ELISA - Science method

Detecting avian leukosis virus (ALV) from egg albumin using ELISA can be challenging due to several factors:

To overcome these challenges, several strategies can be employed:

Coating a microplate with viruses for a whole-virus enzyme-linked immunosorbent assay (ELISA) requires precision and adherence to a standardized protocol to ensure reproducibility and accuracy. Here is a detailed, step-by-step guide on how to coat a microplate with viruses for this purpose:

By following these detailed steps, you ensure that the virus is properly adhered to the microplate, maximizing the sensitivity and specificity of your whole-virus ELISA. Each step, from the preparation of the virus to the final storage of coated plates, is designed to maintain the functional integrity of the viral antigens and provide reliable, reproducible assay results.

Check out this protocol list; it might provide additional insights for resolving the issue.

Kais Khudhair al Hadrawi Phan Thi Lam Hong thanks very helpful

Hello Muhammed Zidan Khan

You may use Liquid Chromatography-Mass Spectrometry (LC-MS) where in you use liquid chromatography for the physical separation by segregating the molecules based on their chemical attributes such as size, polarity and affinity for the stationary phase, followed by identification and quantification of the separated molecules using their mass-to-charge ratios by mass spectrometry. LC-MS can target a wide range of biomarkers from metabolites to proteins and peptides. LC–MS is a bioanalytical method for quantitative analysis in biological fluids like serum, plasma, urine, etc.

For tissue homogenates, you may use Western Blot. However, the data produced with Western Blot is semi-quantitative. This is because it provides a relative comparison of protein levels, but not an absolute measure of quantity.

Best.

Thank you for your reply and valuable suggestions Stephan Steinke

In enzyme-linked immunosorbent assays (ELISA), the cutoff value is typically determined to classify test results as positive or negative. The cutoff value is calculated based on the optical density (OD) readings of samples and control standards. Here's a general guideline on how to calculate the cutoff value in ELISA:

It's important to note that the cutoff value determination may vary depending on factors such as assay variability, assay validation, and specific experimental requirements. Additionally, some commercial ELISA kits may provide pre-defined cutoff values based on extensive validation studies.

Always refer to the specific instructions provided with the ELISA kit or assay protocol for guidance on calculating and interpreting cutoff values tailored to your experimental setup.

Malcolm Nobre thank you very much for your reply! It's clear now.

Hello Prince Dee

For the attached THP-1 cells which have been differentiated, wash the cells gently with pre-cooled PBS, and detach the cells using 0.25% trypsin. Centrifuge the cell suspension for 5 min at 1000×g. Discard the medium and wash the cell pellet three times with pre-cooled PBS. You may add 150-250ul of precooled PBS per 1×10^6 cells to keep the cells suspended. Carry out repeated freeze-thaw cycles by freezing the cell suspension in the freezer and then thawing the cell suspension at room temperature or 37℃ until the cells are fully lysed. This method of lysis will cause the cells to swell and ultimately break as ice crystals form during the freezing process and then contract during thawing. Centrifuge the resultant lysed cells for 10 min at 1500×g at 2-8℃. Discard the pellet and collect the supernatant to carry out the assay. Add protease inhibitor such as PMSF at 0.1 - 1mM in the final volume during cell lysis to prevent protein degradation.

Please note: Do not use RIPA lysis buffer or UREA lysis buffer since there is a possibility of causing deviation due to the introduced chemical substances.

No. But you need to perform the total protein assay (Bicinchoninic acid (BCA) assay) to determine the protein concentration of your lysate. The volume of each sample can be normalized to deliver the same amount of total protein for the assay.

Good Luck!

You could find out how omitting the stop solution works in your system by repeating the experiment. In brief, set up your reactions in duplicate. Add the stop solution to one set and omit it from the other set. Get all the readings and compare the data. From there, you can determine the impact of omitting the solution and apply those findings to your current data set.

We don't know of any kits for pTDP-43 at the moment, but there are a few suppliers of ELISAs for TDP-43 which claim rat reactivity, including Abbexa, MyBiosource, and St Johns Laboratory.

Hi

Sorry for the delay in reply but got very tied up editing a thesis.

If you have access to pure spike protein then I suggest the following method. The reason for this suggestion is to keep assay as direct as possible to prevent potential artifacts.

