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129-428: Western blot technique uses three elements to achieve its task of separating a specific protein from a complex: separation by size, transfer of protein to a solid support, and marking target protein using a primary and secondary antibody to visualize. A synthetic or animal-derived antibody (known as the primary antibody ) is created that recognizes and binds to a specific target protein. The electrophoresis membrane

258-554: A "bull's eye" or "target-like" pattern, but this clearing only happens in 19% of cases in endemic areas of the United States. The rash may feel warm, usually is not itchy, is rarely tender or painful, and takes up to four weeks to resolve if untreated. The Lyme rash is often accompanied by symptoms of a flu-like illness, including fatigue, headache, body aches, fever, and chills [though usually neither nausea nor upper-respiratory problems]. These symptoms may also appear without

387-413: A CCD camera equipped with appropriate emission filters which captures a digital image of the western blot and allows further data analysis such as molecular weight analysis and a quantitative western blot analysis. Fluorescence is considered to be one of the best methods for quantification but is less sensitive than chemiluminescence. One major difference between nitrocellulose and PVDF membranes relates to

516-405: A dilute solution of protein – typically 3–5% bovine serum albumin (BSA) or non-fat dry milk (both are inexpensive) in tris-buffered saline (TBS) or I-Block, with a minute percentage (0.1%) of detergent such as Tween 20 or Triton X-100 . Although non-fat dry milk is preferred due to its availability, an appropriate blocking solution is needed as not all proteins in milk are compatible with all

645-571: A general sense of feeling unwell , and changes in personality. Lyme encephalopathy is controversial in the US and has not been reported in Europe. Problems such as depression and fibromyalgia are as common in people with Lyme disease as in the general population. There is no compelling evidence that Lyme disease causes psychiatric disorders, behavioral disorders (e.g. ADHD ), or developmental disorders (e.g. autism ). Acrodermatitis chronica atrophicans

774-406: A high background in the blots. A high background in the blots could be avoided by fixing these issues. It has been claimed that a variety of odd and unequal bands, including black dots, white spots or bands, and curving bands, have occurred. The block dots are removed from the blots by effective blocking. White patches develop as a result of bubbles between the membrane and gel. White bands appear in

903-411: A host species or immune cell culture is exposed to the protein of interest (or a part thereof). Normally, this is part of the immune response, whereas here they are harvested and used as sensitive and specific detection tools that bind the protein directly. After blocking, a solution of primary antibody (generally between 0.5 and 5 micrograms/mL) diluted in either PBS or TBST wash buffer is incubated with

1032-406: A membrane. Size approximations are taken by comparing the stained bands to that of the marker or ladder loaded during electrophoresis. The process is commonly repeated for a structural protein, such as actin or tubulin , that should not change between samples. The amount of target protein is normalized to the structural protein to control between groups. A superior strategy is the normalization to

1161-402: A near-infrared fluorophore-linked antibody. The light produced from the excitation of a fluorescent dye is static, making fluorescent detection a more precise and accurate measure of the difference in the signal produced by labeled antibodies bound to proteins on a western blot. Proteins can be accurately quantified because the signal generated by the different amounts of proteins on the membranes

1290-411: A negatively charged group). Proteins that have been separated can be cut out of the gel and then analysed by mass spectrometry , which identifies their molecular weight. There may be a weak or absent signal in the band for a number of reasons related to the amount of antibody and antigen used. This problem might be resolved by using the ideal antigen and antibody concentrations and dilutions specified in

1419-628: A number of molecular recognition applications. Affimer technology has been commercialised and developed by Avacta Life Sciences, who are developing Affimer binders as reagents for research, diagnostic and therapeutic applications. Synthetic antibodies have shown their utility in a number of applications. Their use within the field of research lies predominantly in the life sciences as reagents for protein capture and as protein inhibitors. Within diagnostics they have been utilised in applications ranging from infection and cancer screening to mycotoxin detection in grain samples. Synthetic antibodies are currently

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1548-430: A positive result would likely be false, possibly causing unnecessary treatment. In some cases, when history, signs, and symptoms are strongly suggestive of early disseminated Lyme disease, empiric treatment may be started and reevaluated as laboratory test results become available. Tests for antibodies in the blood by ELISA and Western blot is the most widely used method for Lyme diagnosis. A two-tiered protocol

1677-519: A protein analysis step such as the detection of low- or post-translationally modified proteins. Additionally, they can be based on the selection of antibodies since the quality of the antibodies plays a significant role in the detection of proteins specifically. On account of the presence of these kinds of problems, a variety of improvements are being produced in the fields of preparation of cell lysate and blotting procedures to build up reliable results. Moreover, to achieve more sensitive analysis and overcome

1806-400: A protein can be derived from the size and colour intensity of a protein band on the blot membrane. In addition, applying a dilution series of a purified protein of known concentrations can be used to allow a more precise estimate of protein concentration. The western blot is routinely used for verification of protein production after cloning . It is also used in medical diagnostics, e.g., in

1935-478: A range of experimental conditions. Recombinant antibodies are monoclonal antibodies generated in vitro using synthetic genes. Recombinant antibody technology involves recovering the antibody genes from the source cells, amplifying and cloning the genes into an appropriate vector, introducing the vector into a host, and achieving expression of adequate amounts of functional antibody. Recombinant antibodies can be cloned from any species of antibody-producing animal, if

2064-438: A rare peripheral neuropathy was described that included numbness, tingling, or burning starting at the feet or hands and over time possibly moving up the limbs. In a later analysis that discovered poor documentation of this manifestation, experts wondered if it exists at all in the US or is merely very rare. A neurologic syndrome called Lyme encephalopathy is associated with subtle memory and cognitive difficulties, insomnia ,

2193-528: A rash or linger after the rash has disappeared. Lyme can progress to later stages without a rash or these symptoms. People with high fever for more than two days or whose other symptoms of viral-like illness do not improve despite antibiotic treatment for Lyme disease, or who have abnormally low levels of white or red cells or platelets in the blood, should be investigated for possible coinfection with other tick-borne diseases such as ehrlichiosis and babesiosis . Not everyone with Lyme disease has all

