Monkey anti-PEG IgM ELISA Kit

1. Sample collection and storage

1 Serum,

Whole Blood Samples Hold at Room Temperature 2 Hour or 2-8°C Overnight.
1000×g Centrifugation 20 Minutes, take the supernatant.
Can be tested immediately, or frozen in packages according to one-time usage - 20°C Or -80°C.

2 Plasma,

Anticoagulants recommended EDTA-Na2/K2, after sample collection 30 Within minutes 2-8°C,
1000×g Centrifugation 15 Minutes, take the supernatant.
Can be tested immediately, or frozen in packages according to one-time usage -20°C Or -80°C.
Please refer to the sample preparation guidelines for the use and selection of other anticoagulants.

3 Tissue samples,

Tissue samples are generally made into tissue homogenates, and the processing methods are as follows:

1 Placing the target tissue on ice, using pre-cooled PBS Buffer (0.01M, pH=7.4) Wash to remove residual blood, weigh and use later.

2 Grinding the tissue homogenate with the lysate on ice.
The volume of lysate added depends on the weight of the tissue.
Generally, each 1g Use of tissue fragments 9ml Lysate.
It is also recommended to add protease inhibitors to the lysate, such as 1mM PMSF.

3 Can be reused for ultrasonic crushing or repeated freezing and thawing for further treatment (In the process of ultrasonic crushing, ice bath is needed to cool down;
Repeated freeze-thaw method can be repeated 2 Time).

4 Dissolving the prepared homogenate in 5000×g Centrifugation 5 Minutes, retain the supernatant for detection.
Or frozen in packages according to one-time usage -20°C Or -80°C.

5 According to the experimental needs, the total protein concentration of the tissue homogenate sample can be measured first, To facilitate data analysis, recommended BCA Law.
The total protein concentration is generally adjusted to 1-3mg/ml Used for ELISA Detection.
Certain tissue samples, such as liver, kidney, and pancreas, contain higher concentrations of endogenous peroxygenase, When the sample concentration is high, it will react with the chromogenic substrate, resulting in false positives.
Can try to use 1%H2O2 Inactivation 15min Re-test.

Note: Commonly used lysate PBS Buffer, or use moderate strength RIPA Lysate.
Using RIPA When lysing solution, PH The value needs to be adjusted to PH7.3, avoid using containing NP-40, Triton X-100 And DTT The components will seriously inhibit the work of the kit.
Recommended use 50mM Tris+0.9%NaCL+0.1% SDS,PH 7.3.

4 Cell culture supernatant,

Collecting the supernatant, 2-8°C, 2500rpm Centrifugation 5min, the clarified cell culture supernatant was collected.
Use immediately for testing, or dispense in a single-use amount -80°C Freeze for later use.

5 Cell lysate,

1 Collection and lysis of suspended cells: 2-8°C, 2500rpm Centrifugation 5min, collecting the cells.
Add the pre-cooled PBS Gently mix and clean, 2-8°C, 2500rpm Centrifugation 5min, collecting the cells.
join 0.5-1ml Cell lysate and appropriate amount of protease inhibitor (Such as PMSF, working concentration 1mmol/L), placed on ice, lysed 30min-1h, Or cooperate with ultrasonic crushing.

2 Collection and lysis of adherent cells: aspirate the supernatant and add pre-cooled PBS Wash three times.
join 5-1ml Cell lysate and appropriate amount of protease inhibitor (Such as PMSF, working concentration 1mmol/L), gently scrape off the adherent cells with a cell scraper.
The cell suspension was transferred to a centrifuge tube, placed on ice, and lysed 30min-1h, or cooperate with ultrasonic crushing.

3 During the cell lysis process, the centrifuge tube can be blown with the gun tip or shaken intermittently to fully lyse the protein, Appears slimy yes DNA, can be disrupted using ultrasonic DNA.
(Or with ultrasound 3-5mm Probe, Power 150-300W, Sonicated samples on ice, working 1-2 Seconds, stop 30 Seconds, 3~5 A cycle.)

