Information

20.1: Practical Applications of Monoclonal and Polyclonal Antibodies - Biology


Learning Objectives

  • Compare the method of development, use, and characteristics of monoclonal and polyclonal antibodies
  • Explain the nature of antibody cross-reactivity and why this is less of a problem with monoclonal antibodies

clinical focus - part 1

In an unfortunate incident, a healthcare worker struggling with addiction was caught stealing syringes of painkillers and replacing them with syringes filled with unknown substances. The hospital immediately fired the employee and had him arrested; however, two patients that he had worked with later tested positive for HIV.

While there was no proof that the infections originated from the tainted syringes, the hospital’s public health physician took immediate steps to determine whether any other patients had been put at risk. Although the worker had only been employed for a short time, it was determined that he had come into contact with more than 1300 patients. The hospital decided to contact all of these patients and have them tested for HIV.

Exercise (PageIndex{1})

  1. Why does the hospital feel it is necessary to test every patient for HIV?
  2. What types of tests can be used to determine if a patient has HIV?

In addition to being crucial for our normal immune response, antibodies provide powerful tools for research and diagnostic purposes. The high specificity of antibodies makes them an excellent tool for detecting and quantifying a broad array of targets, from drugs to serum proteins to microorganisms. With in vitro assays, antibodies can be used to precipitate soluble antigens, agglutinate (clump) cells, opsonize and kill bacteria with the assistance of complement, and neutralize drugs, toxins, and viruses.

An antibody’s specificity results from the antigen-binding site formed within the variable regions—regions of the antibody that have unique patterns of amino acids that can only bind to target antigens with a molecular sequence that provides complementary charges and noncovalent bonds. There are limitations to antibody specificity, however. Some antigens are so chemically similar that cross-reactivity occurs; in other words, antibodies raised against one antigen bind to a chemically similar but different antigen. Consider an antigen that consists of a single protein with multiple epitopes (Figure (PageIndex{1})). This single protein may stimulate the production of many different antibodies, some of which may bind to chemically identical epitopes on other proteins.

Cross-reactivity is more likely to occur between antibodies and antigens that have low affinity or avidity. Affinity, which can be determined experimentally, is a measure of the binding strength between an antibody's binding site and an epitope, whereas avidity is the total strength of all the interactions in an antibody-antigen complex (which may have more than one bonding site). Avidity is influenced by affinity as well as the structural arrangements of the epitope and the variable regions of the antibody. If an antibody has a high affinity/avidity for a specific antigen, it is less likely to cross-react with an antigen for which it has a lower affinity/avidity.

Exercise (PageIndex{2})

  1. What property makes antibodies useful for research and clinical diagnosis?
  2. What is cross-reactivity and why does it occur?

Producing Polyclonal Antibodies

Antibodies used for research and diagnostic purposes are often obtained by injecting a lab animal such as a rabbit or a goat with a specific antigen. Within a few weeks, the animal’s immune system will produce high levels of antibodies specific for the antigen. These antibodies can be harvested in an antiserum, which is whole serum collected from an animal following exposure to an antigen. Because most antigens are complex structures with multiple epitopes, they result in the production of multiple antibodies in the lab animal. This so-called polyclonal antibody response is also typical of the response to infection by the human immune system. Antiserum drawn from an animal will thus contain antibodies from multiple clones of B cells, with each B cell responding to a specific epitope on the antigen (Figure (PageIndex{2})).

Lab animals are usually injected at least twice with antigen when being used to produce antiserum. The second injection will activate memory cells that make class IgG antibodies against the antigen. The memory cells also undergo affinity maturation, resulting in a pool of antibodies with higher average affinity. Affinity maturation occurs because of mutations in the immunoglobulin gene variable regions, resulting in B cells with slightly altered antigen-binding sites. On re-exposure to the antigen, those B cells capable of producing antibody with higher affinity antigen-binding sites will be stimulated to proliferate and produce more antibody than their lower-affinity peers. An adjuvant, which is a chemical that provokes a generalized activation of the immune system that stimulates greater antibody production, is often mixed with the antigen prior to injection.

Antiserum obtained from animals will not only contain antibodies against the antigen artificially introduced in the laboratory, but it will also contain antibodies to any other antigens to which the animal has been exposed during its lifetime. For this reason, antisera must first be “purified” to remove other antibodies before using the antibodies for research or diagnostic assays.

Clinical Uses of Polyclonal Antisera

Polyclonal antisera are used in many clinical tests that are designed to determine whether a patient is producing antibodies in response to a particular pathogen. While these tests are certainly powerful diagnostic tools, they have their limitations, because they are an indirect means of determining whether a particular pathogen is present. Tests based on a polyclonal response can sometimes lead to a false-positive result—in other words, a test that confirms the presence of an antigen that is, in fact, not present. Antibody-based tests can also result in a false-negative result, which occurs when the test fails to detect an antibody that is, in fact, present.

The accuracy of antibody tests can be described in terms of test sensitivity and test specificity. Test sensitivity is the probability of getting a positive test result when the patient is indeed infected. If a test has high sensitivity, the probability of a false negative is low. Test specificity, on the other hand, is the probability of getting a negative test result when the patient is not infected. If a test has high specificity, the probability of a false positive is low.

False positives often occur due to cross-reactivity, which can occur when epitopes from a different pathogen are similar to those found on the pathogen being tested for. For this reason, antibody-based tests are often used only as screening tests; if the results are positive, other confirmatory tests are used to make sure that the results were not a false positive.

For example, a blood sample from a patient suspected of having hepatitis C can be screened for the virus using antibodies that bind to antigens on hepatitis C virus. If the patient is indeed infected with hepatitis C virus, the antibodies will bind to the antigens, yielding a positive test result. If the patient is not infected with hepatitic C virus, the antibodies will generally not bind to anything and the test should be negative; however, a false positive may occur if the patient has been previously infected by any of a variety of pathogens that elicit antibodies that cross-react with the hepatitis C virus antigens. Antibody tests for hepatitis C have high sensitivity (a low probability of a false negative) but low specificity (a high probability of a false positive). Thus, patients who test positive must have a second, confirmatory test to rule out the possibility of a false positive. The confirmatory test is a more expensive and time-consuming test that directly tests for the presence of hepatitis C viral RNA in the blood. Only after the confirmatory test comes back positive can the patient be definitively diagnosed with a hepatitis C infection. Antibody-based tests can result in a false negative if, for any reason, the patient’s immune system has not produced detectable levels of antibodies. For some diseases, it may take several weeks following infection before the immune system produces enough antibodies to cross the detection threshold of the assay. In immunocompromised patients, the immune system may not be capable of producing a detectable level of antibodies.