Method:

1. Biotinylate the spike protein (kits are available from Thermo Fisher).

2. Coat plate with LPS. Control contain no LPS. Overnight at 4 C.

3. Block plate with 25 mg/ml casein in PBS (2 hours at room temperature).

4. Add biotinylated spike protein in PBS containing 0.05 % Tween 20. Incubate for 2 hours at room temperature.

5. Wash the plate 4 times with PBS containing 0.05 % Tween 20.

6. Add Avidin peroxidase conjugate (Sigma Aldrich/Thermo Fisher and many other reputable suppliers) as per manufacturer's instructions diluted in PBS containing 0.05 % Tween 20.

7. Wash the plate at least 4 times with PBS containing 0.05 % Tween 20.

8. Add substrate. Incubate. Stop and read plate.

For optimization with do checkerboard dilution ranges of LPS and the biotinylated spike protein.

A decade late to respond but for any other scientists coming across this question - there is now an ELISA for high five cells HCP detection. Company is called Krishgen Biosystems.

For everyone interested in this question: We couldn't find out the reason why this ELISA failed except for the standards and have sent it back to the company. Obviously neither the Lysis Buffer nor the amount of Urea was the problem. Maybe it wasn't compatible with HEK-Lysates. But that is just an assumption.

Ahmed Jehanli thank you for your suggestion. I treated the nitrocellulose with 1% BSA after lining the capture antibodies. My conjugated quantum dot-detection antibody contained 1% BSA too.

Is it because the concentration of the

detection antibody is too high that caused non-specific happened?

Dear Corinne,

I am having a similar issue. I wonder, what was your conclusion for this situation?

Thank you very much for giving me the answer.

It helped me a lot! Aitsana Maslakova Deepdarshan Urs Aarya Chitransh

SDS will destroy the protein and will prevent the adsorption of the protein (to the plate or to another protein). As mentioned, try dissolving it first in the carbonate buffer. 50% to wash the pellet from what?

As nanobodies are the usually the product of genetic engineering and phage- or ribosome display selection, the are usually constructed with specific tags for selection, detection and purification, e.g. His-tag, Myc-tag or Flag-tag. Check with the provider of your nanobodies to see whether this is the case for the ones you are using. If this is the case, you can use commercial antibodies against those tags for detection or, in a sandwich ELISA format, for immobilization.

Hello Arkaitz Mucientes

I agree with Luis Mata

As far as possible use large volumes because they are easier to pipette accurately than small ones. Pipetting small amount (3ul for the form #2) accurately is not easy and will likely contribute to statistical error in your results. Factors that impact air pressure or pipette tip function can lead to volume errors, particularly when you work with small volumes.

On the other hand, when you work with large volume and pipette 24µl (for the form #1), you may easily and accurately pipette this amount, which would yield much tighter error bars.

Best.

If you have a cytometry or Luminex system you may look up for bead based assays which will provide you better sensitivity and accuracy.

Best wishes,

Hello Naile Merve Guven,

It would not be proper to give you the cell number because it will vary with cell type. So, you will have to perform a pilot experiment to decide the optimum cell density for a multi- well plate format which you will be using for the actual experiment.

In the pilot experiment, you may seed different cell densities and choose the optimum cell density that gives you 80% confluency in 24 hours. For instance, for HeLa cells in a 96-well plate format you may seed cells in the range of 5000 to 25000 cells per well in 200ul, and choose that cell density which gives you 80% confluency after 24 hours.

After having obtained the optimum seeding density from the pilot experiment, you may seed cells at that optimum cell density in whichever plate format you decide, and after 24 hours you may carry out the treatment.

After treatment, you may lyse the cells with RIPA buffer and determine the total protein of the lysate using BCA assay with different dilutions of the lysate.

Best.

Hello Naile Merve Guven

It depends on your protocol. If you are going to use cell lysate, then you will have to perform the total protein assay to determine the protein concentration of your sample using BCA assay, and normalize the volume of each sample that can deliver the same amount of total protein for ELISA. Usually, total protein concentration of the cell lysate should be at least 1mg/ml.

However, if you are going to use supernatant (without cells) for the ELISA test, then just use equal volumes and perform ELISA.

Best.

Hello Aya Al-rawashdeh

If you have enough wells, you may run both the undiluted as well as the diluted samples (1:2) in the same 96-well plate. I would suggest you also include one more dilution (1:4) in the run.