4 Complete cracking or ultrasonic crushing, 2-8°C, 10000rpm Centrifugation 10min, the supernatant is moved into EP In tubes, immediately used for testing, or dispensed into one-time usage -80°C Freeze for later use.

Note: The precautions are the same as for tissue samples.

6 Other biological samples

2-8°C, 1000×g Centrifuge samples 20 Minutes.
Collect the supernatant for immediate detection, or press The primary usage amount is divided into -80°C Freeze for later use.

Other precautions for samples

1 The blood collection tubes shall be disposable endotoxin-free tubes.
Avoid the use of hemolyzed, hyperlipidemic samples.

2 Optimal storage conditions of samples: 2-8°C Storage should be less than 5 Day, -20°C Should not exceed 6 Months, -80°C Should not exceed 2 Years, beyond the above time should be kept in liquid In nitrogen.
In order to reduce ice crystals when frozen specimens are thawed (0°C) Damage to the sample shall be adopted 15-25°C Quickly melting in a water bath, centrifuging to remove the precipitate after melting, Mix well and use for testing.

3 The detection range of the kit is not equivalent to the concentration range of the test substance in the sample.
If the concentration of the test substance in the sample is too high or too low, please dilute or concentrate the sample appropriately.

4 If the sample tested is special and there is no reference data, it is recommended to do pre-experiment to verify its validity.

2. Recommended sample dilution plan

Serum / Matrix components in plasma can affect test results and need to be diluted with sample diluent at least 2 Times (1/2)!

If your model group sample requires a different dilution ratio Please refer to the general dilution protocol below (This protocol is a dilution protocol that does not set up double wells for testing.
When you need to set up a double hole, please add the sample and diluent volume x Number of complex holes):

Dilution 2 Times (1/2): One-step dilution.
Take 60ul Sample addition 60ul In the sample dilution, mix gently.
Dilution 5 Times (1/5): One-step dilution.
Take 24ul Sample addition 96ul In the sample dilution, mix gently.

Dilution 10 Times (1/10): One-step dilution.
Take 12ul Sample addition 108ul In the sample dilution, mix gently.
Dilution 20 Times (1/20): One-step dilution.
Take 6ul Sample addition 114ul In the sample dilution, mix gently.

Dilution 50 Times (1/50): One-step dilution.
Take 3ul Samples and 47ul Normal saline (I.e 0.9% Sodium chloride) join 100ul In the sample dilution, mix gently.
Dilution 100 Times (1/100): One-step dilution.
Take 3ul Samples and 177ul Normal saline addition 120ul In the sample dilution, mix gently.

Dilution 1000 Times (1/1000): Two-step dilution, you can dilute first 50 Times (This step is entirely diluted with saline), re-dilution 20 Times.
Gently mix well.

Dilution 10000 Times (1/10000): Two-step dilution, you can dilute first 100 Times (This step is entirely diluted with saline), re-dilution 100 Times.
Gently mix well.

Dilution 100000 Times (1/100000): Three-step dilution, you can dilute first 50 Times, re-dilute 20 Times (The first two steps are all diluted with normal saline), final dilution 100 Times.
Gently mix well.

Note: The amount of liquid taken during each dilution step is not less than 3ul, the dilution factor is not more than 100 Times.
Each step of dilution should be mixed evenly to avoid foaming.

3. Reagent preparation before testing

ahead of schedule 20 Minutes remove the kit from the refrigerator and equilibrate to room temperature (18-25°C).
If the kit needs to be used multiple times, please take out only the enzyme-labeled slats and standards required for this experiment, and store the remaining enzyme-labeled slats and standards according to the specified conditions.

1 Lotion, :

With deionized water or distilled water (The recommended resistivity is 18MΩ Ultrapure water) Will 30ml Concentrated wash (48T For 15ml) Diluted to 750ml(48T For 375ml) And mix well.
Or according to the requirements of the experiment, take an appropriate amount of concentrated washing liquid and dilute to 25 Double volume and mix well, return the unused solution 2-8°C.