Another limitation of using antibody production as an indicator of disease is that antibodies in the blood will persist long after the infection has been cleared. Depending on the type of infection, antibodies will be present for many months; sometimes, they may be present for the remainder of the patient’s life. Thus, a positive antibody-based test only means that the patient was infected at some point in time; it does not prove that the infection is active.

In addition to their role in diagnosis, polyclonal antisera can activate complement, detect the presence of bacteria in clinical and food industry settings, and perform a wide array of precipitation reactions that can detect and quantify serum proteins, viruses, or other antigens. However, with the many specificities of antibody present in a polyclonal antiserum, there is a significant likelihood that the antiserum will cross-react with antigens to which the individual was never exposed. Therefore, we must always account for the possibility of false-positive results when working with a polyclonal antiserum.

Exercise (PageIndex{3})

  1. What is a false positive and what are some reasons that false positives occur?
  2. What is a false negative and what are some reasons that false positives occur?
  3. If a patient tests negative on a highly sensitive test, what is the likelihood that the person is infected with the pathogen?

Producing Monoclonal Antibodies

Some types of assays require better antibody specificity and affinity than can be obtained using a polyclonal antiserum. To attain this high specificity, all of the antibodies must bind with high affinity to a single epitope. This high specificity can be provided by monoclonal antibodies (mAbs). Table (PageIndex{1}) compares some of the important characteristics of monoclonal and polyclonal antibodies.

Unlike polyclonal antibodies, which are produced in live animals, monoclonal antibodies are produced in vitro using tissue-culture techniques. mAbs are produced by immunizing an animal, often a mouse, multiple times with a specific antigen. B cells from the spleen of the immunized animal are then removed. Since normal B cells are unable to proliferate forever, they are fused with immortal, cancerous B cells called myeloma cells, to yield hybridoma cells. All of the cells are then placed in a selective medium that allows only the hybridomas to grow; unfused myeloma cells cannot grow, and any unfused B cells die off. The hybridomas, which are capable of growing continuously in culture while producing antibodies, are then screened for the desired mAb. Those producing the desired mAb are grown in tissue culture; the culture medium is harvested periodically and mAbs are purified from the medium. This is a very expensive and time-consuming process. It may take weeks of culturing and many liters of media to provide enough mAbs for an experiment or to treat a single patient. mAbs are expensive (Figure (PageIndex{3})).

Table (PageIndex{1}): Characteristics of Polyclonal and Monoclonal Antibodies
Monoclonal AntibodiesPolyclonal Antibodies
Expensive productionInexpensive production
Long production timeRapid production
Large quantities of specific antibodiesLarge quantities of nonspecific antibodies
Recognize a single epitope on an antigenRecognize multiple epitopes on an antigen
Production is continuous and uniform once the hybridoma is madeDifferent batches vary in composition

Clinical Uses of Monoclonal Antibodies

Since the most common methods for producing monoclonal antibodies use mouse cells, it is necessary to create humanized monoclonal antibodies for human clinical use. Mouse antibodies cannot be injected repeatedly into humans, because the immune system will recognize them as being foreign and will respond to them with neutralizing antibodies. This problem can be minimized by genetically engineering the antibody in the mouse B cell. The variable regions of the mouse light and heavy chain genes are ligated to human constant regions, and the chimeric gene is then transferred into a host cell. This allows production of a mAb that is mostly “human” with only the antigen-binding site being of mouse origin.

Humanized mAbs have been successfully used to treat cancer with minimal side effects. For example, the humanized monoclonal antibody drug Herceptin has been helpful for the treatment of some types of breast cancer. There have also been a few preliminary trials of humanized mAb for the treatment of infectious diseases, but none of these treatments are currently in use. In some cases, mAbs have proven too specific to treat infectious diseases, because they recognize some serovars of a pathogen but not others. Using a cocktail of multiple mAbs that target different strains of the pathogen can address this problem. However, the great cost associated with mAb production is another challenge that has prevented mAbs from becoming practical for use in treating microbial infections.1

One promising technology for inexpensive mAbs is the use of genetically engineered plants to produce antibodies (or plantibodies). This technology transforms plant cells into antibody factories rather than relying on tissue culture cells, which are expensive and technically demanding. In some cases, it may even be possible to deliver these antibodies by having patients eat the plants rather than by extracting and injecting the antibodies. For example, in 2013, a research group cloned antibody genes into plants that had the ability to neutralize an important toxin from bacteria that can cause severe gastrointestinal disease.2 Eating the plants could potentially deliver the antibodies directly to the toxin.

Exercise (PageIndex{4})

  1. How are humanized monoclonal antibodies produced?
  2. What does the “monoclonal” of monoclonal antibodies mean?

USING MONOCLONAL ANTIBODIES TO COMBAT EBOLA

During the 2014–2015 Ebola outbreak in West Africa, a few Ebola-infected patients were treated with ZMapp, a drug that had been shown to be effective in trials done in rhesus macaques only a few months before.3 ZMapp is a combination of three mAbs produced by incorporating the antibody genes into tobacco plants using a viral vector. By using three mAbs, the drug is effective across multiple strains of the virus. Unfortunately, there was only enough ZMapp to treat a tiny number of patients.

While the current technology is not adequate for producing large quantities of ZMapp, it does show that plantibodies—plant-produced mAbs—are feasible for clinical use, potentially cost effective, and worth further development. The last several years have seen an explosion in the number of new mAb-based drugs for the treatment of cancer and infectious diseases; however, the widespread use of such drugs is currently inhibited by their exorbitant cost, especially in underdeveloped parts of the world, where a single dose might cost more than the patient’s lifetime income. Developing methods for cloning antibody genes into plants could reduce costs dramatically.