Run the standards as well as the samples (both undiluted and diluted) in triplicates. In case the wells are not sufficient, you may use another 96-well plate. Sample volumes are usually between 50-100μl; however, this can vary from assay to assay. I suggest you use 100ul of undiluted sample and for the diluted sample (1:2), add 50ul of the supernatant to 50ul of fresh media. Samples that generate values which are higher than the highest standard need to be repeated using a higher dilution factor.

Note that sample values should fall within the assay’s dynamic range. Values outside the range of the standard curve are usually non-linear, and because of this property it is not possible to extrapolate a value correctly using this curve.

You may also note that a standard curve should be run for every plate which means each plate should have a standard curve. If you cannot afford to run the standard curve for every plate because of limited supply or cost, at least you should run one standard curve with each set of plates for that day. If you are using serum-containing medium, it is necessary that you run an uncultured medium blank to obtain the baseline signals which can then be subtracted from the cultured media sample data.

Good Luck!

You may find this article useful ( ). Basically depending on the insulin preparation and amino acid sequence (analogues) you can get variations in potency (IU) for the same mass (SI).

1. how do you determine the total amount of drug, free amount of drug and nanoparticles weight?

From the uv calibration curve of your drug you will get amount of drug loaded by subtracting total amount of drug by Free drug. This total amount is the initial concentration of drug you have added in your chitosan nanoparticles and the free drug can be find out by (supernatant absorbance + intercept/ slope).

2. I have obtained a standard curve from the ELISA test. The dilution factor is 5 and the dilution of standards is in pg/ml. Based on the y=mx+c equation, I am able to find the concentration of the supernatant. However, I am not sure the unit of it? is it pg/ml as well?

The unit of concentration you have found from the calibration curve is same the concentration of drug solution you prepared for calibration curve study i.e. pg/ml.

After finding free drug from above formula you have to multiply that free drug concentration by dillution factor to get your actual free drug in supernatant.

Now you need amount of you nanoparticles in the formulation you can get it by freez dryng nps and and weigh the pellet.

DRUG LOADING CAPACITY = (total drug weight -free drug weight) /weight of nps(g)

for % * by 100

Hello,

I recommend the CFT too. It is good and has international standardization. I would also add the SAT (serum agglutination test). In addition, I would test the camels after a month to see if these antibodies' levels are increasing, decreasing, or maybe they have already disappeared entirely. During brucellosis, the antibodies mostly persist, and in most cases, their levels increase. It would also be helpful to see if these antibodies are IgG or IgM. A rivanol or 2-mercaptoethanol test is very helpful for this purpose.

The occurrence of false negatives and false positives in your results is intricately linked to the sensitivity and specificity of the diagnostic protocol. One effective strategy for optimization involves utilizing known positives and known negatives during the experimental procedure. Subsequently, the development of a Receiver Operating Characteristic (ROC) curve can aid in identifying an optimal cutoff point. This is the point at which both sensitivity and specificity approach 100%. We trust this suggestion proves beneficial to your work.

Assuming the peptide functions by binding. The target protein could be immobilized on a plate or transferred to a blot. Then the peptide (likely also with fluorescent / chemiluminescent tags) could be applied to bind to the protein for detection.

May be inactive at that temp and interval.i assuming it not sure.

Dear Shimos A. Alshareef

ELISA may be run in a qualitative or quantitative format.

Qualitative results provide a simple positive or negative result (yes or no) for a sample. The cut-off between positive and negative is determined by the analyst and may be statistical.

For diagnostic tests that produce a numerical result, the point above which test results are classified as positive is called the cut-off. These are usually established by testing a large number of infected and non-infected animals and selecting the value that maximizes the sensitivity and specificity of the test. For diagnostic kits the cut-off is established by the kit manufacturer. You may want to refer to the article attached below.

In quantitative ELISA, the optical density of the sample is compared to a standard curve, which is typically a serial dilution of a known concentration solution of the target molecule. For instance, if a test sample shows an absorbance of 1.0, the point on the standard curve that gives an OD = 1.0 will be of the same analyte concentration as the sample.

So in this case, the samples showing values 11 and above are considered positive.

Best.

thank you!

