate ( 100x ）
120uL
4℃/-20℃
Enzyme conjugate dilution
12mL
4℃/-20℃
Concentrated wash 25×
20mL
4℃/-20℃
Chromogenic substrate solution ( TMB ）
10mL
4℃/-20℃ (protected from light)
Reaction stop solution
6mL
4℃/-20℃
Sealing plate coating
2
normal temperature
Product Instructions
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normal temperature

1 After opening the package, please check whether all items are complete in time. The batch numbers of all reagents are shown on the label.
2 Salt precipitation will occur when the concentrated washing liquid is stored at low temperature. When diluted, it can be heated in a water bath to help dissolve.
3 The newly opened wells of the enzyme labeled plate may contain a little water-like substance, which is a normal phenomenon and will not have any impact on the experimental results.
4 All kit components have undergone formulation and quality control tests to successfully function as a kit. Do not mix or replace reagents or materials from other kits, performance cannot be guaranteed if used alone or replaced.
General Notes 1 This kit is intended for laboratory research and development only, not for use in humans or animals.
2 Reagents should be treated as hazardous substances and should be handled carefully and properly disposed of.
3 Gloves, lab coats and protective glasses should always be worn to avoid skin and eye contact with the termination solution and TMB 。 In case of accidental contact, please wash thoroughly with water.
4 The sample should be clear and transparent, and the suspended solids should be removed by centrifugation. Hemolysis of the sample will affect the results, so hemolyzed samples should not be used.
5 , after sample collection, if 1 Testing within weeks can be stored at 4°C , if it cannot be detected in time, please pack it according to the one-time usage amount and freeze it in -20°C （ 1 Within months), or -80°C （ 3-6 Test within a month) to avoid repeated freezing and thawing. Keep the sample at room temperature prior to the experiment.
6 Before using the kit, please make sure that all components are dissolved and mixed. If the reconstituted standard is not used, please discard it.
7 , concentrated biotinylated antibody, the volume of concentrated enzyme conjugate is small, may be dispersed in various parts of the tube during transportation, please 1000×g Centrifugation 1 Minutes to allow the liquid of the tube wall or cap to deposit to the bottom of the tube. Pipette carefully before use 4-5 The solution was mixed once. Standard, biotinylated antibody working solution and enzyme conjugate working solution should be prepared according to the required dosage, and the corresponding diluent should be used to prepare without confusion.
8 The concentrated washing liquid taken out of the refrigerator may have crystals. This is a normal phenomenon. The crystals can be completely dissolved in a water bath or incubator before preparing the washing liquid (the heating temperature should not exceed 40°C ）。 The wash liquid should be at room temperature when used.
9 , sample addition needs to be quick, and it is best to control each sample addition within 10 Within minutes, in order to ensure the accuracy of the experiment, it is recommended to use a double hole. Maintaining a consistent sequence of addition from well to well when pipetting reagents will ensure the same incubation time for all wells.
10 During the washing process, the washing liquid remaining in the reaction hole should be patted dry on absorbent paper. Do not put the filter paper directly into the reaction hole to absorb water. Before reading, pay attention to removing the residual liquid and fingerprints at the bottom, so as not to affect the reading of the microplate reader.
11 A color developer, TMB Direct exposure to bright light should be avoided during storage and use. After adding the substrate, pay attention to the color change in the reaction well. If the gradient is obvious, please terminate the reaction in advance to avoid too dark color affecting the reading of the microplate reader.
12 The test tubes and reagents used during the experiment are disposable, and it is strictly forbidden to reuse them, otherwise the experimental results will be affected.
13 During the experiment, please wear a laboratory coat and latex gloves for protection, especially when testing blood or other body fluid samples, please follow the national biological laboratory safety protection regulations.
14 Kit components of different batch numbers cannot be mixed (except washing liquid and reaction stop liquid).
15 The enzyme labeling strip in the kit is a detachable plate, please use it in batches according to the experimental requirements.
16 The kit may not be suitable for the detection of some special experimental samples whose validity is uncertain , for example , Gene knockout experiments, etc . Certain native or recombinant proteins , Include prokaryotic and eukaryotic recombinant proteins , Possibly due to mismatch with the detection antibody and capture antibody used in this product , Without being detected.
17 This kit is not compared with similar kits of other manufacturers or products with different methods to detect the same target substance , Therefore, it is not excluded that the test results are inconsistent
18 Due to the existing conditions and scientific and technological level, it is not yet possible to comprehensively identify and analyze all raw materials. This product may have certain quality and technical risks.
19 The final experimental results are closely related to the effectiveness of the reagents, the relevant operations of the experimenter and the experimental environment at that time. Please be sure to prepare sufficient samples to be tested.
20 In order to ensure the accuracy of the experimental results, please strictly follow the instructions and do not mix it with reagents from other companies.
Storage Temp. Unopened kit, sealed storage at 2-8 ℃, shelf life 6 months
Test Range 0.31-20 ng/mL