Key Concepts and Summary

  • Antibodies bind with high specificity to antigens used to challenge the immune system, but they may also show cross-reactivity by binding to other antigens that share chemical properties with the original antigen.
  • Injection of an antigen into an animal will result in a polyclonal antibody response in which different antibodies are produced that react with the various epitopes on the antigen.
  • Polyclonal antisera are useful for some types of laboratory assays, but other assays require more specificity. Diagnostic tests that use polyclonal antisera are typically only used for screening because of the possibility of false-positive and false-negative results.
  • Monoclonal antibodies provide higher specificity than polyclonal antisera because they bind to a single epitope and usually have high affinity.
  • Monoclonal antibodies are typically produced by culturing antibody-secreting hybridomas derived from mice. mAbs are currently used to treat cancer, but their exorbitant cost has prevented them from being used more widely to treat infectious diseases. Still, their potential for laboratory and clinical use is driving the development of new, cost-effective solutions such as plantibodies.

Multiple Choice

For many uses in the laboratory, polyclonal antibodies work well, but for some types of assays, they lack sufficient ________ because they cross-react with inappropriate antigens.

A. specificity
B. sensitivity
C. accuracy
D. reactivity

A

How are monoclonal antibodies produced?

A. Antibody-producing B cells from a mouse are fused with myeloma cells and then the cells are grown in tissue culture.
B. A mouse is injected with an antigen and then antibodies are harvested from its serum.
C. They are produced by the human immune system as a natural response to an infection.
D. They are produced by a mouse’s immune system as a natural response to an infection.

A

Fill in the Blank

When we inject an animal with the same antigen a second time a few weeks after the first, ________ takes place, which means the antibodies produced after the second injection will on average bind the antigen more tightly.

affinity maturation

When using mAbs to treat disease in humans, the mAbs must first be ________ by replacing the mouse constant region DNA with human constant region DNA.

humanized

If we used normal mouse mAbs to treat human disease, multiple doses would cause the patient to respond with ________ against the mouse antibodies.

neutralizing antibodies

A polyclonal response to an infection occurs because most antigens have multiple ________,

epitopes

Short Answer

Describe two reasons why polyclonal antibodies are more likely to exhibit cross-reactivity than monoclonal antibodies.

Critical Thinking

Suppose you were screening produce in a grocery store for the presence of E. coli contamination. Would it be better to use a polyclonal anti-E. coli antiserum or a mAb against an E. coli membrane protein? Explain.

Footnotes

  1. 1 Saylor, Carolyn, Ekaterina Dadachova and Arturo Casadevall, “Monoclonal Antibody-Based Therapies for Microbial Diseases,” Vaccine 27 (2009): G38-G46.
  2. 2 Nakanishi, Katsuhiro et al., “Production of Hybrid-IgG/IgA Plantibodies with Neutralizing Activity against Shiga Toxin 1,” PloS One 8, no. 11 (2013): e80712.
  3. 3 Qiu, Xiangguo et al., “Reversion of Advanced Ebola Virus Disease in Nonhuman Primates with ZMapp,” Nature 514 (2014): 47–53.

Critical Steps in the Production of Polyclonal and Monoclonal Antibodies: Evaluation and Recommendations

Marlies Leenaars, Ph.D., Animal Welfare Officer, and Coenraad F. M. Hendriksen, D.V.M., Ph.D., Animal Welfare Officer and Senior Scientist, are with the Netherlands Vaccine Institute, Bilthoven, The Netherlands.

Marlies Leenaars, Ph.D., Animal Welfare Officer, and Coenraad F. M. Hendriksen, D.V.M., Ph.D., Animal Welfare Officer and Senior Scientist, are with the Netherlands Vaccine Institute, Bilthoven, The Netherlands.

Marlies Leenaars, Coenraad F. M. Hendriksen, Critical Steps in the Production of Polyclonal and Monoclonal Antibodies: Evaluation and Recommendations, ILAR Journal, Volume 46, Issue 3, 2005, Pages 269–279, https://doi.org/10.1093/ilar.46.3.269


Abstract

Antibodies are host proteins that comprise one of the principal effectors of the adaptive immune system. Their utility has been harnessed as they have been and continue to be used extensively as a diagnostic and research reagent. They are also becoming an important therapeutic tool in the clinician's armamentarium to treat disease. Antibodies are utilized for analysis, purification, and enrichment, and to mediate or modulate physiological responses. This overview of the structure and function of polyclonal and monoclonal antibodies describes features that distinguish one from the other. A limited review of their use as specific research, diagnostic, and therapeutic reagents and a list of printed and electronic resources that can be utilized to garner additional information on these topics are also included.


Monoclonal Antibodies

An antibody is a protein produced by the body's immune system in response to antigens, which are harmful substances. Antigens include bacteria, fungi, parasites, viruses, chemicals, and other substances the immune system identifies as foreign. Sometimes the body mistakenly identifies normal tissues as foreign and produces antibodies against the tissue. This is the underlying cause of autoimmune conditions such as rheumatoid arthritis and multiple sclerosis or MS.

Antibodies are naturally produced by the immune system. However, scientists can produce antibodies in the lab that mimic the action of the immune system. These man-made (synthetic) antibodies act against proteins that attack normal tissues in people with autoimmune disorders. Man-made antibodies are produced by introducing human genes that produce antibodies into mice or another suitable mammal. The mice then are vaccinated with the antigen that scientists want to produce antibodies against. This causes the immune cells of the mice to produce the desired human antibody. The term monoclonal antibody means that the man-made antibody is synthesized from cloned immune cells, and the identical monoclonal antibody produced binds to one type of antigen. Polyclonal antibodies are synthesized from different immune cells and the antibodies produced bind to multiple antigens.

As of October 2020, drug companies Regeneron and Eli Lilly were conducting clinical trials on two monoclonal antibody therapy cocktails for bridge treatment of the coronavirus disease COVID-19. Early results are promising, but there is far from enough data to show whether monoclonal antibody therapy is broadly useful against the SARS-CoV-2 virus that causes COVID-19.