You can detect the presence of bacteriocins by well diffusion method. In this method, after bacterium growth in the selective medium, make centrifugation to get supernatant. inoculate the sensitive bacteria in suitable medium, make holes with a diameter of 6 to 8 mm with a sterile cork borer and introduce about 50 -200 µL/well. incubate the plates under suitable conditions depending upon the sensitive microorganism. No growth occurs in the presence of bacteriocins and the inhibition zone depends on bacteriocin concentration.

Hi,

My guess is that the His ELISA kit you are using is a sandwich ELISA, whereas the HA kit is a competitive ELISA. In competitive ELISA the assay signal is opposite to the concentration of the analyte (inversely proportional).

A target analyte that has a detection moiety (eg, a conjugated fluorescent dye) can be added, competing for binding sites on the antibody. A low amount of binding would indicate a high level of target analyte in the sample.

The signal inversely correlates with the amount of analyte such that, if the concentration of the target analyte is high, then the reference signal is diminished through its competitive binding to a limited amount of labelled antibody.

The background signal of Trx with your antibodies can be checked if you coat with Trx only and measure the signal.

Although, it seems more likely that you have the problem that ELISA and Western Blot are not necessarily identical assays, so you could get signal in Western Blot but not ELISA for various reasons, e.g. differences in binding native and denatured target protein. Your fusion protein could also bind the plate surface in a preferred orientation like the hydrophilic Trx towards the solution, so accessibility to the target protein may be limited or less epitopes available for binding.

You have to standardize your blocking material and its percentage.

Here are some suggestions for you:

1. Try different percentage of blocking solution (1%, 3% and 5%) at the same time in a single plate.

2. Try different blocking materials (BSA, Skimmed milk or casein)

3. Add at least 200uL of solution to block the wells (300uL capacity)

4. Wash your plate 3-5times after blocking

It has certainly been observed many times that some antisera are more effective against denatured protein (such as western blots or ELISA) and others against native protein. This is due to the nature of the primary epitopes the pABs react against. There are certainly many other possible reasons and A.B Bayazid gave a good list of them

Dear Malcolm Nobre, I am planning a 24-hour cell stimulation. I'll try to do it with your suggestion. Thank you very much!

Robert Adolf Brinzer I will try that. Thank you!

Hi Donis, did you figure out this problem? The same thing happened in our study. We tested mice plasma with novusbio elisa kit.

Hello Bushra Hameed

A standard curve is a curve that may be used to determine the concentration of target protein in the sample.

In ELISA technique, if one must quantify the concentration of an antigen or an antibody in a sample, one must plot a standard curve.

One can plot a standard curve by using the known concentrations of the standard reference antigen against the optical density for each concentration. For this purpose, you may use a series of serial dilution of a standard protein (for instance, BSA) of one known concentration across a range of concentrations near the expected unknown concentration.

Then the unknown concentration of the antigen can be interpolated from the linear part of the standard curve to calculate the amount of antigen present in the sample.

A typical standard curve will have a sigmoidal shape in which the higher concentrations of the standard dilutions will reach a plateau in absorbance. It is the relatively long linear region of the curve that makes the ELISA results accurate and reproducible. So, the calculation of the unknown antigen in the sample is carried out through extrapolating the linear part of the standard curve.

Best.

Hi Madison! I have saved my ELISA standard for mouse IL-6 in the -80 several times without seeing any weakening in the signal. The longest I have saved a standard, however, was only for a couple of weeks (and only thawing it 2 times total). I'm sure storage temperature and freeze-thaw cycles play a role. Hope this helps!

It can happen if the tube is old, and/or liquid is stored for an extended period of time - better change solutions every week/month or so, depending on usage. Likely it will not affect plate wash unless the amount of plastic is very extensive, just prepare new PBS in a new bottle.

Deplete the serum using protein A to get rid of IgG prior to the ELISA app. Collect the flow-through fraction resulting from the spin column or cartridge and dilute with the proper buffer and volume...

Hi. Thanks you

I think you're saying you're getting an unacceptably high background reading from your negative control. It's good that you're concerned about this problem. It is common, especially for human blood samples.

If you are using human blood, be sure to take proper bloodborne pathogen precautions, including your vaccinations and PPE.

Consider using serum rather than plasma. And for human samples you should heat the sera to kill viruses.

You should be diluting your samples in blocker solution. For ELISA you probably can start with a 1:1000 dilution and work toward 1:1000000.