2322-561: A related syndrome called southern tick-associated rash illness , which resembles a mild form of Lyme disease. On the West Coast of the United States , the main vector is the western black-legged tick ( Ixodes pacificus ). The tendency of this tick species to feed predominantly on host species such as the Western Fence Lizard that are resistant to Borrelia infection appears to diminish transmission of Lyme disease in

2451-728: A tick or a bite, and the EM rash need not look like a bull's eye (most EM rashes in the U.S. do not) or be accompanied by any other symptoms. In the U.S., Lyme is most common in the New England and Mid-Atlantic states and parts of Wisconsin and Minnesota , but it is expanding into other areas. Several bordering areas of Canada also have high Lyme risk. In the absence of an EM rash or history of tick exposure, Lyme diagnosis depends on laboratory confirmation. The bacteria that cause Lyme disease are difficult to observe directly in body tissues and also difficult and too time-consuming to grow in

2580-516: A tick quickly (within 24 hours), you can greatly reduce your chances of getting Lyme disease." In Europe, Ixodes ricinus ticks may spread the bacteria more quickly. In North America, the bacterial species Borrelia burgdorferi and B. mayonii cause Lyme disease. In Europe and Asia, Borrelia afzelii , Borrelia garinii , B. spielmanii and four other species also cause the disease. The disease does not appear to be transmissible between people, by other animals nor through food. Diagnosis

2709-567: A tick salivary protein that appears to have immunosuppressive effects that enhance infection. Successful infection of the mammalian host depends on bacterial expression of OspC. Tick bites often go unnoticed because of the small size of the tick in its nymphal stage, as well as tick secretions that prevent the host from feeling any itch or pain from the bite. However, transmission is quite rare, with only about 1.2 to 1.4 percent of recognized tick bites resulting in Lyme disease. While B. burgdorferi

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2838-422: A total body skin examination for EM rashes and asked whether EM-type rashes had manifested within the last 1–2 months. Presence of an EM rash and recent tick exposure (i.e., being outdoors in a likely tick habitat where Lyme is common , within 30 days of the appearance of the rash) are sufficient for Lyme diagnosis; no laboratory confirmation is needed or recommended. Most people who get infected do not remember

2967-529: A week afterwards. The rash is typically neither itchy nor painful. Approximately 70–80% of infected people develop a rash. Early diagnosis can be difficult. Other early symptoms may include fever, headaches and tiredness . If untreated, symptoms may include loss of the ability to move one or both sides of the face , joint pains , severe headaches with neck stiffness or heart palpitations . Months to years later, repeated episodes of joint pain and swelling may occur. Occasionally, shooting pains or tingling in

3096-601: Is borrelial lymphocytoma , a purplish lump that develops on the ear lobe, nipple, or scrotum . Lyme arthritis occurs in up to 60% of untreated people, typically starting about six months after infection. It usually affects only one or a few joints, often a knee or possibly the hip , other large joints, or the temporomandibular joint . Usually, large joint effusion and swelling occur, but only mild or moderate pain. Without treatment, swelling and pain typically resolve over time, but periodically return. Baker's cysts may form and rupture. In early US studies of Lyme disease,

3225-453: Is a chronic skin disorder observed primarily in Europe among the elderly. It begins as a reddish-blue patch of discolored skin, often on the backs of the hands or feet. The lesion slowly atrophies over several weeks or months, with the skin becoming first thin and wrinkled and then, if untreated, completely dry and hairless. It is also associated with peripheral neuropathy. Lyme disease is caused by spirochetes , gram-negative bacteria from

3354-559: Is a play on the Southern blot , a technique for DNA detection named after its inventor, English biologist Edwin Southern . Similarly, detection of RNA is termed as northern blot . The term "western blot" was given by W. Neal Burnette in 1981, although the method itself was independently invented in 1979 by Jaime Renart, Jakob Reiser, and George Stark at Stanford University , and by Harry Towbin, Theophil Staehelin, and Julian Gordon at

3483-440: Is added. The stained bands then indicate the proteins to which the patient's serum contains antibody. A western blot is also used as the definitive test for variant Creutzfeldt–Jakob disease , a type of prion disease linked to the consumption of contaminated beef from cattle with bovine spongiform encephalopathy (BSE, commonly referred to as 'mad cow disease'). Another application is in the diagnosis of tularemia . An evaluation of

3612-399: Is applied along the gel, proteins migrate through it at different speeds dependent on their size. These different rates of advancement (different electrophoretic mobilities ) separate into bands within each lane . Protein bands can then be compared to the ladder bands, allowing estimation of the protein's molecular weight. It is also possible to use a two-dimensional gel which spreads

3741-525: Is based on a combination of symptoms, history of tick exposure and possibly testing for specific antibodies in the blood. Blood tests are often falsely negative in the early stages of the disease. Testing of individual ticks is not typically useful. Prevention includes efforts to prevent tick bites by wearing clothing to cover the arms and legs and using DEET or picaridin -based insect repellents . Using pesticides to reduce tick numbers may also be effective. Ticks can be removed using tweezers . If

3870-446: Is classified as a zoonosis , as it is transmitted to humans from a natural reservoir among small mammals and birds by ticks that feed on both sets of hosts . Hard-bodied ticks of the genus Ixodes are the vectors of Lyme disease (also the vector for Babesia ). Most infections are caused by ticks in the nymphal stage , because they are very small, thus may feed for long periods of time undetected. Nymphal ticks are generally

3999-411: Is composed of gel electrophoresis to separate native proteins by 3-D structure or denatured proteins by the length of the polypeptide, followed by an electrophoretic transfer onto a membrane (mostly PVDF or nitrocellulose ) and an immunostaining procedure to visualize a certain protein on the blot membrane. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) is generally used for

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4128-487: Is crucial to describe different experimental circumstances or parameters in order to increase the repeatability and precision of WB. To increase WB repeatability, a minimum reporting criteria is thus required. Two-dimensional SDS-PAGE uses the principles and techniques outlined above. 2-D SDS-PAGE, as the name suggests, involves the migration of polypeptides in 2 dimensions. For example, in the first dimension, polypeptides are separated according to isoelectric point , while in