List and types of monoclonal antibodies (FDA approved)

Here is a list of examples some FDA-approved monoclonal antibody drugs.

  • abciximab (Reopro)
  • adalimumab (Humira, Amjevita)
  • alefacept (Amevive)
  • alemtuzumab (Campath)
  • basiliximab (Simulect)
  • belimumab (Benlysta)
  • bezlotoxumab (Zinplava)
  • canakinumab (Ilaris)
  • certolizumab pegol (Cimzia)
  • cetuximab (Erbitux) (Zenapax, Zinbryta)
  • denosumab (Prolia, Xgeva)
  • efalizumab (Raptiva)
  • golimumab (Simponi, Simponi Aria) (Remicade)
  • ipilimumab (Yervoy)
  • ixekizumab (Taltz)
  • natalizumab (Tysabri)
  • nivolumab (Opdivo)
  • olaratumab (Lartruvo)
  • omalizumab (Xolair)
  • palivizumab (Synagis)
  • panitumumab (Vectibix)
  • pembrolizumab (Keytruda)
  • rituximab (Rituxan)
  • tocilizumab (Actemra)
  • trastuzumab (Herceptin)
  • secukinumab (Cosentyx)
  • ustekinumab (Stelara)

Each monoclonal antibody listed above has a role in treating a targeted disease (for example, basiliximab treats transplant rejection while belimumab treats systemic lupus erythematosus).

SLIDESHOW

What are the uses for monoclonal antibodies?

The use of monoclonal antibodies to treat diseases is called immunotherapy therapy because each type of monoclonal antibody will target a specific targeted antigen in the body.

Uses for monoclonal antibodies include:

In these conditions the monoclonal antibody targets and interferes with the action of a chemical or receptor that is involved in the development of the condition that is being treated. For example, a monoclonal antibody used for treating cancer may block a receptor that cancer cells use for preventing the immune system from the destroying the cancer cell. Blocking this receptor allows the immune system to recognize cancer cells and destroy them.

The monoclonal antibodies for the COVID-19 pandemic coronavirus may soon reach the market late in 2020 under emergency use authorization from the FDA, according to the magazine Science, but the manufacturers were still in talks with the agency about this matter as of this update.


Abstract

For several decades, polyclonal and monoclonal antibodies, and more recently recombinant antibodies and fragments thereof have been used for pesticide residue analysis and biomedical applications. Antibodies are being used now to treat cancer, and have been produced in plants for protection against viral and fungal pathogens, as well as for pesticide resistance. This article comprises a review of anti-fungal monoclonal, polyclonal and recombinant antibodies used in plant pathology over the last two decades. The antigens used for animal immunizations, the specificity of the antibodies thereby obtained, and a few examples of practical applications of these antibodies such as immunodiagnostics, immunofluorescence, fungal growth inhibition, aerobiology and glycobiology studies are reviewed.


What makes an antibody monoclonal, polyclonal, or recombinant?

When you’re shopping for antibodies, there are so many factors to consider. For example, will it work in my cell or tissue model? Has it been tested in the application I want to use? Sometimes it’s a struggle to find what you need because your options are limited, but in other instances there may be several reagents that seem like they could work in your experiment.

What if you’re looking at a monoclonal, polyclonal, or recombinant antibody against the same target? What’s the difference? Is one better than the other or are they all designed for different purposes? It’s a complicated question, but one definitely worth asking.

A polyclonal antibody is a heterogeneous mixture of antibodies derived from distinct B- lymphocyte populations that detect different epitopes within the same immunogen. Polyclonal antibodies are typically produced in rabbits but can also be made in ungulates (sheep, goat, horse, etc.), rodents, and chickens, depending on the researchers’ need. Production of polyclonals typically involves collection of blood from the immunized animal, isolation of the immunoglobulin fraction, and affinity purification to remove non-specific antibody populations. Rabbits offer significant advantages over other species due to their larger antibody diversity and ability to generate immune responses to a wide range of antigens including small molecules and peptides with or without post-translational modifications.

A monoclonal antibody is a homogeneous antibody derived from a single B-cell clone which detects a single epitope within the immunogen. All monoclonal antibodies begin as a pool of polyclonal antibodies but are isolated through a selection or cloning process to identify and expand the desired monovalent clone. Monoclonal antibodies are typically produced from rodent hosts, rabbits and camelids, and are produced via a variety of methods depending on the species and type of antibody desired (as detailed below). Traditionally, monoclonal antibodies are generated via stable clones of immortalized B-cells either by injecting and collecting ascites from a mouse or culturing the antibody expressing B-cells and collecting the supernatant. More recently, newer techniques have enabled recombinant cloning of the immunoglobulin heavy and light chain genes from immunoreactive B-cells for expression and production of recombinant monoclonal antibodies in mammalian cell lines.

As mentioned above, there are many ways to produce an antibody. Generally polyclonal antibodies are purified from the serum of the immunized animal. Monoclonal antibodies can be manufactured using a number of different strategies. Most monoclonal antibodies are produced by culturing the antibody-producing B-cell hybridoma and collecting the supernatant off the cells. Antibodies generated this way can be used with or without additional purification depending on the titer and affinity of the antibody. Alternate methods include injection of the B-cell hybridoma in to the peritoneal cavity of a suitable host and collecting the ascites fluid after a period of time. This method has the advantage of producing large quantities of antibody rapidly, but has the disadvantage of requiring an animal host and may require an additional purification step to remove lipids and other debris. Alternatively, the heavy and light immunoglobulin chains of a monoclonal antibody can be cloned in to an expression vector, transfected in to a mammalian cell line suited for antibody production and expressed ectopically for collection and purification. Recombinant production of antibodies is a growing trend among antibody manufacturers due to its inherent consistency and the ability to genetically engineer the antibody.

Regardless of the clonality or production method, all antibodies must be properly validated in the intended application prior to use in an experiment. Even the best antibody, regardless of clonality, host species or isotype, can yield spurious or misleading results when used incorrectly.

