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Why do antibody tests target different antibodies?

Why do antibody tests target different antibodies?


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I've noticed in various COVID-19 lateral flow assays, they seem to target different antibodies (S1, S2, NP). What are the benefits or downsides of choosing different antibodies? I know that some have a higher chance of cross-reactivity, but why wouldn't all tests just use the ones with the lowest cross-reactivity?


Interim Guidelines for COVID-19 Antibody Testing

Healthcare providers considering serologic testing of persons with history of possible coronavirus disease 2019 (COVID-19) or public health officials and other researchers conducting investigations involving serologic tests.

Serologic methods have public health and clinical utility for monitoring and responding to the COVID-19 pandemic, and caring for patients, respectively.

  • Serologic testing does not replace virologic testing and should not be used to establish the presence or absence of acute SARS-CoV-2 infection.
  • Serologic tests can vary in their individual performance characteristics tests that have received Emergency Use Authorization (EUA) external icon should be used for public health and clinical purposes.
  • Serologic tests yielding qualitative or semi-quantitative results have been issued EUAs there currently is no recognized public health or clinical indication for preferential use of semi-quantitative tests.
  • Virus-based neutralization assays are currently not authorized for emergency use by the FDA, although an enzyme-linked immunosorbent assay (ELISA)-based competitive neutralization test for qualitative detection of total neutralizing antibodies has been issued an EUA. Neutralization assays currently are being used as possible surrogates of protection in epidemiological and clinical studies.
  • Antibody testing is not currently recommended to assess for immunity to COVID-19 following COVID-19 vaccination or to assess the need for vaccination in an unvaccinated person. Since vaccines induce antibodies to specific viral protein targets, post-vaccination serologic test results will be negative in persons without history of previous natural infection if the test used does not detect antibodies induced by the vaccine.
  • Unvaccinated persons who have tested antibody positive within 3 months before or immediately following an exposure to someone with suspected or confirmed COVID-19 and who have remained asymptomatic since the current COVID-19 exposure do not need to quarantine, provided there is limited or no contact with persons at high risk for severe COVID-19 illness, including older adults and persons with certain medical conditions.

Fluorescent Immunodetection for ICC: Step by Step

IF-ICC follows some basic protocol steps, shown here for indirect staining:

Figure 2. Fluorescent immunodetection

Optimizing some steps of an immunofluorescence protocol may be needed in order to obtain the best results based on factors that include subcellular location of the target and antigen characteristics.

Sample Preparation:

Regardless of cell growth format, successful IF-ICC imaging starts with harvesting healthy samples at the appropriate culture density.

Suspension cell:

Cell types may be washed to remove media and resuspended, then stained in small volume tubes before mounting onto slides for microscopic examination. Suspensions should first be examined microscopically for morphology and should not exceed the recommended confluency/titer for the cell line in order to be deemed suitable for immunocytochemistry. Healthy, subconfluent cultures will not be excessively turbid nor demonstrate yellowing indicating media acidification. Cells should appear bright and rounded by microscopic examination. Percent viability may be determined by hemocytometer and trypan blue exclusion. Following washing, cells should be resuspended at 1 -2 x 10 6 cells/mL, whether for ICC staining in solution, or smear application for subsequent staining on slides specially treated to enhanced adhesion of cells.

When adherent cells are cultured (and perhaps treated) to examine the effect on target expression by IF-ICC, cells may be seeded onto chamber slides, which are slides optimized for optical imaging with single- or multiple-chamber superstructures affixed that allow the addition of media for culture. Chamber slides permit transition directly from cell culture to probing and imaging. Adherent cell types may also be seeded onto microscope slide coverslips immersed in media-filled wells, which are subsequently stained and mounted onto slides for imaging.

Regardless of culture surface and vessel approach, cell growth on coverslips or in slide chambers should be carefully monitored, so that cells can be removed from culture and fixed for immunocytochemistry before they are overgrown. Cells are often imaged when confluency (the amount of surface area covered by cells) is in the 50-80% range. If cell viability must be definitively determined prior to immunostaining, some researchers have used dyes such as ethidium homodimer, a nuclear marker that cannot cross the intact cell membranes of live cells—so costaining with DAPI, which fluoresces in the nuclei of both viable and nonviable cells, can help to quantify percent viability as the number of ethidium-stained nuclei/number of dual-stained nuclei. Alternatively, commercial live/dead cell kits are available that often employ calcein and propidium iodide to distinguish live from dead cells, respectively.