4257-453: Is different from chronic Lyme disease , a term no longer supported by scientists and used in different ways by different groups. Some healthcare providers claim that chronic Lyme is caused by persistent infection, but this is not believed to be true because no evidence of persistent infection can be found after standard treatment. As of 2023 , clinical trials of proposed human vaccines for Lyme disease were being carried out, but no vaccine

4386-403: Is extensively used in biochemistry for the qualitative detection of single proteins and protein-modifications (such as post-translational modifications ). At least 8–9% of all protein-related publications are estimated to apply western blots. It is used as a general method to identify the presence of a specific single protein within a complex mixture of proteins. A semi-quantitative estimation of

4515-400: Is incubated with the membrane in a manner similar to that for the primary antibody in a two-step process, and then is ready for direct detection after a series of wash steps. After the unbound probes are washed away, the western blot is ready for detection of the probes that are labeled and bound to the protein of interest. In practical terms, not all westerns reveal protein only at one band in

4644-426: Is influenced by the protein preparation, which is composed of protein extraction and purification processes. To achieve efficient protein extraction, a proper homogenization method needs to be chosen due to the fact that it is responsible for bursting the cell membrane and releasing the intracellular components. Besides that, the ideal lysis buffer is needed to acquire substantial amounts of target protein content because

4773-418: Is measured in a static state, as compared to chemiluminescence, in which light is measured in a dynamic state. A third alternative is to use a radioactive label rather than an enzyme coupled to the secondary antibody, such as labeling an antibody-binding protein like Staphylococcus Protein A or Streptavidin with a radioactive isotope of iodine. Since other methods are safer, quicker, and cheaper, this method

4902-410: Is most associated with ticks hosted by white-tailed deer and white-footed mice , Borrelia afzelii is most frequently detected in rodent-feeding vector ticks, and Borrelia garinii and Borrelia valaisiana appear to be associated with birds. Both rodents and birds are competent reservoir hosts for B. burgdorferi sensu stricto . The resistance of a genospecies of Lyme disease spirochetes to

5031-514: Is now rarely used; however, an advantage of this approach is the sensitivity of auto-radiography-based imaging, which enables highly accurate protein quantification when combined with optical software (e.g. Optiquant). Historically, the probing process was performed in two steps because of the relative ease of producing primary and secondary antibodies in separate processes. This gives researchers and corporations huge advantages in terms of flexibility, reduction of cost, and adds an amplification step to

5160-565: Is prohibited under WADA's List of Prohibited Substances and Methods. The western blot technique was used during the 2014 FIFA World Cup in the anti-doping campaign for that event. In total, over 1000 samples were collected and analysed by Reichel, et al. in the WADA accredited Laboratory of Lausanne , Switzerland . Recent research utilizing the western blot technique showed an improved detection of EPO in blood and urine based on novel Velum SAR precast horizontal gels optimized for routine analysis. With

5289-429: Is proportional to the amount of horseradish peroxidase-conjugated secondary antibody, and therefore, indirectly measures the presence of the target protein. A sensitive sheet of photographic film is placed against the membrane, and exposure to the light from the reaction creates an image of the antibodies bound to the blot. A cheaper but less sensitive approach utilizes a 4-chloronaphthol stain with 1% hydrogen peroxide ;

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5418-574: Is recommended by the Centers for Disease Control and Prevention (CDC): the sensitive ELISA test is performed first, and if it is positive or equivocal, then the more specific Western blot is run. The immune system takes some time to produce antibodies in quantity. After Lyme infection onset, antibodies of types IgM and IgG usually can first be detected respectively at 2–4 weeks and 4–6 weeks, and peak at 6–8 weeks. When an EM rash first appears, detectable antibodies may not be present. Therefore, it

5547-632: Is recommended that testing not be performed and diagnosis be based on the presence of the EM rash. Up to 30 days after suspected Lyme infection onset, infection can be confirmed by detection of IgM or IgG antibodies; after that, it is recommended that only IgG antibodies be considered. A positive IgM and negative IgG test result after the first month of infection is generally indicative of a false-positive result. The number of IgM antibodies usually collapses 4–6 months after infection, while IgG antibodies can remain detectable for years. Other tests may be used in neuroborreliosis cases. In Europe, neuroborreliosis

5676-626: Is the most common disease spread by ticks in the Northern Hemisphere. Infections are most common in the spring and early summer. Lyme disease was diagnosed as a separate condition for the first time in 1975 in Lyme, Connecticut . It was originally mistaken for juvenile rheumatoid arthritis . The bacterium involved was first described in 1981 by Willy Burgdorfer . People with persistent symptoms after appropriate treatments are said to have Post-Treatment Lyme Disease Syndrome (PTLDS). PTLDS

5805-406: Is usually caused by Borrelia garinii and almost always involves lymphocytic pleocytosis , i.e. the densities of lymphocytes (infection-fighting cells) and protein in the cerebrospinal fluid (CSF) typically rise to characteristically abnormal levels, while glucose level remains normal. Additionally, the immune system produces antibodies against Lyme inside the intrathecal space, which contains

5934-652: Is washed in a solution containing the primary antibody, before excess antibody is washed off. A secondary antibody is added which recognizes and binds to the primary antibody. The secondary antibody is visualized through various methods such as staining , immunofluorescence , and radioactivity, allowing indirect detection of the specific target protein. Other related techniques include dot blot analysis, quantitative dot blot , immunohistochemistry and immunocytochemistry , where antibodies are used to detect proteins in tissues and cells by immunostaining , and enzyme-linked immunosorbent assay (ELISA). The name western blot

6063-545: The Friedrich Miescher Institute in Basel , Switzerland . The Towbin group also used secondary antibodies for detection, thus resembling the actual method that is almost universally used today. Between 1979 and 2019 "it has been mentioned in the titles, abstracts, and keywords of more than 400,000 PubMed -listed publications" and may still be the most used protein-analytical technique. The western blot

6192-448: The HIV test or BSE -Test. The confirmatory HIV test employs a western blot to detect anti-HIV antibody in a human serum sample. Proteins from known HIV -infected cells are separated and blotted on a membrane as above. Then, the serum to be tested is applied in the primary antibody incubation step; free antibody is washed away, and a secondary anti-human antibody linked to an enzyme signal