  1. Collection and purification from ascites or cultured cell supernatant
  2. Recombinant expression of heavy and light chains in mammalian cell lines

Rabbit mAbs: High
Mouse mAbs: Moderate

Related Resources and Additional Information:

Weber et al. (2017) From rabbit antibody repertoires to rabbit monoclonal antibodies. Experimental and Molecular Medicine. Vol. 49: e305

Rajewski, Klaus (1996) Clonal selection and learning in the antibody system. Nature. Vol. 381: 751-758.

Cheung et al. (2012) A proteomics approach for the identification and cloning of monoclonal antibodies from serum. Nature Biotechnology. Vol. 10(5): 447-454

Ascoli and Aggeler (2018) Overlooked benefits of using polyclonal antibodies. BioTechniques 65(3): 127-136


Dengue Virus

Dengue (breakbone fever) is a mosquito-borne infection caused by Dengue virus (DENV), a Flavivirus (Others see West Nile Virus (WNV), Japanese Encephalitis Virus (JEV), Yellow Fever Virus (YFV) and Zika Virus (ZIKV).) that is characterized by fever, severe headache, muscle and joint pain, nausea and vomiting, eye pain, and rash. Severe forms of the disease, dengue hemorrhagic fever and dengue shock syndrome, principally affect children.

Dengue virus is transmitted by female mosquitoes mainly of the species Aedes aegypti and, to a lesser extent, Ae. albopictus. This mosquito also transmits chikungunya, yellow fever and Zika infection. Dengue is widespread throughout the tropics, with local variations in risk influenced by rainfall, temperature and unplanned rapid urbanization.

The first record of a case of probable dengue fever is in a Chinese medical encyclopedia from the Jin Dynasty (265–420 AD) which referred to a “water poison” associated with flying insects. The first recognized Dengue epidemics occurred almost simultaneously in Asia, Africa, and North America in the 1780s, shortly after the identification and naming of the disease in 1779. The first confirmed case report dates from 1789 and is by Benjamin Rush, who coined the term "breakbone fever" because of the symptoms of myalgia and arthralgia. The viral etiology and the transmission by mosquitoes were only deciphered in the 20th century. The socioeconomic impact of World War II resulted in increased spread globally. Nowadays, about 2.5 billion people, or 40% of the world’s population, live in areas where there is a risk of dengue transmission. Dengue spread to more than 100 countries in Asia, the Pacific, the Americas, Africa, and the Caribbean.

Browse All Dengue Virus Related Products

2. Signs and Symptoms

After being bitten by a mosquito carrying the dengue virus, the incubation period ranges from 3 to 14 (usually 4 to 7) days before the signs and symptoms of dengue appear. Dengue occurs in two forms:

Dengue Fever:
Dengue fever is a severe, flu-like illness that affects infants, young children and adults, but seldom causes death. Dengue should be suspected when a high fever (40°C/104°F) is accompanied by 2 of the following symptoms: severe headache, pain behind the eyes, muscle and joint pains, nausea, vomiting, swollen glands or rash. Symptoms usually last for 2–7 days, after an incubation period of 4–10 days after the bite from an infected mosquito.

Recognition of Dengue fever:
? Sudden onset of high fever
? Severe headache (mostly in the forehead)
? Pain behind the eyes which worsens with eye movement
? Body aches and joint pains
? Nausea or vomiting

Dengue Haemorrhagic Fever:
Dengue hemorrhagic fever (DHF) is a potentially deadly complication due to plasma leaking, fluid accumulation, respiratory distress, severe bleeding, or organ impairment. Warning signs occur 3–7 days after the first symptoms in conjunction with a decrease in temperature (below 38°C/100°F) and include: severe abdominal pain, persistent vomiting, and rapid breathing, bleeding gums, fatigue, restlessness and blood in vomit. The next 24–48 hours of the critical stage can be lethal proper medical care is needed to avoid complications and risk of death.

Recognition of Dengue Haemorrhagic Fever (DHF):
? Symptoms similar to dengue fever plus, any one of the following:
? Severe and continuous pain in abdomen
? Bleeding from the nose, mouth and gums or skin bruising
? Frequent vomiting with or without blood
? Black stools, like coal tar
? Excessive thirst (dry mouth)
? Pale, cold skin
? Restlessness, or sleepiness

3. About Dengue Virus

Dengue virus (DENV), a member of the family Flaviviridae, is a major human pathogen transmitted by mosquitoes. There are four antigenically different serotypes of the virus (although there is report of 2013 that a fifth serotype has been found): These four subtypes are different strains of dengue virus that have 60-80% homology between each other. The major difference for humans lies in subtle differences in the surface proteins of the different dengue subtypes. Infection induces long-life protection against the infecting serotype, but it gives only a short time cross protective immunity against the other types. The first infection cause mostly minor disease, but secondary infections has been reported to cause severe diseases (DHF or DSS) in both children and adults. This phenomenon is called Antibody-Dependent Enhancement.

Each of the four serotypes of DENV (DENV-1, DENV-2, DENV-3 and DENV-4) is capable of causing the full spectrum of clinical manifestations following DENV infection, ranging from an asymptomatic infection to dengue fever (DF) and the most severe disease, dengue haemorrhagic fever (DHF) and dengue shock syndrome (DSS).

DENV is a 50-nm virus enveloped with a lipid membrane (Figure 1a). There are 180 identical copies of the envelope (E) protein attached to the surface of the viral membrane by a short transmembrane segment. The virus has a genome of about 11000 bases that encodes a single large polyprotein that is subsequently cleaved into several structural and non-structural mature peptides. The 11-kb positive sense RNA genome encodes 3 structural proteins (capsid, prM, and E) and 7 nonstructural proteins (NS1, NS2a, NS2b, NS3, NS4a, NS4b, and NS5) (Figure 1a). The virus particle consists of an RNA-capsid protein complex, surrounded by a bilayer lipid membrane. The proteins present on the surface of the dengue immature virus are the E and prM. The mature virus surface, on the other hand, contains E and M proteins (a cleaved derivative of prM).

Figure 1. Structure of Dengue Virus particle. The surface protein (a) and Envelope glycoproteins are showed (b,c).