You can reduce non-specific binding by adding things to your blocker solution. Starting with a base of PBS, blockers usually contain a protein like casein or albumin at 0.1% (1 mg/mL) and a nonionic detergent like Tween at 0.01%. You can try using a different protein. You can safely increase the protein 10-fold. Depending on the ELISA format, you may want to include 1% control serum in your diluent and blocker as a blocker protein. Increasing the detergent (up to 10x) may decrease your overall signal, but it may increase signal/noise ratio. And sometimes it helps to add 0.1M glycine.

ELISA is easier, more sensitive and more accurate than a western blot. Do duplicates or triplicates for your samples so your results to be more reliable.

Yes, the equation you have provided is correct for calculating the percentage difference in reduction of resazurin between treated and control cells in the resazurin assay. This is a standard way to quantify the results.

Regarding your second question - you are absolutely right that the positive control wells (cells + resazurin but no drug) should show more metabolic activity and reduce more resazurin (thus give higher absorbance readings) compared to the drug-treated wells.

Some things to troubleshoot:

- Make sure the positive control cells are healthy and actively proliferating/metabolizing. Old, dense or contaminated cultures could give low readings.

- Use the optimal cell seeding density. Too low density can reduce metabolic activity.

- Allow sufficient incubation time for the cells to reduce resazurin before measuring absorbance.

- There could be an issue with resazurin stock solution if the positive control wells turn brightly pink. The conversion to fluorescent resorufin indicates reduction, but there should still be enough oxidized resazurin left to allow quantification. Make fresh stock.

- carefully confirm there are no pipetting errors mixing cells, drugs, resazurin etc.

I would first repeat the assay with fresh optimized positive control cells + new resazurin to see if gives expected higher readings compared to treated cells.

Dear Daniel,

your working conditions seem not optimal. In case you are developing an ELISA test, please try different conditions, regarding Ag con., primary and secondary Ab dilutions. If you are working with a commercial ELISA, the problem could be from washing; so you may need extra washing steps.

You can also try using 0.03 M urea in the serum diluent and incubate the plates at 37C for 1 hr then wash and add the secondary Ab. This may reduce the cross reactivity. Cross reactivity is often observe with canine sera.

Hope that is useful.

Elfadil

Finding specific research reagents like ELISA kits in regions where they may not be readily available can be challenging, but there are several options you can explore:

Before purchasing any kits, it's crucial to verify the quality and authenticity of the products and ensure that they meet your specific research needs. Additionally, be prepared for potential delays in shipping and customs clearance when ordering from international suppliers.

I agree with Elliot J. Glotfelty. Try first to obtain a positive response to LPS dilutions after you assured the viability of your cells.

Saif Wahid Thank you very much for your valuable suggestions! I do have some follow up question of your suggestions

Suggestions 1 and 2.. They kit does not come with a homogenization buffer, the used have to prepare the buffer. Did you mean to say homogenize with less concentrated guanidine buffer instead of the suggested 5M concentration? Because after homogenization, samples were diluted to final concentration of 0.08M guanidine in sample

As for suggestion 3, I ran another trial with BSA in the dilution buffer however with no success. I will try another run with BSA in the homogenization buffer. What concentration of BSA should I use? If you could suggest, that would be big help! Thanks!

Dear Prof. Saima Mukhtar

The kits that are labeled as “research purpose only” are exempt from regulatory requirements and approvals that would be needed for clinical diagnosis or patient management. They are supposed to be used only in research and discovery work. Since there are no guidelines for such “research purpose only” kits, the extent of manufacturing quality compliance, assay characterization and documentation will vary considerably across different vendors.

On the other hand, for the diagnostic kits, pre-market regulatory clearance is required by the FDA. The review of analytical and clinical validity of a diagnostic kit is done prior to marketing and thereby its use on patient specimens in clinical diagnosis where in the diagnostic kit identifies, measures, or predicts the presence or absence of a clinical condition or predisposition in a patient.

The “research purpose only” kits which have no regulatory compliance, no manufacturer dependent specifications, the kit inserts being provided have variable degrees of information, and a wide variety of sample matrix (e.g., animal/human fluids, tissues, culture media) being present, cannot be used for diagnostic purpose.

So, I feel you should not make an attempt to translate the results obtained from kit labelled as “research purpose only” to diagnostic values.

Best.