6321-461: The areas of the body served by nerves connected to the affected roots , e.g. limb(s) or part(s) of trunk. The pain is often described as unlike any other previously felt, excruciating, migrating, worse at night, rarely symmetrical, and often accompanied by extreme sleep disturbance. Mononeuritis multiplex is an inflammation causing similar symptoms in one or more unrelated peripheral nerves. Rarely, early neuroborreliosis may involve inflammation of

6450-432: The bacteriolytic activities of the alternative complement pathway of various host species may determine its reservoir host association. Budding research has suggested that B. burgdorferi sensu lato may also be able to form enzootic cycle among lizard populations; this was previously assumed not to be possible in major areas containing populations of lizards, such as California. Except for one study in Europe, much of

6579-468: The brain or spinal cord , with symptoms such as confusion, abnormal gait, ocular movements, or speech , impaired movement , impaired motor planning , or shaking . In North America, facial palsy is the typical early neuroborreliosis presentation, occurring in 5–10% of untreated people, in about 75% of cases accompanied by lymphocytic meningitis. Lyme radiculopathy is reported half as frequently, but many cases may be unrecognized. In European adults,

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6708-480: The cerebrospinal fluid (CSF) and may be accompanied for several weeks by variable headache and, less commonly, usually mild meningitis signs such as inability to flex the neck fully and intolerance to bright lights but typically no or only very low fever. After several months neuroborreliosis can also present otolaryngological symptoms . Up to 76.5% of them present as tinnitus , the most common symptom. Vertigo and dizziness (53.7%) and hearing loss (16.7%) were

6837-485: The extracellular matrix , which may interfere with the function of immune factors. Exposure to the Borrelia bacterium during Lyme disease possibly causes a long-lived and damaging inflammatory response , a form of pathogen-induced autoimmune disease. The production of this reaction might be due to a form of molecular mimicry , where Borrelia avoids being killed by the immune system by resembling normal parts of

6966-947: The genus Borrelia . Spirochetes are surrounded by peptidoglycan and flagella . The Lyme-related Borrelia species are collectively known as Borrelia burgdorferi sensu lato , and show a great deal of genetic diversity . B. burgdorferi sensu lato is a species complex made up of 20 accepted and three proposed genospecies. Eight species are known to cause Lyme disease: B. mayonii (found in North America), B. burgdorferi sensu stricto (found in North America and Europe), B. afzelii , B. garinii, B. spielmanii, and B. lusitaniae (all found in Eurasia ). Some studies have also proposed that B. valaisiana may sometimes infect humans, but this species does not seem to be an important cause of disease. Three stages occur in

7095-424: The white-footed mouse , and occasionally birds. Areas where Lyme is common are expanding. Within the tick midgut, the Borrelia ' s outer surface protein A (OspA) binds to the tick receptor for OspA, known as TROSPA. When the tick feeds, the Borrelia downregulates OspA and upregulates OspC, another surface protein. After the bacteria migrate from the midgut to the salivary glands, OspC binds to Salp15,

7224-513: The CSF. Demonstration by lumbar puncture and CSF analysis of pleocytosis and intrathecal antibody production are required for definite diagnosis of neuroborreliosis in Europe (except in cases of peripheral neuropathy associated with acrodermatitis chronica atrophicans , which usually is caused by Borrelia afzelii and confirmed by blood antibody tests). In North America, neuroborreliosis is caused by Borrelia burgdorferi and may not be accompanied by

7353-466: The Northern Hemisphere because the nymphal stage of the tick is responsible for most cases. 80% of Lyme infections begin with a rash of some sort at the site of a tick bite, often near skin folds such as the armpit , groin , back of the knee , or the trunk under clothing straps, or in children's hair, ears, or neck. Most people who get infected do not remember seeing a tick or a bite. The rash appears typically one or two weeks (range 3–32 days) after

7482-464: The PNS, electromyography and nerve conduction studies can be used to monitor objectively the response to treatment. In Lyme carditis, electrocardiograms are used to evidence heart conduction abnormalities, while echocardiography may show myocardial dysfunction. Biopsy and confirmation of Borrelia cells in myocardial tissue may be used in specific cases but are usually not done because of risk of

7611-618: The West. Transmission can occur across the placenta during pregnancy and as with a number of other spirochetal diseases, adverse pregnancy outcomes are possible with untreated infection; prompt treatment with antibiotics reduces or eliminates this risk. There is no scientific evidence to support Lyme disease transmission via blood transfusion, sexual contact, or breast milk. Ticks that transmit B. burgdorferi to humans can also carry and transmit several other microbes, such as Babesia microti and Anaplasma phagocytophilum , which cause

7740-548: The ability of each to support "stripping" antibodies off and reusing the membrane for subsequent antibody probes. While there are well-established protocols available for stripping nitrocellulose membranes, the sturdier PVDF allows for easier stripping, and for more reuse before background noise limits experiments. Another difference is that, unlike nitrocellulose, PVDF must be soaked in 95% ethanol, isopropanol or methanol before use. PVDF membranes also tend to be thicker and more resistant to damage during use. In order to ensure that

7869-406: The adoption of the horizontal SAR-PAGE in combination with the precast film-supported Velum SAR gels the discriminatory capacity of micro-dose application of rEPO was significantly enhanced. For medication development, the identification of therapeutic targets, and biological research, it is essential to comprehend where proteins are located within a cell. The subcellular locations of proteins inside

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7998-413: The aid of subcellular fractionation methods, WB continues to be an important fundamental method for the investigation and comprehension of protein localization. Due to their various epitopes, antibodies have gained interest in both basic and clinical research. The foundation of antibody characterization and validation is epitope mapping. The procedure of identifying an antibody's binding sites (epitopes) on

8127-682: The appropriate oligonucleotide primers or hybridization probes are available. The ability to manipulate the antibody genes make it possible to generate new antibodies and antibody fragments, such as Fab fragments and scFv in vitro. This can be done at the level of the whole combining site by making new combinations of H and L chains. It can also be done by mutating individual CDRs . Display libraries, commonly expressed in phage or yeast, can be analysed to select for desirable characteristics arising from such changes in antibody sequence. These molecules typically differ in structure to that of an antibody and can be generated either from nucleic acids , as in