4. Life Cycle of Dengue Virus

The dengue viral replication process begins when the virus attaches to a human skin cell (Figure 2). After this attachment, the skin cell's membrane folds around the virus and forms a pouch that seals around the virus particle. This pouch is called an endosome. A cell normally uses endosomes to take in large molecules and particles from outside the cell for nourishment. By hijacking this normal cell process, the dengue virus is able to enter a host cell. Once the virus has entered a host cell, the virus penetrates deeper into the cell while still inside the endosome. Researchers have learned that two conditions are needed for the dengue virus to exit the endosome: The endosome must be deep inside the cell where the environment is acidic. The endosomal membrane must gain a negative charge. These two conditions allow the virus envelope to fuse with the endosomal membrane, and that process releases the dengue nucleocapsid into the cytoplasm of the cell. In the cytoplasm, the nucleocapsid opens to uncoat the viral genome. This process releases the viral RNA into the cytoplasm. The viral RNA then hijacks the host cell's machinery to replicate itself. The virus uses ribosomes on the host's rough endoplasmic reticulum (ER) to translate the viral RNA and produce the viral polypeptide. This polypeptide is then cut to form the ten dengue proteins. The newly synthesized viral RNA is enclosed in the C proteins, forming a nucleocapid. The nucleocapsid enters the rough ER and is enveloped in the ER membrane and surrounded by the M and E proteins. This step adds the viral envelope and protective outer layer. The immature viruses travel through the Golgi apparatus complex, where the viruses mature and convert into their infectious form. The mature dengue viruses are then released from the cell and can go on to infect other cells.

Figure 2. Life cycle of Dengue virus

5. Diagnosis, Treatment and Prevention

Diagnosis:
Dengue can be diagnosed by isolation of the virus, by serological tests, or by molecular methods. Diagnosis of acute (on-going) or recent dengue infection can be established by testing serum samples during the first 5 days of symptoms and/or early convalescent phase (more than 5 days of symptoms). Acute infection with dengue virus is confirmed when the virus is isolated from serum or autopsy tissue specimens, or the specific dengue virus genome is identified by RT–PCR from serum or plasma, cerebrospinal fluid, or autopsy tissue specimens during an acute febrile illness. Acute infections can also be laboratory confirmed by identification of dengue viral antigen or RNA in autopsy tissue specimens by immunofluorescence or immunohistochemical analysis, or by seroconversion from negative to positive IgM antibody to dengue or demonstration of a fourfold or greater increase in IgG antibody titers in paired (acute and convalescent) serum specimens.

Treatment:
There is no specific treatment for dengue fever.

For severe dengue, medical care by physicians and nurses experienced with the effects and progression of the disease can save lives – decreasing mortality rates from more than 20% to less than 1%. Maintenance of the patient's body fluid volume is critical to severe dengue care.

Prevention:
In late 2015 and early 2016, the first dengue vaccine, Dengvaxia (CYD-TDV) by Sanofi Pasteur, was registered in several countries for use in individuals 9-45 years of age living in endemic areas. At present, the main method to control or prevent the transmission of dengue virus is to combat vector mosquitoes.


Producing Monoclonal Antibodies

Some types of assays require better antibody specificity and affinity than can be obtained using a polyclonal antiserum. To attain this high specificity, all of the antibodies must bind with high affinity to a single epitope. This high specificity can be provided by monoclonal antibodies (mAbs). Table 1 compares some of the important characteristics of monoclonal and polyclonal antibodies.

Table 1. Characteristics of Polyclonal and Monoclonal Antibodies
Monoclonal Antibodies Polyclonal Antibodies
Expensive production Inexpensive production
Long production time Rapid production
Large quantities of specific antibodies Large quantities of nonspecific antibodies
Recognize a single epitope on an antigen Recognize multiple epitopes on an antigen
Production is continuous and uniform once the hybridoma is made Different batches vary in composition

Unlike polyclonal antibodies, which are produced in live animals, monoclonal antibodies are produced in vitro using tissue-culture techniques. mAbs are produced by immunizing an animal, often a mouse, multiple times with a specific antigen. B cells from the spleen of the immunized animal are then removed. Since normal B cells are unable to proliferate forever, they are fused with immortal, cancerous B cells called myeloma cells, to yield hybridoma cells. All of the cells are then placed in a selective medium that allows only the hybridomas to grow unfused myeloma cells cannot grow, and any unfused B cells die off. The hybridomas, which are capable of growing continuously in culture while producing antibodies, are then screened for the desired mAb. Those producing the desired mAb are grown in tissue culture the culture medium is harvested periodically and mAbs are purified from the medium. This is a very expensive and time-consuming process. It may take weeks of culturing and many liters of media to provide enough mAbs for an experiment or to treat a single patient. mAbs are expensive (Figure 3).

Figure 3. Monoclonal antibodies (mAbs) are produced by introducing an antigen to a mouse and then fusing polyclonal B cells from the mouse’s spleen to myeloma cells. The resulting hybridoma cells are cultured and continue to produce antibodies to the antigen. Hybridomas producing the desired mAb are then grown in large numbers on a selective medium that is periodically harvested to obtain the desired mAbs.


Results and Discussion

In vitro expression and purification of membrane proteins by IVT-HMB

To facilitate analyses of the sera, we developed a novel in vitro approach for simultaneous expression and capture of each of the membrane protein targets. We optimized a commercially available in vitro protein translation system and included unmodified tosylactivated magnetic beads in the reaction to yield the method IVT-HMB ( i n v itro translation in the presence of hydrophobic magnetic beads Fig. 1a). Tosylated beads are typically used to capture protein via covalent modification of the bead surface through replacement of the tosyl leaving group with a capturing ligand. Rather, we found that unmodified beads allowed non-covalent capture of each of the 17 membrane protein targets at high purity (Fig. 1b,c). Using IVT-HMB, we obtained 15 of the 17 targets in full-length form (Fig. 1b). The remaining two targets were captured in multiple truncated forms that in total represented most of the length of each target sequence (Fig. 1c). The capture process may be based on bead surface adherence to hydrophobic residues that are normally buried in the membrane or that lie internal to the protein’s tertiary structure, but are exposed during the in vitro synthesis events.