Fixation:

Fixation and permeabilization may be achieved in a single step with organic fixatives like alcohol and acetone. Organic solvents should, however, not be used when lipid integrity must be maintained, as when the target antigen is associated with the membrane. Although formaldehyde (often sold and used in its polymeric form paraformaldehyde) is a common choice that halts decomposition and fixes proteins in place, crosslinking aldehydes can hinder antibody access to some antigens, and should therefore be used at the lowest concentration that effectively preserves structure after 10-20 minutes incubation, typically between 1-4% for (para)formaldehyde. NOTE: Because it is a source of autofluorescence, it’s best to avoid glutaraldehyde in IF fixation protocols.

Antigen Retrieval:

Some target epitopes may be masked by being complexed in a crowded cellular microenvironment, or by fixation techniques. Antigen retrieval techniques have been demonstrated to enhance access of antibodies to certain antigen targets, but should be used with extreme caution and pretested when working with cells on slides, as the heat or chemical conditions to which they expose cells are typically too harsh for ICC samples.

Permeabilization:

Triton-X or Tween-20 detergents are commonly used to effectively permeabilize fixed cells, but these detergents indiscriminately dissolve lipids and can be more destructive than saponin, a plant-based glycoside that perforates the membrane by selectively dissolving cholesterols, leaving organelle membranes essentially intact. If the target of interest is located within membrane bound structures inside the cell, more robust detergents such as Triton-X, Tween-20, the Brij detergents, or NP-40 are more likely to provide antibody access. Saponin is a gentler permeabilizer that preserves the integrity of surface antigens, but may not be suitable for permeabilizing intracellular membranes to provide antibody access to targets in the nucleus or other organelles. Because saponin’s effects may be reversible and therefore lost to washes, its use should be maintained throughout the staining protocol to ensure antibody access.

Blocking:

A solution of up to 5% normal serum from the species in which the secondary antibody is raised will effectively block its nonspecific binding. When serum is not available, an approach that adapts to any secondary is to use a more general protein blocker like bovine serum albumin (BSA) at a similar dilution in PBS. Some labs use a combination of serum and BSA to achieve both antibody host-specific and general blocking. BSA is often supplied lyophilized and reconstituted to liquid, so it should be filtered and/or regularly examined for particulates that may deposit on samples, creating staining artefact. Blocking may be maintained throughout the immunofluorescence protocol by diluting antibodies in the blocking buffer.


  1. IgG provides long term protection because it persists for months and years after the prescence of the antigen that has triggered their production.
  2. IgG protect against bacteris, viruses, neutralise bacterial toxins, trigger compliment protein systems and bind antigens to enhance the effectiveness of phagocytosis.
  3. Main function of IgA is to bind antigens on microbes before they invade tissues. It aggregates the antigens and keeps them in the secretions so when the secretion is expelled, so is the antigen.
  4. IgA are also first defense for mucosal surfaces such as the intestines, nose, and lungs.
  5. IgM is involved in the ABO blood group antigens on the surface of RBCs.
  6. IgM enhance ingestions of cells by phagocytosis.
  7. IgE bind to mast cells and basophils wich participate in the immune response.
  8. Some scientists think that IgE’s purpose is to stop parasites.
  9. IgD is present on the surface of B cells and plays a role in the induction of antibody production.

10 thoughts on &ldquoAntibody- Structure, Classes and Functions&rdquo

If IgG is low in its strength ” so to speak” does that mean the virus and or bacterial infection has surpassed it in the fight to protect the individual….

Does all the antibody classes have the same number of constant regions. If no, please explain to me why with vivid structure of the different antibody classes. It’s an assignment that I need to submit. Please


Video summary of secondary antibodies

Secondary antibodies are used for the indirect detection of a target to which a specific primary antibody is first bound. The secondary antibody must have specificity both for the antibody species as well as the isotype of the primary antibody being used. Also, a secondary antibody generally has a detectable tag or other label facilitating detection or purification.