If crystals are formed in the concentrated washing liquid, they may be 40°C Heating in water bath (The heating temperature should not exceed 50°C), until the crystals are completely dissolved, mix well and use.
The prepared lotion is best used on the same day.
If you can't use it up, you can keep it in 2-8°C, not more than 48 Hours.

2 Standard, :

1 Standard quality control in 1000×g Centrifugation 1 Minutes, allowing the liquid to flow to the bottom of the tube.
Marked as Zero tube.

2 Gradient dilution: separately 7 1 EP Tubes, respectively labeled as 1/2, 1/4, 1/8, 1/16, 1/32, 1/64 And blank.
First in each EP Add separately into the tube 0.3ml Sample dilution of.
Take again 0.3ml Zero tube Standard solution is added to 1/2 Tube, mix thoroughly.
Take again 0.3ml 1/2 Tube standard solution to 1/4 Tube, mix thoroughly.
Take again 0.3ml 1/4 Tube standard solution to 1/8 Tube, mix thoroughly, and so on.
attention Blank EP Only the sample dilution is in the tube.
At this point, from Zero tube Tube to blank Take care of this 8 1 EP The concentration of the standard in the tube is 20ng/ml, 10ng/ml, 5ng/ml, 2.5ng/ml, 1.25ng/ml, 0.625ng/ml, 0.312ng/ml, 0ng/ml.

Note: Dissolved tube zero standard, please store in 2-8°C, and in 12 Use within hours.
For other diluted gradient standard working solution, please refer to 2 Use within hours.

3 HRP- Antigen Working Solution:

Pre-experiment 30 Ready within minutes, ready-to-use, not suitable for long-term storage.

1 Calculate the total volume of working fluid required: 100ul/ Hole × Number of holes.
(It is best to prepare more than the total volume 100ul-200ul Amount of)

2 1000×g Low speed centrifugation 1 Minutes, will concentrate HRP- The antigen was collected to the bottom of the tube.

3 Press with antigen diluent 1/100 Dilution and concentration HRP- Antigen, mix well.
(If will 10ul concentrate HRP- Antigen Addition 990ul In antigen dilution)

4. Operation steps

When diluting samples and reagents, they should be thoroughly mixed.
It is recommended that a standard curve be plotted for each test.

1 Set standard wells, sample wells, blank wells and record their positions.
In order to reduce the experimental error, it is recommended to set up double holes for standards and samples.
Wash before adding standards and samples board 2 Times.

2 Sample addition: Add to the standard well 100ul Each gradient standard was added to the sample well 100ul Moderately diluted sample to be tested, added to the blank well 100ul Sample dilution.
Apply a laminating film and apply it in 37°C Static incubation 90 Minutes.
(Add the solution to the bottom of the plate and gently shake it Mix evenly and avoid contact with the pipe wall and blistering as much as possible.)

3 Wash board 3 Time: Remove the coating film, suck off or shake off the liquid in the enzyme labeled plate, and pat it on clean absorbent paper 2-3 Times.
Add wash buffer per well 350ul, immersion 1 Minutes, discard the liquid in the hole and pat it on absorbent paper 2-3 Times.
Repeat this plate washing step 3 Times.

4 Add HRP- Antigen Working Solution: Add to each well 100ul HRP- Antigen Working Fluid.
Apply a laminating film, 37°C Static incubation 30 Minutes.

5 Wash board 5 Time: Remove the coating and wash the plate with wash buffer 5 Time, method reference steps 3.

6 Add TMB Chromogenic substrate: Add to each well 90ul TMB Chromogenic substrate, coated with film, in 37°C Static incubation in the dark 10-20 Minutes.
Turn on the plate reader to preheat 15min.
(Note: Do not use the formulation HRP Loading tank for the conjugate.
Color development According to the actual change of color, the reaction time can be shortened or extended, but it cannot exceed 30 Minutes.
When appear in the standard hole Better blue gradient The reaction may be terminated.
The color rendering intensity is not easy to be too weak or too strong.
Please refer to the relevant documents and QR codes on the second page of the manual for accurate control of color rendering methods)

7 Add reaction stop solution: After color development, Hole Inner Liquid not Can Abandon Drop, add to each well 50ul Reaction stop solution.
The color will instantly change from blue to yellow.
Sequence and addition of stop solution TMB The order of the substrates is the same.