(a) Schematic of the IVT-HMB method for expression and purification of membrane proteins. (b) Generation of full-length proteins for 15 targets in the form of fusions to green fluorescent protein (GFP). (c) Generation of partial-length proteins for F. tularensis FTT0759 (305 amino acids) and ASFV CD2v (360 amino acids) in the form of fusions to thioredoxin. Shown are SDS-PAGE visualized by Coomassie stain (left panel of b) or silver stain (remaining panels in b,c) and containing, per lane, magnetic-bead purified fractions from 10 μL of IVT-HMB. Molecular weight (MW) in kDa is indicated, and arrowheads indicate the predicted migration position. BSA, bovine serum albumin.

Towards characterization of polyclonal sera, the IVT-HMB approach effectively simplified antigen preparation by precluding the need to use endogenous protein or to purify detergent-solubilized or urea-denatured membrane protein, since the protein can be directly used without a separate elution step. Although the IVT-HMB protein is not expected to be natively-folded, as GFP fluorescence of the bead-bound protein was not detectable above empty vector controls, this protein is suitable for evaluation of polyclonal immune responses since a significant proportion of polyclonal antibody species recognizes linear epitopes 26 . The resulting yields of 5–20 μg of membrane protein per 500 μL of IVT-HMB reaction were sufficient to allow completion of ELISA and Western analyses of the sera from 5 mice. Other unique advantages of the method include eliminating any need for tagging the target protein or including a capturing ligand on the beads. Limitations of the current IVT-HMB method may be in identifying conformationally-specific monoclonal antibodies and antibodies for antigens that are highly modified post-translationally. However, we expect that IVT-HMB can be adapted to in vitro systems that provide such modifications 27 .

Antibody production by genetic immunization using DNA-gold micronanoplexes

Biolistic immunization with the gene gun uses a burst of gas to propel DNA-bound gold particles to the dermal tissues (Fig. 2a). This method of genetic immunization leads to direct transfection of dendritic cells 28 and in vivo expression of the encoded protein in both the dermal tissues and the lymph nodes 28,29 . Although biolistic immunization is more technically challenging than immunization by DNA needle injection, biolistic delivery effects a Th2 response, which is specifically associated with antibody production, in comparison to the Th1 response favored upon DNA injection 30 .

(a) Overview of DNA-gold micronanoplex bullet production. (b,c) Genetic immunization vectors pCMVi-LSrCOMPTT (b) and pCMVi-UB 35 (c). (d) Representative Westerns on IVT-HMB target protein using immunizations with pCMVi-LSrCOMPTT, except for (*) which used pCMVi-UB. Positive immunoblots were for 5 out of 5 mice and using a serum dilution of 1:2000 unless noted. Individual Western and ELISA results are in Supplementary Fig. 1a–q.

The DNA-gold particles used in this study were micronanoplexes (Fig. 2a) 31 . Micronanoplexes are complexes of two differently-sized gold particles: micron-sized (1–2 μm) gold particles that are coated with polyethylenimine (microgold-PEI Fig. 2a) 32 , and nanometer-sized gold particles that are generated upon modification with cysteamine and DNA (DNA-nanogold Fig. 2a) 31,33 . The micron-sized particles allow dermal penetration, and the nanometer-sized particles provide a high surface area for DNA binding. Micronanoplexes allow an order of magnitude higher DNA-binding capacity compared to micron-sized particles alone 31 . Micronanoplexes have previously been used to express luciferase in mice 31 and to identify vaccine antigen candidates that provided protection against the bioterrorism agent Burkholderia mallei 34 . The results presented here represent the first described application of DNA-gold micronanoplexes to antibody production.

To generate polyclonal antibodies, each antigen-encoding gene was cloned as a full-length open reading frame with its natural codon usage into the novel vector pCMVi-LSrCOMPTT (Fig. 2b), and several targets were also cloned into the vaccine construct pCMVi-UB (Fig. 2c) 35 . pCMVi-LSrCOMPTT is based on vectors that were used to produce antibodies against proteins lacking transmembrane domains 15 . pCMVi-LSrCOMPTT allows expression of the target membrane protein as a fusion with a 174-residue sequence containing the following four immune-stimulating domains (Fig. 2b). “LS” is the 24 amino acid secretion leader sequence from the human α1-antitrypsin gene 36 , which allows targeting of the fused protein to the plasma membrane 37 . To our knowledge, this work is the first reported use of the LS sequence for generation of antibodies against membrane proteins. “r” is a randomly-generated 23 amino acid peptide that was previously shown to be immunogenic 15 . “COMP” is the 45 amino acid cartilage oligomeric matrix protein assembly domain from the pentameric rat matrix protein 38 . “TT” is a 50 amino acid sequence containing tetanus toxoid epitopes, which has been used to overcome humoral tolerance 39 . All plasmids used in this study and their sequences are available from the PSI:Biology-Materials Repository at DNASU 40 (Supplementary Table 2).

A typical immunization schedule was applied and consisted of a double prime plus 2–4 genetic boosts until target-specific ELISA titers of 1:1000 were achieved, in comparison to naïve mice (Supplementary Fig. 1a–q). Antibody specificity was confirmed in Western blots (Fig. 2d and Supplementary Fig. 1a–q). Biolistic immunization using pCMVi-LSrCOMPTT yielded antibodies for 12 membrane proteins, including 11 of 14 targets from F. tularensis and one of three targets from ASFV (Fig. 2d). Using altered adjuvants, improved titers were obtained with fewer boosts (Supplementary Fig. 2a–k), which suggests the possibility of further enhancement upon systematic investigations.

For nine targets that yielded negative immunoblots at a serum dilution of ≤1:500, the corresponding genes were cloned into pCMVi-UB (Fig. 2c) 35 , which allows expression of the target as a fusion with mouse ubiquitin. pCMVi-UB has been used previously to evaluate protective antigens in vaccine studies (reviewed in 41 ) but has also yielded strong antibody responses 35,42 . The ubiquitin sequence contains the Gly76Ala mutation to prevent de-ubiquitination 43 . The expressed protein is not deliberately membrane-directed and may be expected to undergo MHC I presentation. We found that pCMVi-UB yielded a high response for the p54 envelope protein from ASFV (Fig. 2d and Supplementary Fig. 3a,b). ELISA responses for the other eight targets were not appreciably higher than in the presence of negative control IVT-HMB products (Supplementary Fig. 3a, dashed lines). The lack of measurable response for these eight targets is not an unbiased predictor of antibody generation by pCMVi-UB, since the antigens tested also yielded moderate or no reactivity in pCMVi-LSrCOMPTT.