8256-461: The arms and legs may develop. Lyme disease is transmitted to humans by the bites of infected ticks of the genus Ixodes . In the United States, ticks of concern are usually of the Ixodes scapularis type. According to the Centers for Disease Control and Prevention , "In most cases, a tick must be attached for 36 to 48 hours or more before the Lyme disease bacterium can be transmitted. If you remove

8385-404: The bacteria to survive and eventually spread throughout the body. Days to weeks following the tick bite, the spirochetes spread via the bloodstream to joints, heart, nervous system, and distant skin sites, where their presence gives rise to the variety of symptoms of the disseminated disease. The spread of B. burgdorferi is aided by the attachment of the host protease plasmin to the surface of

8514-399: The bite and expands 2–3 cm per day up to a diameter of 5–70 cm (median is 16 cm). The rash is usually circular or oval, red or bluish, and may have an elevated or darker center. This rash is termed an Erythema Migrans (EM) which translates as "Migrating Redness." In about 79% of cases in Europe, this rash gradually clears from the center toward the edges possibly forming

8643-427: The blots when main and secondary antibodies are present in significant concentrations. Because of the high voltage used during the gel run and the rapid protein migration, smiley bands appear in the blots. The strange bands in the blot are resolved by resolving these problems. During the western blotting, there could be several problems related to the different steps of this procedure. Those problems could originate from

8772-495: The body that bear no relation to the original tick bite. Transient muscle pains and joint pains are also common. In about 10–15% of untreated people, Lyme causes neurological problems known as neuroborreliosis . Early neuroborreliosis typically appears 4–6 weeks (range 1–12 weeks) after the tick bite and involves some combination of lymphocytic meningitis, cranial neuritis, radiculopathy, and/or mononeuritis multiplex. Lymphocytic meningitis causes characteristic changes in

8901-621: The body's tissues. Chronic symptoms from an autoimmune reaction could explain why some symptoms persist even after the spirochetes have been eliminated from the body. This hypothesis may explain why chronic arthritis persists after antibiotic therapy, similar to rheumatic fever , but its wider application is controversial. Lyme disease is diagnosed based on symptoms, objective physical findings (such as erythema migrans (EM) rash, facial palsy , or arthritis ), history of possible exposure to infected ticks , and possibly laboratory tests . People with symptoms of early Lyme disease should have

9030-443: The buffer is leading the process of protein solubilization and preventing protein degradation. After completing the sample preparation, the protein content is ready to be separated by the utilization of gel electrophoresis. The proteins of the sample are separated using gel electrophoresis . Separation of proteins may be by isoelectric point (pI), molecular weight , electric charge, or a combination of these factors. The nature of

9159-447: The case of aptamers , or from non-immunoglobulin protein scaffolds / peptide aptamers , into which hypervariable loops are inserted to form the antigen binding site. Constraining the hypervariable binding loop at both ends within the protein scaffold improves the binding affinity and specificity of the synthetic antibody to levels comparable to or exceeding that of a natural antibody. Common advantages of these molecules compared to use of

9288-422: The case of HGA—can be treated with the doxycycline prescribed for Lyme. Persistent fever or compatible anomalous laboratory findings may be indicative of a co-infection. B. burgdorferi can spread throughout the body during the course of the disease, and has been found in the skin, heart, joints, peripheral nervous system, and central nervous system. B. Burgdorferi does not produce toxins. Therefore, many of

9417-426: The cell and their functions are closely related. The relationship between protein function and localization suggests that when proteins move, their functions may change or acquire new characteristics. A protein's subcellular placement can be determined using a variety of methods. Numerous efficient and reliable computational tools and strategies have been created and used to identify protein subcellular localization. With

9546-409: The data implicating lizards is based on DNA detection of the spirochete and has not demonstrated that lizards are able to infect ticks feeding upon them. As some experiments suggest lizards are refractory to infection with Borrelia , it appears likely their involvement in the enzootic cycle is more complex and species-specific. In Europe, the main vector is Ixodes ricinus , which is also called

9675-544: The denaturing electrophoretic separation of proteins. Sodium dodecyl sulfate (SDS) is generally used as a buffer (as well as in the gel) in order to give all proteins present a uniform negative charge, since proteins can be positively, negatively, or neutrally charged. Prior to electrophoresis, protein samples are often boiled to denature the proteins present. This ensures that proteins are separated based on size and prevents proteases (enzymes that break down proteins) from degrading samples. Following electrophoretic separation,

9804-419: The detection bands. The protein in the dilute solution attaches to the membrane in all places where the target proteins have not attached. Thus, when the antibody is added, it cannot bind to the membrane, and therefore the only available binding site is the specific target protein. This reduces background in the final product of the western blot, leading to clearer results, and eliminates false positives. During

9933-458: The detection process, the membrane is "probed" for the protein of interest with a modified antibody which is linked to a reporter enzyme; when exposed to an appropriate substrate, this enzyme drives a colorimetric reaction and produces a colour. For a variety of reasons, this traditionally takes place in a two-step process, although there are now one-step detection methods available for certain applications. The primary antibodies are generated when

10062-438: The detection process. Given the advent of high-throughput protein analysis and lower limits of detection, however, there has been interest in developing one-step probing systems that would allow the process to occur faster and with fewer consumables. This requires a probe antibody which both recognizes the protein of interest and contains a detectable label, probes which are often available for known protein tags . The primary probe

10191-439: The diseases babesiosis and human granulocytic anaplasmosis (HGA), respectively. Among people with early Lyme disease, depending on their location, 2–12% will also have HGA and 2–10% will have babesiosis. Ticks in certain regions also transmit viruses that cause tick-borne encephalitis and Powassan virus disease . Co-infections of Lyme disease may not require additional treatment, since they may resolve on their own or—as in

10320-406: The fastest growing class of therapeutics. Lyme disease Lyme disease , also known as Lyme borreliosis , is a tick-borne disease caused by species of Borrelia bacteria, transmitted by blood-feeding ticks in the genus Ixodes . The most common sign of infection is an expanding red rash , known as erythema migrans (EM), which appears at the site of the tick bite about