Indirect detection of the target antigen using secondary antibodies requires more steps than direct detection using primary antibodies. However, the advantage of indirect detection is increased sensitivity due to the signal amplification from multiple secondary antibodies binding to a single primary antibody. In addition, a given secondary antibody can be used with any primary antibody of the same type and host species, making it an infinitely more versatile reagent than individually labeled primary antibodies. Secondary antibodies with specificity for the primary antibodies of common species are commercially available pre-conjugated with many of the common labels, including fluorescent and enzyme conjugate options.

Difference between direct and indirect protein detection in an assay involving detection with specific antibodies (e.g., an immunoassay). The surface on which the target protein is bound and immobilized is either a membrane (western blot) or a microplate well (ELISA).

Indirect detection of antigens using secondary antibodies offers other advantages besides diversity, flexibility, and versatility:

  • Allow for the use of the same primary antibody with different secondary antibodies depending on the application.
  • In some cases the same secondary antibody can be used across applications (i.e. fluorescent western blot and immunofluorescence) to validate target antigen detection.
  • Secondary antibodies also offer the ability to perform multiplexing or multi-labeling in experiments.

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Guidelines for detection of ANA

A positive ANA result in conjunction with clinical findings is diagnostic therefore frequently asked by the clinician in case of suspected CTD. Since different ANA are associated with one or other CTD a systematic approach has to be followed while performing these tests. Therefore initially screening is carried out usually by IF-ANA/ELISA and if positive more specific tests are performed based on clinical findings and IF-ANA staining patterns (table ​ (table3 3 ).

Autoantibody to dsDNA is specific and diagnostic for SLE and levels are elevated during active disease. Therefore in a case of suspected SLE if homogenous pattern is observed on IF-ANA further tests i.e. CLIF, ELISA, blotting tests etc. may be done to confirm dsDNA. Similarly anti-Sm is highly specific for SLE and needs confirmation by other tests i.e. Blotting etc. but is present in only 10% of SLE cases.

Anti-SSA/Ro antibody although more common in Sjogren's syndrome but can also be found in 30% cases of SLE with cutaneous involvement. Therefore if IF-ANA shows speckled/peripheral pattern further tests i.e. Blotting, MIA are required for detection of anti-SSA/Ro antibody. Clinical significance and detection methods for anti-SSB/La are similar to that for anti-SSA/Ro except that it is less common and may indicate minor course of disease. While presence of these two autoantibodies supports Sjogren's syndrome they are not much needed for diagnosis. Anti-Scl-70 autoantibody found in scleroderma (SS) gives a fine speckled staining pattern on IF-ANA and can be confirmed by immunodiffusion techniques but its detection is also not a necessity for diagnosis.

Antinucleolar antibodies are a group of autoantibodies which give nucleolar staining pattern. Most common of these are anti-PM-Scl, anti-RNA polymerase I-III and anti-U3-RNP (antifibrillarin). Although seen in scleroderma and polymyositis (PM) their detection is also not widely practiced [24].

A protocol generally followed by the clinicians and step by step approach to detect all these autoantibodies has been described in figure ​ figure2. 2 . A summary of certain other guidelines [24,61] to be considered are:

Algorithmic approach for ANA testing.

- ANA testing is not helpful in confirming a diagnosis of rheumatoid arthritis or osteoarthritis therefore should not be used in such conditions.

- ANA testing is not recommended to evaluate fatigue, back pain or other musculoskeletal pain unless accompanied by one or more of the clinical features in favor of a CTD.

- ANA testing should usually be ordered only once.

- Positive ANA tests do not need to be repeated.

- Negative tests need to be repeated only if there is a strong suspicion of an evolving CTD or a change in the patient's illness suggesting the diagnosis should be revised.

- A positive ANA test is important only in conjunction with clinical evaluation and in the absence of symptoms and signs of a CTD a positive ANA test only confounds the diagnosis. A positive ANA test can also be seen in healthy individuals, particularly the elderly or in a wide range of diseases other than CTD, where it has no diagnostic or prognostic value.

Recommendations in the guidelines may further evolve over time, as newer analytic methods and additional clinical research yield important results.


Are they identical? Is one more effective than the other?