Boosts with protein from IVT-HMB

We further noted that boosting with antigen from IVT-HMB reactions yielded higher ELISA titers (Supplementary Fig. 2f,j,k). However, this reactivity may be due in part to irrelevant proteins (Supplementary Fig. 3c), possibly those visible as background bands in gels containing captured antigen (Fig. 1b,c). It is also not apparent whether the captured antigen contains folded domains. Nevertheless, for the eight pCMVi-UB constructs that lacked a DNA response (Supplementary Fig. 3a), target-specific reactivity was not observed in immunoblots following two boosts with IVT-HMB protein.

Method appraisal

Importantly, success with this genetic immunization approach generally correlated with previous reports of target-specific immunogenicity in infected and immunized hosts (Supplementary Table 1, column “Reported immunogenicity”). The exceptions were CD2v, for which host recognition has been described for only a highly glycosylated form 44 , and F. tularensis FupB, for which antibodies were raised in rats from a recombinant fragment 45 . The efficiency of our method (71%) also approaches that described for non-transmembrane proteins, which was 84% (n = 100) using biolistic immunization with micron-sized gold and codon optimization 15 , and 78% (n = 18 proteins) or 83% (n = 6), respectively, using tail or limb vein injection of DNA 46 .

Our approach is expected to have wide applicability, given that robust antibodies were generated for more than half of the targets from two disparate hosts and across a structurally diverse set of membrane proteins. The targets included ten α-helical, seven β-barrel, and two lipidated proteins (Supplementary Table 1). Of interest will be whether this method can be adapted to better recognize targets that are poorly immunogenic by any approach, such as for ion channels that consist mainly of membrane-buried α-helices 47 and for highly conserved targets whose autoreactive B cells would likely be removed by tolerance checkpoints 48 . Indeed, our approach yielded no measurable serum reactivity for the two most hydrophobic targets, FTT0759 and the CapC subunit of the capsule biosynthesis complex. This result is consistent with the well-known poor immunogenicity of hydrophobic regions, and is usually attributed to the low structural complexity of these stretches. It will be of interest to determine whether improved recognition can be achieved by the use of codon-optimized genes or by co-immunization of subunits that normally exist as part of a membrane protein complex (F. tularensis CapBCA 49 ). Further method development may be guided by improved understanding of the membrane protein expression pathways undertaken by these targets upon immunization as well as the mechanisms of immune recognition upon biolistic immunization, which have generally focused on cellular, not humoral, immune responses and have not been specifically explored for integral membrane proteins.

Antibody-based characterization of membrane-targeting upon recombinant expression

To support structural studies of membrane proteins involved in pathogenesis, we used the antibodies to characterize membrane-targeting of several proteins from F. tularensis upon recombinant expression in Escherichia coli. Because several targets could not be purified when expressed as fusions with purification tags, we analyzed expression of targets that lacked purification tags. This tag-free approach evaluated whether membrane-targeting alone could be achieved in E. coli using the native F. tularensis protein sequences and independently of other F. tularensis-specific factors.

Western analyses using antibodies against four tag-less targets confirmed their expression in vitro (Fig. 3a, lane “IVT-HMB, target”). In contrast, in vivo expression in E. coli was undetectable for the outer membrane efflux protein (OMEP Fig. 3a, lane “Total protein, target”). PilQ was poorly expressed in vivo (Fig. 3a, lane “Total protein, target”), and little of this membrane protein appeared to be membrane-directed as indicated by detergent solubility (Fig. 3a, lane “DDM, target” DDM = n-dodecyl β-D-maltoside). These results suggest that OMEP and PilQ may require alternative strategies to generate these proteins for structural analyses. In comparison, in vivo expression was more apparent for the virulence determinants FopA and FTT1525 (Fig. 3a, lanes “Total protein, target”). Importantly, the antibody reagents generated in this study allowed membrane-targeting of FopA and FTT1525 to be demonstrated for the first time, by detection of these proteins in DDM-solubilized fractions (Fig. 3a, lanes “DDM, target”). These immunoblots additionally demonstrate the target specificity of some of the non-enriched sera obtained in this study, as evidenced by the lack of additional bands that represent proteins from host cells (Fig. 3a, lane “BL21(DE3)”) or proteins from IVT-HMB reactions (Fig. 3a, lane “IVT-HMB, no DNA”). Use of the remaining sera in future studies will require the application of appropriate controls 50 to avoid misinterpretation of data.

(a) Immunoblots probed with 1:2000 dilution of serum. IVT-HMB samples contain 100–200 ng of target protein, or beads from an equivalent reaction volume lacking template DNA. Total protein and detergent-solubilized samples contain protein from 16 μL of culture in which the target protein or an unrelated target was expressed. DDM, n-dodecyl β-D-maltoside. The unrelated targets used in the blots were, from top to bottom: PilQ, OMEP, FTT1525, and TolC. (b) Western analyses of the membrane fraction, cytosolic fraction, and detergent-solubilized proteins of the membrane fraction upon expression of F. tularensis BamA containing the signal sequence (ss) from E. coli BamA. The BamA protein is identified (yellow triangles). Lanes contain protein from the equivalent of 100 μL of culture. Serum dilution was 1:500. LDAO, N,N-dimethyldodecylamine N-oxide.

Similar analyses with purification fractions of a recombinant form of the BamA major subunit of the F. tularensis β-barrel assembly machine indicated virtually no membrane-targeting in E. coli (Fig. 3b, lanes “LDAO” and “DDM” LDAO = N,N-dimethyldodecylamine N-oxide). Evidence of strong expression (Fig. 3b, lane “membrane”) suggests that recombinant BamA may be a candidate for refolding from inclusion bodies, towards structural investigations.


Production and characterization of monoclonal and polyclonal antibodies against the mycotoxin cyclopiazonic acid

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