10449-497: The first manifestation of Lyme disease. Lyme carditis in 19–87% of people adversely impacts the heart's electrical conduction system, causing atrioventricular block that often manifests as heart rhythms that alternate within minutes between abnormally slow and abnormally fast. In 10–15% of people, Lyme causes myocardial complications such as cardiomegaly , left ventricular dysfunction, or congestive heart failure. Another skin condition, found in Europe but not in North America,

10578-541: The gel – the greater the acrylamide concentration, the better the resolution of lower molecular weight proteins. The lower the acrylamide concentration, the better the resolution of higher molecular weight proteins. Proteins travel only in one dimension along the gel for most blots. Samples are loaded into wells in the gel. One lane is usually reserved for a marker or ladder , which is a commercially available mixture of proteins of known molecular weights, typically stained so as to form visible, coloured bands. When voltage

10707-533: The high molecular weight region because some proteins form dimers, trimers, and multimers; this issue might be solved by heating the sample for longer periods of time. Proteins with post-translational modifications (PTMs) or numerous isoforms cause several bands to appear at various molecular weight areas. PTMs can be removed from a specimen using specific chemicals, which also remove extra bands. Strong antibody concentrations, inadequate blocking, inadequate washing, and excessive exposure time during imaging can result in

10836-514: The laboratory. The most widely used tests look instead for presence of antibodies against those bacteria in the blood. A positive antibody test result does not by itself prove active infection but can confirm an infection that is suspected because of symptoms, objective findings, and history of tick exposure in a person. Because as many as 5–20% of the normal population have antibodies against Lyme, people without history and symptoms suggestive of Lyme disease should not be tested for Lyme antibodies:

10965-415: The life cycle of a tick - larva, nymph, and adult. During the nymph stage, ticks most frequently transmit Lyme disease and are usually most active in late spring and early summer in regions where the climate is mild. During the adult stage, Lyme disease transmission is less common because adult ticks are less likely to bite humans and tend to be larger in size, so can be easily seen and removed. Lyme disease

11094-748: The lower limbs may occur. In European children, the most common manifestations are facial palsy (in 55%), other cranial neuritis, and lymphocytic meningitis (in 27%). In about 4–10% of untreated cases in the United States and 0.3–4% of untreated cases in Europe, typically between June and December, about one month (range 4 days to 7 months) after the tick bite, the infection may cause heart complications known as Lyme carditis . Symptoms may include heart palpitations (in 69% of people), dizziness , fainting , shortness of breath , and chest pain . Other symptoms of Lyme disease may also be present, such as EM rash, joint aches , facial palsy , headaches , or radicular pain . In some people, however, carditis may be

11223-445: The membrane and protein. Nitrocellulose membranes are cheaper than PVDF, but are far more fragile and cannot withstand repeated probings. Total protein staining allows the total protein that has been successfully transferred to the membrane to be visualised, allowing the user to check the uniformity of protein transfer and to perform subsequent normalization of the target protein with the actual protein amount per lane. Normalization with

11352-448: The membrane under gentle agitation for typically an hour at room temperature, or overnight at 4 ° C. It can also be incubated at different temperatures, with lesser temperatures being associated with more binding, both specific (to the target protein, the "signal") and non-specific ("noise"). Following incubation, the membrane is washed several times in wash buffer to remove unbound primary antibody, and thereby minimize background. Typically,

11481-428: The membrane will be stained with a secondary antibody that recognizes the first antibody staining, which can then be used for detection by a variety of methods. The gel electrophoresis step is included in western blot analysis to resolve the issue of the cross-reactivity of antibodies. As a significant step in conducting a western blot, sample preparation has to be done effectively since the interpretation of this assay

11610-401: The membrane. Nevertheless, post-antibody stainings have been described as well. Since the membrane has been chosen for its ability to bind protein and as both antibodies and the target are proteins, steps must be taken to prevent the interactions between the membrane and the antibody used for detection of the target protein. Blocking of non-specific binding is achieved by placing the membrane in

11739-442: The most common presentation is a combination of lymphocytic meningitis and radiculopathy known as Bannwarth syndrome , accompanied in 36-89% of cases by facial palsy. In this syndrome, radicular pain tends to start in the same body region as the initial erythema migrans rash, if there was one, and precedes possible facial palsy and other impaired movement . In extreme cases, permanent impairment of motor or sensory function of

11868-403: The negatively charged SDS, effectively becoming anionic , and migrate towards the positively charged (higher voltage) anode (usually having a red wire) through the acrylamide mesh of the gel. Smaller proteins migrate faster through this mesh, and the proteins are thus separated according to size (usually measured in kilodaltons, kDa ). The concentration of acrylamide determines the resolution of

11997-466: The negatively charged proteins from the gel towards the positively charged anode, and into the PVDF or NC membrane. The proteins move from within the gel onto the membrane while maintaining the organization they had within the gel. An older method of transfer involves placing a membrane on top of the gel, and a stack of filter papers on top of that. The entire stack is placed in a buffer solution which moves up

12126-484: The next most common symptoms. In children, partial loss of vision may also occur. Cranial neuritis is an inflammation of cranial nerves . When due to Lyme, it most typically causes facial palsy , impairing blinking, smiling, and chewing on one or both sides of the face. It may also cause intermittent double vision . Lyme radiculopathy is an inflammation of spinal nerve roots that often causes pain and less often weakness , numbness , or altered sensation in

12255-474: The paper by capillary action , bringing the proteins with it. In practice this method is not commonly used due to the lengthy procedure time. As a result of either transfer process, the proteins are exposed on a thin membrane layer for detection. Both varieties of membrane are chosen for their non-specific protein binding properties (i.e. binds all proteins equally well). Protein binding is based upon hydrophobic interactions, as well as charged interactions between

12384-608: The problems associated with western blotting, several different techniques have been developed and utilized, such as far-western blotting , diffusion blotting, single-cell resolution western blotting , and automated microfluidic western blotting . Researchers use different software to process and align image-sections for elegant presentation of western blot results. Popular tools include Sciugo , Microsoft PowerPoint , Adobe Illustrator and GIMP . Synthetic antibody Synthetic antibodies are affinity reagents generated entirely in vitro, thus completely eliminating animals from