Yes, you can normalize your progesterone data to a housekeeping gene using qPCR. Here's how you can do it:

Regarding combining methods like housekeeping gene normalization and percentage change:

Here are some suggestions based on my experience with ELISAs:

1. Check the specificity of your reagents: Make sure that all of your reagents, especially the primary and secondary antibodies, are specifically reacting with the intended target (i.e., IgM) and not cross-reacting with other proteins in the sample. Cross-reactivity can cause false positives or negatives.

2. Optimize the concentration and incubation time of reagents: As you've already tried, optimizing the concentration and incubation time of reagents can help improve the assay's sensitivity and specificity. You might want to try different concentrations of the reagents and compare their performance. Also, consider using appropriate controls (e.g., negative controls, positive controls) to monitor the assay's performance.

3. Use appropriate blocking agents: Non-specific binding can be a common issue in ELISAs. In addition to blocking buffers, you can try adding milk or bovine serum albumin (BSA) to the blocking solution to reduce non-specific binding.

4. Consider using a different detection method: Instead of using streptavidin-HRP, which can sometimes give rise to non-specific signals, you could switch to a different detection method like alkaline phosphatase (ALP) or horseradish peroxidase (HRP)-conjugated secondary antibodies. ALP tends to produce less background than HRP and can be a good alternative.

5. Ensure proper washing steps: Washing steps are crucial in removing unbound material and reducing non-specific signals. Be sure to perform thorough washes after each step, and use appropriate wash buffers (e.g., PBS-Tween).

6. Validate your protocol with known samples: Test your optimized protocol with a set of known samples (both positive and negative controls) to ensure it's performing correctly. This will help you identify any issues before proceeding with unknown samples.

7. Consider consulting experts or literature: If you continue to encounter problems, reach out to experts in the field or consult relevant literature to gain insights into successful Mac ELISA protocols for detecting IgM in mice infected with T. marneffei.

8. Evaluate the possibility of sample contamination: Sample contamination can introduce false positives or negatives into your results. Make sure to handle samples properly, and consider testing your samples for potential contaminants.

9. Assess the impact of dilution factors: Diluting samples can affect the accuracy of the assay. Try comparing the results of undiluted and diluted samples to determine whether dilution is causing issues.

10. Monitor assay performance over time: ELISA performance can degrade over time due to reagent degradation or changes in experimental conditions. Regularly assess the assay's performance and make adjustments as needed.

By addressing these points, you may be able to improve the reliability and accuracy of your Mac ELISA protocol for detecting IgM in mice infected with T. marneffei. Good luck with your research!

Moangsom, I have had this problem before. It is not due to Hook effect because you doing Indirect ELISA. It probably is because of some High affinity and low concentration of IgA. Also, look for ELISA interference and false negative results.

you can find here more:

Thanks a lot

Interpretation of the Avidityof ELISA according to manufactory prochure of the ELISA machine

For complex samples such as cell extracts, the sandwich ELISA may be more sensitive than the indirect ELISA because it captures all of the antigen onto the surface, no matter how little of the antigen is present, until the capture antibody is saturated, and everything else is washed away.

For the indirect ELISA, the sensitivity is limited by the proportion of the sample that consists of the antigen of interest. There is a limited binding capacity of the surface, and everything in the sample competes for the limited surface.

So, for the indirect ELISA, you probably need to use higher antigen concentrations for the standard curve than you would for the sandwich ELISA.

Dear,

The number of cells is depend on perticular assay which you want to perform. Examples, if you want to perform Scratch assay then you need more number of cells and if you wish to do colony formation assay then you need less number of cell (600 cells per well in 6-well culture plate).

You can visist Manufacturer company of your culture plates regarding this.. they provide cell number on thier websites. refere cell number to respective culture plates.

Third, you can refer literature where you can find cell number for respective culture plates and fourth, you can optimise yourself. When diameter of plate is decrease, cell number will be also decrease.

As of my concern, I used to seed 7 thousands, 30 thousands, 50 thousands and 1 lacs cells per well in 96, 48, 24 and 12 well plates respectively.

Thank you

I agree with Hüseyin Besir that ELISA is not an appropriate method for measuring the Kd (equilibrium dissociation constant) of an interaction because it is not an equilibrium method. That is because of the washing steps required to remove unbound components.