2

It depends, not really a black and white answer.

For the most part, the antibodies that you form from getting vaccinated are the same kind of antibodies you would get from a natural infection. One difference is that certain types of vaccines only show the immune system part of the relevant virus. Because of that, the immune system doesn’t form as many different types of antibodies as it would in the course of a natural infection. For example the Pfizer covid 19 mRNA vaccine, only a certain part of the viral protein is used to trigger a strong immune response. So, someone who had naturally been infected with the virus might have some additional antibody types not found in someone who had been successfully vaccinated.

However not all antibodies produced by natural infection are effective. Genetic variability and age will also affect the quality of antibodies produced. Ideally, a specific vaccine is designed to trigger a strong response so in this case a vaccine might be more effective. Reverse can be true also from infection. We can't say for sure without long term data.

Edit: Wow this blew up overnight. Thank you guys for the awards!

Yes, thank you. I think it's also important to mention in this time of anti-vaxers that contracting many illnesses can leave you with long-term or chronic problems permanently. Many people use what you said to justify a "it's better to build immunity the NATURAL way" attitude. Both my in-laws (asthma and post-polio, among others) and my daughter (fibromyalgia, chronic fatigue and autoimmune) were left with chronic illnesses that have drastically impacted their quality and length of life as a result of diseases.

Thanks for the straightforward answer.

Thank you for a really clear answer!

I have heard that you can get the virus twice, but not if you get the vaccine. Wouldn't having better antibodies from being actually infected last longer/forever vs having vaccine antibodies? Kind of confused how that works.

There’s also “clonal selection” which is like rapid evolution for your antibodies. Antibodies produced by the body in response to an infection will proceed to make various flavors of the same antibody, some better some worse, and the body makes more of the successful variants. But I’d guess that the body will make variations of the same antibodies for the vaccine and a normal infection, so at some point, I’m not sure if there’s a point in comparing antibodies. Each body will have a slightly different functioning set of antibodies that are subsequently committed to long term storage.

In fact you can actually test whether someone received the vaccine or was infected with the virus. For instance, many antibody tests look for antibodies directed at the capsid (the virus shell) rather than the spike protein. So if you took this particular test youɽ be negative if you received a US approved vaccine but positive if you were infected by SARS-CoV 2.

2 5 2 & 51 More

Thank you very much for your reply! Iɽ never heard of the narcolepsy bit before. I'm gonna go down the Google rabbit hole, thanks again!

Reference for your central claim that Abs from a vaccine are more numerous?

Also, some viruses actively target the immune system. For example, measles can infect white blood cells and kill them, including cells that make antibodies. The resultant immunosuppression lasts a couple of years. The vaccine therefore protects against measles-induced immunosuppression.

This is wrong. Generally natural infections give a much stronger immune response and memory.

How long did it take to figure out the narcolepsy side effect and correct for it? Also, was this a permanent effect or only for a duration following the vaccine?

Is there for any auto immune response a mix of various antibodies? I always imagined that there was exactly one antibody per virus/bacteria strain.

a vaccination triggers a kind of antibodies (IgG) that are particularly effective at fighting the virus in the internal organs.

there is a different kind of antibody (IgA) that is present in the mucosa of the respiratory tract. a vaccination does not trigger a similarly effective response here

as a result, a vaccination might trigger an immune response that is good at preventing a severe multi-organ infection, but not good at preventing a lighter infection in the respiratory tract.

as a result, vaccinated people might still be able to contract a light infection that is harmless to themselves (a minor cold), but still very dangerous for those around them.

in case of covid, how strong that effect actually is, and how it develops over time remains to be seen. it's also possible that there are huge differences between the different vaccines that are currently being developed.

edit: this is obviously a massive simplification. don't quote me on it.

In my case, the 1st shot of the vaccine (taken December 5th) resulted in two weeks in a positive test for IgM and negative for IgG (this was a qualitative test, not quant, sadly). I have since taken the 2nd shot last Saturday, will wait a few days and take a proper quant test.

The vaccine manufacturer says that they have observed the proper levels of IgG on weeks 5-6.

What about IgM? Or are those just the memory ab’s that sounds the alarm?

Can you give us any sources?