12513-451: The process. There are two types of membrane: nitrocellulose (NC) or polyvinylidene difluoride (PVDF ). NC membrane has high affinity for protein and its retention abilities. However, NC is brittle, and does not allow the blot to be used for re-probing, whereas PVDF membrane allows the blot to be re-probed. The most commonly used method for transferring the proteins is called electroblotting . Electroblotting uses an electric current to pull

12642-795: The production process. Synthetic antibodies include recombinant antibodies , nucleic acid aptamers and non-immunoglobulin protein scaffolds. As a consequence of their in vitro manufacturing method the antigen recognition site of synthetic antibodies can be engineered to any desired target and may extend beyond the typical immune repertoire offered by natural antibodies. Synthetic antibodies are being developed for use in research, diagnostic and therapeutic applications. Synthetic antibodies can be used in all applications where traditional monoclonal or polyclonal antibodies are used and offer many inherent advantages over animal-derived antibodies, including comparatively low production costs, reagent reproducibility and increased affinity, specificity and stability across

12771-408: The protein bands of interest (see image above). The importance of radioactive detections methods is declining due to its hazardous radiation , because it is very expensive, health and safety risks are high, and ECL (enhanced chemiluminescence) provides a useful alternative. The fluorescently labeled probe is excited by light and the emission of the excitation is then detected by a photosensor such as

12900-400: The proteins are transferred to a membrane (typically nitrocellulose or PVDF). The membrane is often then stained with Ponceau S in order to visualize the proteins on the blot and ensure a proper transfer occurred. Next the proteins are blocked with milk (or other blocking agents) to prevent non-specific antibody binding, and then stained with antibodies specific to the target protein. Lastly,

13029-404: The proteins from a single sample out in two dimensions. Proteins are separated according to isoelectric point ( pH at which they have a neutral net charge) in the first dimension, and according to their molecular weight in the second dimension. To make the proteins accessible to antibody detection, they are moved from within the gel onto a membrane, a solid support, which is an essential part of

13158-512: The reaction of peroxide radicals with 4-chloronaphthol produces a dark purple stain that can be photographed without using specialized photographic film. As with the ELISPOT and ELISA procedures, the enzyme can be provided with a substrate molecule that will be converted by the enzyme to a coloured reaction product that will be visible on the membrane (see the figure below with blue bands). Another method of secondary antibody detection utilizes

13287-442: The relative amount of protein staining and quantifies the results in terms of optical density. Newer software allows further data analysis such as molecular weight analysis if appropriate standards are used. Radioactive labels do not require enzyme substrates, but rather, allow the placement of medical X-ray film directly against the western blot, which develops as it is exposed to the label and creates dark regions which correspond to

13416-602: The removed tick is full of blood a single dose of doxycycline may be used to prevent the development of infection but is not generally recommended since the development of infection is rare. If an infection develops, a number of antibiotics are effective, including doxycycline, amoxicillin and cefuroxime . Standard treatment usually lasts for two or three weeks. Some people develop a fever and muscle and joint pains from treatment, which may last for one or two days. In those who develop persistent symptoms, long-term antibiotic therapy has not been found to be useful. Lyme disease

13545-478: The results of Western blots are reproducible, it is important to report the various parameters mentioned above, including specimen preparation, the concentration of protein used for loading, the percentage of gel and running condition, various transfer methods, attempting to block conditions, the concentration of antibodies, and identification and quantitative determination methods. Many of the articles that have been published don't cover all of these variables. Hence, it

13674-565: The same CSF signs; they confirm a diagnosis of central nervous system (CNS) neuroborreliosis if positive, but do not exclude it if negative. American guidelines consider CSF analysis optional when symptoms appear to be confined to the peripheral nervous system (PNS), e.g. facial palsy without overt meningitis symptoms. Unlike blood and intrathecal antibody tests, CSF pleocytosis tests revert to normal after infection ends and therefore can be used as objective markers of treatment success and inform decisions on whether to retreat. In infection involving

13803-537: The second dimension, polypeptides are separated according to their molecular weight . The isoelectric point of a given protein is determined by the relative number of positively (e.g. lysine, arginine) and negatively (e.g. glutamate, aspartate) charged amino acids, with negatively charged amino acids contributing to a low isoelectric point and positively charged amino acids contributing to a high isoelectric point. Samples could also be separated first under nonreducing conditions using SDS-PAGE, and under reducing conditions in

13932-438: The second dimension, which breaks apart disulfide bonds that hold subunits together. SDS-PAGE might also be coupled with urea-PAGE for a 2-dimensional gel. In principle, this method allows for the separation of all cellular proteins on a single large gel. A major advantage of this method is that it often distinguishes between different isoforms of a particular protein – e.g. a protein that has been phosphorylated (by addition of

14061-405: The secondary antibody. This converts the soluble dye into an insoluble form of a different colour that precipitates next to the enzyme and thereby stains the membrane. Development of the blot is then stopped by washing away the soluble dye. Protein levels are evaluated through densitometry (how intense the stain is) or spectrophotometry . Chemiluminescent detection methods depend on incubation of

14190-580: The separation depends on the treatment of the sample and the nature of the gel. By far the most common type of gel electrophoresis employs polyacrylamide gels and buffers loaded with sodium dodecyl sulfate (SDS). SDS-PAGE (SDS-polyacrylamide gel electrophoresis) maintains polypeptides in a denatured state once they have been treated with strong reducing agents to remove secondary and tertiary structure (e.g. disulfide bonds [S-S] to sulfhydryl groups [SH and SH]) and thus allows separation of proteins by their molecular mass . Sampled proteins become covered in

14319-601: The sheep tick or castor bean tick. In China, Ixodes persulcatus (the taiga tick) is probably the most important vector. In North America, the black-legged tick or deer tick ( Ixodes scapularis ) is the main vector on the East Coast. The lone star tick ( Amblyomma americanum ), which is found throughout the Southeastern United States as far west as Texas , is unlikely to transmit the Lyme disease spirochetes , though it may be implicated in

14448-412: The signal, allowing the detection of proteins of a much lower concentration than would be visible by SDS-PAGE alone. Horseradish peroxidase is commonly linked to secondary antibodies to allow the detection of the target protein by chemiluminescence . The chemiluminescent substrate is cleaved by horseradish peroxidase, resulting in the production of luminescence . Therefore, the production of luminescence