Hello Philip Samaan

Storage of tissue culture supernatant containing protein of interest at room temperature often leads to protein degradation and/or inactivity. After obtaining the supernatant from PBMC culture, instead of letting it sit at room temperature, you should have kept the supernatant on ice or in the refrigerator at 4 degree C which I assume must have been within reach from your workstation. After completion of your other experiment, you could have transferred the supernatant to -80-degree C. At room temperature proteins start degrading with time.

As far as TNFa is concerned, it will be affected if you let it sit at room temperature for more than 1 hour. Please refer to the articles attached below.

Perforin is not a cytokine. It is a 60-70kDa glycoprotein which is a cytotoxic molecule. I am not certain about the stability of perforin at room temperature, but I am sure that it would also start degrading at room temperature with time.

Best.

The use of rapid diagnostic immunochromatographic technique (ICT) methods are prevalent in developing countries, while ELISA and molecular testing are recognized as more widely accepted for their enhanced accuracy. Rapid diagnostic tests for viral infections have been available since the 1990s, primarily designed for emergency diagnostics, home-based testing, and field surveys. Furthermore, in several deprived regions, rapid diagnostic tests are used as a means to identify infections caused by the hepatitis B virus (HBV), hepatitis C virus (HCV), and human immunodeficiency virus (HIV). This method serves as a solution to address the scarcity of financial resources and medical equipment. Nevertheless, a significant issue regarding the utilization of rapid screening tests revolves around the necessity for these tests to demonstrate an acceptable level of sensitivity and a sufficient level of specificity to reduce the occurrence of false results.

Reference: Al-Matary, A. M., & Al Gashaa, F. A. S. (2022). Comparison of different rapid screening tests and Elisa for HBV, HCV, and HIV among healthy blood donors and recipients at Jibla University Hospital Yemen. Journal of Medicine and Life. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9762378/

Hi Sepideh,

Biomatik offers an extensive list of over 38,000 high-quality ELISA Kits. Feel free to check out our Insulin and IGF-1 kits here: https://www.biomatik.com/search-results-page?q=%20IGF-1&page=1&rb_categories=ELISA%20Kits&rb_custom_field_4af649a635c21db41b73eafb1def22da=Mouse

Best regards,

Biomatik Team

Welcome dear, I hope my response will be useful to you, Good Luck with your experiments

Thank you very much for all the useful information, very much appreciated.

I use the program Prism GraphPad for that. The data used is only the concentration level of the standard curve and the optical density levels. Then I interpolate the values for the estimation of the concentration of the protein in my samples. After selecting the interpolate option, a parameters window is open, where I select the 4 parameter logistic regression.

I hope this can help you.

Dear Dr Abdul Main

PCR is the gold standard for pathogen detection while ELISA is enzymatic titration. Due to high sensitivity and specificity.

In terms of accuracy, both ELISA and RT-PCR can be highly accurate when performed correctly. ELISA might be a suitable choice if the primary concern is detecting high viral concentrations quickly and cost-effectively. On the other hand, if you need to detect low viral concentrations or require quantitative data, RT-PCR is the more appropriate option despite its higher cost and complexity.

Kind regards

AB Bayazid

https://journals.sagepub.com/doi/10.1177/104063870301500203?url_ver=Z39.88-2003&rfr_id=ori:rid:crossref.org&rfr_dat=cr_pub%20%200pubmed

Thanks!

Dear Dr. Rishabh Tomar

The Index Value is calculated by dividing the sample OD value by the cut-off-OD.

So, Index Value = OD sample / OD cut-off

Let me explain with an example.

Suppose an ELISA for a given test, the Index Values are as follows:

Negative < 1.0

Borderline 1.0 – 1.2

Positive: ≥ 1.2

For the OD cut-off of 0.420, let us consider the following sample OD values.

1. Sample OD: 0.110

2. Sample OD: 0.420

3. Sample OD: 1.220

Therefore, the index values for the above sample ODs would be as follows.

Sample 1: 0.261

Sample 2: 1.0

Sample 3: 2.904

The interpretation would be as follows:

Sample 1: negative

Sample 2: borderline

Sample 3: positive

Regards

Malcolm Nobre

Dear doctor

Go To

Microscopical and serological studies on Sarcocystis infection with first report of S. cruzi in buffaloes (Bubalus bubalis) in Assiut, Egypt

Asmaa M. Metwally, Mahmoud R. Abd Ellah,📷 Amira A. AL-Hosary, and Mosaab A. Omar

J Parasit Dis. 2014 Dec; 38(4): 378–382.