Just to add to what everyone has stated already, infections and vaccinations differ majorly in the type and magnitude of inflammation they generate. This has many downstream effects, especially with antibody production.

During infection, there are three phases of antibody generation. Immediate early, which is predominantly IgM and non-specific early, which CAN be specific for the pathogen and late, which is specific. In a typical vaccination, only the "late" phase usually occurs.

Early and late antibodies can share some of the same specificities, but also have unique targets as well. They may differ in isotype (e.g. IgG1, IgG2, IgA, etc), and will also differ greatly in how they're accessorized. This will effect not their specificity, but how immune cells will recognize antibody:target complexes and respond, which may be pro or anti inflammatory in nature.

With a vaccine, you're likely not getting the benefits of the early antibody response. Is it a big deal? Depends on the pathogen and depends on the epitopes, i.e patterns, generated by the vaccine that select specific antibodies. But against a rapidly mutating, highly infectious virus, or any virulent pathogen for that matter, itɽ be handy to have access to all the tools in the tool chest.


Are they identical? Is one more effective than the other?

2

It depends, not really a black and white answer.

For the most part, the antibodies that you form from getting vaccinated are the same kind of antibodies you would get from a natural infection. One difference is that certain types of vaccines only show the immune system part of the relevant virus. Because of that, the immune system doesn’t form as many different types of antibodies as it would in the course of a natural infection. For example the Pfizer covid 19 mRNA vaccine, only a certain part of the viral protein is used to trigger a strong immune response. So, someone who had naturally been infected with the virus might have some additional antibody types not found in someone who had been successfully vaccinated.

However not all antibodies produced by natural infection are effective. Genetic variability and age will also affect the quality of antibodies produced. Ideally, a specific vaccine is designed to trigger a strong response so in this case a vaccine might be more effective. Reverse can be true also from infection. We can't say for sure without long term data.

Edit: Wow this blew up overnight. Thank you guys for the awards!

Yes, thank you. I think it's also important to mention in this time of anti-vaxers that contracting many illnesses can leave you with long-term or chronic problems permanently. Many people use what you said to justify a "it's better to build immunity the NATURAL way" attitude. Both my in-laws (asthma and post-polio, among others) and my daughter (fibromyalgia, chronic fatigue and autoimmune) were left with chronic illnesses that have drastically impacted their quality and length of life as a result of diseases.

Thanks for the straightforward answer.

Thank you for a really clear answer!

I have heard that you can get the virus twice, but not if you get the vaccine. Wouldn't having better antibodies from being actually infected last longer/forever vs having vaccine antibodies? Kind of confused how that works.

There’s also “clonal selection” which is like rapid evolution for your antibodies. Antibodies produced by the body in response to an infection will proceed to make various flavors of the same antibody, some better some worse, and the body makes more of the successful variants. But I’d guess that the body will make variations of the same antibodies for the vaccine and a normal infection, so at some point, I’m not sure if there’s a point in comparing antibodies. Each body will have a slightly different functioning set of antibodies that are subsequently committed to long term storage.

In fact you can actually test whether someone received the vaccine or was infected with the virus. For instance, many antibody tests look for antibodies directed at the capsid (the virus shell) rather than the spike protein. So if you took this particular test youɽ be negative if you received a US approved vaccine but positive if you were infected by SARS-CoV 2.

2 5 2 & 51 More

Thank you very much for your reply! Iɽ never heard of the narcolepsy bit before. I'm gonna go down the Google rabbit hole, thanks again!

Reference for your central claim that Abs from a vaccine are more numerous?

Also, some viruses actively target the immune system. For example, measles can infect white blood cells and kill them, including cells that make antibodies. The resultant immunosuppression lasts a couple of years. The vaccine therefore protects against measles-induced immunosuppression.

This is wrong. Generally natural infections give a much stronger immune response and memory.

How long did it take to figure out the narcolepsy side effect and correct for it? Also, was this a permanent effect or only for a duration following the vaccine?