8 OD Measurement of values: Immediately use a microplate reader in 450nm Read at OD450 Numerical value.
(If your microplate reader has a selectable correction wavelength, set to 570nm Or 630nm.
The calibration reading is OD450 Minus the value of OD570 Or OD630 The value of.
This method corrects and removes non-chromogenic substances OD Value, Thereby obtaining more accurate detection results.
If the plate reader does not 570nm Or 630nm Wavelength, you can use the original OD450 Value.)

5. Result calculation

1 Take the average of standard and sample replicate wells OD450 Value (using raw OD450 Value or correction reading value), subtract the blank hole OD450 Value, as the calculated value.

2 Taking concentration as the abscissa, OD450 Values are ordinates, and four-parameter equations can be used 4PL Draw a standard curve (Remove the values of blank holes when drawing).
You can also use the mapping software that comes with the plate reader (Such as Thermo FC Model plate reader SkanIt RE Software) Or Curve Expert 1.3 or 1.4 Professional Software (Our website can be downloaded and used for free) Draw the standard curve.

3 The sample's OD450 By substituting the value into the standard curve, the concentration value of the sample can be calculated.
If the sample has been diluted, multiply it by the corresponding dilution factor.

Experimental data and standard curve

This product has been tested by the quality control department and meets the performance requirements of the instruction manual.
(Laboratory humidity is 20%-60%, the temperature is 18°C -25°C.
Before color development TMB Balance to 37°C After adding enzyme labeled plate wells, 37°C Incubate in the dark 15 Minutes.)

Due to differences in specific experimental environment and operation, the following experimental data and standard curves are for reference only, and experimenters need to establish standard curves according to their own experiments.

Precision

In-plate precision: low, medium and high concentration samples are in the same 1 On-block plate detection 20 Times.

Inter-plate precision: Low, medium and high concentration samples are respectively in 3 On-block plate detection 20 Times.

Recovery

Combine a certain amount of Anti-PEG IgM Added to the sample, and the measured values were added to the sample by comparing the measured values with Anti-PEG IgM The expected amounts of were compared to calculate the recovery.

linear

The appropriate concentration will be added Anti-PEG IgM The samples were diluted separately 2 Times, 4 Times, 8 Times to obtain the recovery range.

Stability

The unopened kit is available in 37°C And 2-8°C Stability experiments were performed and stability data were obtained.

Note: The volume of liquid reagent supplied in the reagent bottle is slightly larger than the label indicates. Please use a pipette to measure accurately and dilute accordingly.

Unopened kit, please store in 2-8°C 。 After opening, the storage conditions are shown in the table above:

1. When using different kits, it is necessary to mark them first to prevent the components from being mixed, resulting in experimental failure.

2. After the kit is opened, please refer to the component storage conditions table for the storage conditions of the enzyme plate and standard product (the activity will decrease after moisture).

3. Please use a sterile disposable tip to suck the reagent. After use, the reagent bottle cap must be tightened to prevent microbial contamination and evaporation.

4. When washing the plate manually, add the tip or dropper of the washing liquid and do not touch the hole of the enzyme labeled plate. Inadequate washing or contamination tend to cause false positives and high backgrounds.

5. During the testing process, please prepare the reagents required for the next experiment in advance, and add the reagents to the plate holes in time after washing the plate to prevent the plate holes from drying out and causing testing failure.

6. Do not use reagents from other batches of kits or reagents from other sources for this kit without confirmation.

7. Do not reuse disposable tips to avoid cross-contamination.

8. After the sample addition is completed, the film is applied to prevent the sample from evaporating during the incubation process, and the incubation process is completed at the recommended temperature.

9. Please wear laboratory coats, masks, gloves, etc. during the test, and do a good job of protection. Especially when testing blood or other body fluid samples, please follow the national biological laboratory safety protection regulations.