14577-412: The signs and symptoms of Lyme disease are a consequence of the immune response to spirochete in those tissues. B. burgdorferi is injected into the skin by the bite of an infected Ixodes tick. Tick saliva, which accompanies the spirochete into the skin during the feeding process, contains substances that disrupt the immune response at the site of the bite. This provides a protective environment where

14706-403: The size of a poppy seed and sometimes with a dark head and a translucent body. Or, the nymphal ticks can be darker. The younger larval ticks are very rarely infected. Although deer are the preferred hosts of adult deer ticks, and tick populations are much lower in the absence of deer, ticks generally do not acquire Borrelia from deer, instead they obtain them from infected small mammals such as

14835-516: The so-called "loading control" was based on immunostaining of housekeeping proteins in the classical procedure, but is heading toward total protein staining recently, due to multiple benefits. At least seven different approaches for total protein staining have been described for western blot normalization: Ponceau S , stain-free techniques, Sypro Ruby, Epicocconone , Coomassie R-350 , Amido Black , and Cy5 . In order to avoid noise of signal, total protein staining should be performed before blocking of

14964-512: The species-specific portion of the primary antibody. Therefore, an anti-mouse secondary antibody will bind to almost any mouse-sourced primary antibody, and can be referred to as an 'anti-species' antibody (e.g. anti-mouse, anti-goat etc.). To allow detection of the target protein, the secondary antibody is commonly linked to biotin or a reporter enzyme such as alkaline phosphatase or horseradish peroxidase . This means that several secondary antibodies will bind to one primary antibody and enhance

15093-425: The spirochete can establish infection. The spirochetes multiply and migrate outward within the dermis . The host inflammatory response to the bacteria in the skin causes the characteristic circular EM lesion. Neutrophils , however, which are necessary to eliminate the spirochetes from the skin, fail to appear in necessary numbers in the developing EM lesion because tick saliva inhibits neutrophil function. This allows

15222-469: The spirochete. If untreated, the bacteria may persist in the body for months or even years, despite the production of B. burgdorferi antibodies by the immune system. The spirochetes may avoid the immune response by decreasing expression of surface proteins that are targeted by antibodies, antigenic variation of the VlsE surface protein, inactivating key immune components such as complement , and hiding in

15351-450: The supplier's data sheet. Increasing the exposition period in the detection system's software can address weak bands caused by lower sample and antibody concentrations. When the protein is broken down by proteases, several bands other than predicted bands of low molecular weight might appear. The development of numerous bands can be prevented by properly preparing protein samples with enough protease inhibitors. Multiple bands might show up in

15480-469: The symptoms, and many of these symptoms can also occur with other diseases. Asymptomatic infection exists, but occurs in less than 7% of infected individuals in the United States. Asymptomatic infection may be much more common among those infected in Europe. Within days to weeks after the onset of local infection, the Borrelia bacteria may spread through the lymphatic system or bloodstream. In 10–20% of untreated cases, EM rashes develop at sites across

15609-459: The synthetic antibody family. The Affimer protein scaffold is derived from the cysteine protease inhibitor family of cystatins. Within the protein scaffold there exist two variable peptide loops and a variable N-terminal sequence that provide a high affinity binding surface for the specific target protein. Affimer binders have been produced to a large number of targets including ubiquitin chains, immunoglobulins and C-reactive protein for use in

15738-536: The target protein is referred to as "epitope mapping." Finding the binding epitope of an antibody is essential for the discovery and creation of novel vaccines, diagnostics, and therapeutics. As a result, various methods for mapping antibody epitopes have been created. At this point, western blotting's specificity is the main feature that sets it apart from other epitope mapping techniques. There are several application of western blot for epitope mapping on human skin samples, hemorrhagic disease virus. The western blot method

15867-402: The total protein visualized with trichloroethanol or epicocconone . This practice ensures correction for the amount of total protein on the membrane in case of errors or incomplete transfers. (see western blot normalization ) The colorimetric detection method depends on incubation of the western blot with a substrate that reacts with the reporter enzyme (such as peroxidase ) that is bound to

15996-410: The typical antibody structure include a smaller size, giving improved tissue penetration, rapid generation times of weeks compared to months for natural and recombinant antibodies and cheaper costs. Affimer proteins are small robust affinity reagents, with a molecular weight of 12–14kDa. They are engineered to bind to their target proteins with high affinity and specificity and as such are a member of

16125-412: The wash buffer solution is composed of buffered saline solution with a small percentage of detergent, and sometimes with powdered milk or BSA. After rinsing the membrane to remove unbound primary antibody, the membrane is exposed to another antibody known as the secondary antibody . Antibodies come from animal sources (or animal sourced hybridoma cultures). The secondary antibody recognises and binds to

16254-538: The western blot technique include its use by the World Anti-Doping Agency (WADA). Blood doping is the misuse of certain techniques and/or substances to increase one's red blood cell mass, which allows the body to transport more oxygen to muscles and therefore increase stamina and performance. There are three widely known substances or methods used for blood doping, namely, erythropoietin (EPO), synthetic oxygen carriers and blood transfusions. Each

16383-415: The western blot with a substrate that will luminesce when exposed to the reporter on the secondary antibody. The light is then detected by CCD cameras which capture a digital image of the western blot or photographic film. The use of film for western blot detection is slowly disappearing because of non linearity of the image (non accurate quantification). The image is analysed by densitometry, which evaluates

16512-445: The western blot's ability to detect antibodies against F. tularensis revealed that its sensitivity is almost 100% and the specificity is 99.6%. Some forms of Lyme disease testing employ western blotting. A western blot can also be used as a confirmatory test for Hepatitis B infection and HSV-2 (Herpes Type 2) infection. In veterinary medicine, a western blot is sometimes used to confirm FIV + status in cats. Further applications of

16641-417: Was available. A vaccine, LYMERix, was produced, but discontinued in 2002 due to insufficient demand. There are several vaccines for the prevention of Lyme disease in dogs. Lyme disease can produce a broad range of symptoms. The incubation period is usually one to two weeks, but can be much shorter (days) or much longer (months to years). Lyme symptoms most often occur from the month of May to September in

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