Is there for any auto immune response a mix of various antibodies? I always imagined that there was exactly one antibody per virus/bacteria strain.

a vaccination triggers a kind of antibodies (IgG) that are particularly effective at fighting the virus in the internal organs.

there is a different kind of antibody (IgA) that is present in the mucosa of the respiratory tract. a vaccination does not trigger a similarly effective response here

as a result, a vaccination might trigger an immune response that is good at preventing a severe multi-organ infection, but not good at preventing a lighter infection in the respiratory tract.

as a result, vaccinated people might still be able to contract a light infection that is harmless to themselves (a minor cold), but still very dangerous for those around them.

in case of covid, how strong that effect actually is, and how it develops over time remains to be seen. it's also possible that there are huge differences between the different vaccines that are currently being developed.

edit: this is obviously a massive simplification. don't quote me on it.

In my case, the 1st shot of the vaccine (taken December 5th) resulted in two weeks in a positive test for IgM and negative for IgG (this was a qualitative test, not quant, sadly). I have since taken the 2nd shot last Saturday, will wait a few days and take a proper quant test.

The vaccine manufacturer says that they have observed the proper levels of IgG on weeks 5-6.

What about IgM? Or are those just the memory ab’s that sounds the alarm?

Can you give us any sources?

Just to add to what everyone has stated already, infections and vaccinations differ majorly in the type and magnitude of inflammation they generate. This has many downstream effects, especially with antibody production.

During infection, there are three phases of antibody generation. Immediate early, which is predominantly IgM and non-specific early, which CAN be specific for the pathogen and late, which is specific. In a typical vaccination, only the "late" phase usually occurs.

Early and late antibodies can share some of the same specificities, but also have unique targets as well. They may differ in isotype (e.g. IgG1, IgG2, IgA, etc), and will also differ greatly in how they're accessorized. This will effect not their specificity, but how immune cells will recognize antibody:target complexes and respond, which may be pro or anti inflammatory in nature.

With a vaccine, you're likely not getting the benefits of the early antibody response. Is it a big deal? Depends on the pathogen and depends on the epitopes, i.e patterns, generated by the vaccine that select specific antibodies. But against a rapidly mutating, highly infectious virus, or any virulent pathogen for that matter, itɽ be handy to have access to all the tools in the tool chest.


Negative (antibody test) results mean that either (1) you have not been exposed to the coronavirus, or (2) you were exposed to the virus, but at the time of your test it was too soon for your body to produce antibodies or the level of antibodies present at the time of the test were below the test&rsquos limit of detection. It takes one to three weeks after exposure to the coronavirus to develop antibodies. It is also possible that many weeks or months following an infection, the antibody test may be negative as well. For some infections, antibodies decrease over time, but if the individual is exposed again to the same infectious virus, the body gears up and rapidly produces the needed antibody defenses. We are not yet sure whether SARS-CoV-2 antibodies protect the person against reinfection with the virus in the future.

Positive (antibody test) results mean that you have likely encountered the coronavirus at some point. It is still essential to protect yourself by taking precautions such as physical distancing, hand washing and mask wearing until more is known about immunity to this virus, and how long people infected with it remain contagious to others.


The basic biology of HER2

Human epidermal growth factor receptors (HER/erbB) constitute a family of four cell surface receptors involved in transmission of signals controlling normal cell growth and differentiation. A range of growth factors serve as ligands, but none is specific for the HER2 receptor. HER receptors exist as both monomers and dimers, either homo- or heterodimers. Ligand binding to HERI, HER3 or HER4 induces rapid receptor dimerization, with a marked preference for HER2 as a dimer partner. Moreover, HER2-containing heterodimers generate intracellular signals that are significantly stronger than signals emanating from other HER combinations. In normal cells, few HER2 molecules exist at the cell surface, so few heterodimers are formed and growth signals are relatively weak and controllable. When HER2 is overexpressed multiple HER2 heterodimers are formed and cell signaling is stronger, resulting in enhanced responsiveness to growth factors and malignant growth. This explains why HER2 overexpression is an indicator of poor prognosis in breast tumors and may be predictive of response to treatment. HER2 is a highly specific and promising target for new breast cancer treatments. The recombinant human anti-HER2 monoclonal antibody (rhuMAb-HER2, trastuzumab, Herceptin) induces rapid removal of HER2 from the cell surface, thereby reducing its availability to heterodimers and reducing oncogenicity.



Comments:

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