Published online 2013 Feb 15. doi: 10.1007/s12639-013-0257-x

"Abstract

This study was performed for the purpose of investigating the prevalence and the species composition of Sarcocystis spp. in buffaloes in Assiut province, Egypt. Macroscopically we reported the infection of buffaloes with Sarcocystis fusiformis, while microscopically three Sarcocystis species (Sarcocystis cruzi, Sarcocystis levinei and Sarcocystis hominis) cysts were recognized, and were differentiated by their morphological features using both histopathological sections and electron microscope scanning. Regarding the prevalence of Sarcocystis species among buffaloes in Assiut province, we reported that, using gross examination of 90 buffaloes’ esophagus, only 23 samples out of 90 (25.5 %) were found to be infected; on the other hand, by using microscopical examination, the prevalence was 27.7 % (25 samples out of 90 samples were found to be infected). Using ELISA, 85 samples out of 90 (94.4 %) were found positive, an overall prevalence of 94.4 %. In this work we concluded that customary meat inspection methods in abattoirs in Egypt are insufficient for detecting Sarcocystis infection. Due to the presence of hidden or microscopic cysts, we strongly recommend the use of combined microscopical examination and ELISA for Sarcocystis diagnosis, to avoid human infection of such zoonotic parasite and to control the consequent disease. In addition, this study introduced the first report of S. cruzi in buffaloes in Egypt, and proved the hypothesis that S. cruzi is able to use animals such as water buffalo as intermediate hosts."

Hello,

CUSABIO Human Nuclear factor-kappa B (NF-κB) ELISA Kit may work in this situation. The kit was designed for the quantitative measurement of Human NFKB1 protein in serum, plasma, tissue homogenates, and cell lysates. It has been cited in 16 articles.

You can find more details on:

According to your description, you probably use cellular supernatant. However, in consideration of NFkB protein, cell lysate is recommended. But 24 well culture plates are rarely used if the sample type is cell lysate.

No matter what the sample type is, preliminary experiments are recommended.

I used the AssayGenie protocol and worked perfectly for me. There are 2 codes for mouse IL6. You should be able to find the protocols in the websites.

If you still have trouble, I can send you pictures of the protocol

In my experience a "Blank" well means a well receiving nothing but blocking buffer, sample diluent, and detector. A "Negative control" well receives the blocking buffer, negative sample, and detector. It is probably best to run both. I would not subtract things because you will want to see if there is any changes to your blanks and negative controls, rather then just subtracting them out of the data. Changes to the blank or negative control can show stability, or not, in the assay.

I have used this one - R&D Systems Mouse Somatostatin Quantikine ELISA Kit

Hello Maroš Huličiak

Protein-free blocking buffer does not contain protein. It usually contains polymers such as polyethylene glycol, polyvinyl alcohol, and polyvinylpyrrolidone. The absence of protein from protein-free blocking buffer eliminates problems associated with traditional protein-based blockers, such as cross-reactivity and interference from glycosylated proteins.

Also, protein-free blocking buffer is compatible with antibodies and avidin/biotin-based systems and results in high signal to background ratios. Besides, this type of buffer will eliminate any concern associated with regulatory compliance issues where use of animal source components is restricted.

Best.

Hi Sebastián Castro-Trujillo, Bland-Altman plots are commonly used to assess the agreement between two measurement methods. They can provide valuable insights into the level of agreement and any potential biases between the methods being compared. Considering your situation, it's important to interpret the Bland-Altman plots accurately. If you observe a skew in the plots, it suggests a systematic difference between your in-house method and the commercial ELISA kits. This discrepancy could be due to the different approaches used in determining positive or negative results.

To address the reviewers' concerns, you can explain the fundamental differences in measurement values and highlight that the commercial ELISA kits utilize arbitrary values for classification, while your in-house method uses absolute optical density. This distinction could contribute to the observed differences in the Bland-Altman plots and to further support your argument, you could consider conducting additional analyses, such as comparing the performance of the different methods using other statistical measures like sensitivity, specificity, or diagnostic accuracy. This would provide a more comprehensive evaluation of the diagnostic test and help you present a well-rounded analysis to the reviewers.

homo Arginine Elisa kit.