January 2010
This tumor marker resembles albumin except that it has a prominent carbohydrate chain. The addition of a fucose (red) near the link to the protein makes it a much more specific marker for neoplastic (as opposed to inflammatory) lesions of the liver. Can you guess what it is?
Alpha-fetoprotein (AFP)
AFP is the fetal version of albumin. The fetal liver begins to switch from AFP to albumin soon before birth and, although serum AFP levels are very high in neonates, they reach adult levels by about 6 months of age. (The high levels of AFP in the fetus explain the use of this test – in maternal serum and amniotic fluid – as a prenatal screen for neural tube defect.) As a tumor marker, AFP is primarily used to detect and follow patients with hepatocellular carcinoma, although it may also be expressed by a variety of germ cell tumors. Because AFP levels are elevated in other hepatic disorders, especially chronic viral hepatitis, its usefulness for detection of hepatocellular carcinoma may be limited. Recent findings have shown that the presence of the AFP isoform (called “L3”), with fucose added to the single carbohydrate chain, may be specific for hepatic tumors.
December 2009
This molecule is often used to treat a disorder associated with chronic renal failure but autoantibodies against it can make the disorder worse. Recently, an unrelated agonist has been shown to help patients with this complication. Can you guess what it is?
Erythropoietin
Recombinant human erythropoietin usually successfully treats the anemia associated with chronic renal failure. Rarely, however, autoantibodies may develop against erythropoietin and these can produce red-cell aplasia, a serious condition. European investigators recently reported that a synthetic peptide was able to interact with the receptor for erythropoietin and stimulate red blood cell production in such patients. The peptide is able to stimulate the receptor but is different enough from erythropoietin that it is not recognized by the anti-erythropoietin autoantibodies.
November 2009
This type of display of antibody binding was discussed during the CDID session at the annual CLAS meeting, featured in the new Fall Immunotes on-line now. Can you guess what it is?
Microarray Heatmap
In a technique analogous to the use of differently colored cDNA (made from either test sample or reference mRNA) hybridization to a DNA microarray, antibody binding to multiple antigens can be studied. This is a portion of an array of antigens (a different antigen is in each row) interacted with sera from different animals (a different animal is in each column). The color represents the amount of antibody (using fluorescent-labelled anti-immunoglobulin). The rows and columns are ordered by hierarchical cluster analysis to reveal patterns in the data. Wild type (WT) differs from mutant for a number of antibodies, and tree branches show relationships among samples and antigens.
October 2009
This month’s Clinical Chemistry includes a description of an immunoassay for this protein, which is crucial for B cell survival. Can you guess what it is?
BAFF (B cell-activating factor of the TNF family)
This molecule is a member of the tumor necrosis factor (TNF) family of cytokines and it regulates the growth and survival of B cells. There have been conflicting reports regarding whether levels of this cytokine are elevated in autoimmune disorders and the report in last month’s Clinical Chemistry (by Le Pottier et al) describes how the discrepancies are likely to be do to variability in the way in which different immunoassay antibodies recognize differently glycosylated forms of the protein.
September 2009
The autoantibody producing this ANA pattern was missed by ELISA and delayed the diagnosis in the case which opened our 2009 “Issues in Immunodiagnostics”. Can you guess what it is?
Anti-sp100
Dr. Donald Bloch (a rheumatologist at Massachusetts General Hospital in Boston) argued that the ANA is not a test to establish the diagnosis of a particular autoimmune disease but, rather, a screen for the possibility of autoimmune disease. He was critical of immunoassays that only include some specific antigens, and presented a case in which the ELISA ANA was negative but the immunofluorescent ANA (finally obtained after the patient saw several different rheumatologists) was positive because of this relatively unusual pattern. The nuclear dots are seen because of antibodies to sp100, one of several proteins associated with these nuclear structures, whose exact function is still not known. These autoantibodies may be seen in patients with primary biliary cirrhosis (the diagnosis in the case presented) and may be helpful when the test for anti-mitochondrial antibody is negative (which may occur in a minority of PBC patients). To be fair, Dr. Bloch did not mention whether the anti-mitochondrial antibody test had been performed. Also, this pattern is not specific for PBC. But the case was certainly food for thought about using immunoassay screens for “ANA” in place of indirect immunofluorescence.
August 2009
This molecule was first identified using an antibody but we now know that it functions as a primitive antigen recognition protein. Can you guess what it is?
Toll-like receptor
Toll-like receptors (TLRs) are an evolutionarily ancient family of pattern recognition molecules which play a major role in innate immunity. They recognize bacterial lipoproteins on the surface of the cell and bacterial nucleic acid inside of lysosomes. Shown is the leucine-rich portion that appears on the membrane. This is connected to a cytoplasmic domain that can initiate signaling to activate the cell. In the case of a macrophage, this will allow the cell to produce inflammatory cytokines.
July 2009
The accuracy of immunoassays using antibodies against this molecule is investigated using tandem mass spectrometry in this month’s issue of Clinical Chemistry. Can you guess what it is?
(Free) Thyroxine
Investigators at Georgetown University and Children’s National Medical Cenetr in Washington DC reported in last month’s issue that free T4 determinations by LC/MSMS (after ultrafiltration) correlated better with TSH than did immunoassay free T4 results. They looked at samples from several different populations including pregnant women, a group in which free T4 results have been controversial.
June 2009
Everyone’s been talking about this organism and wondering if there is time to develop a vaccine to stimulate antibodies against it. Can you guess what it is?
H1N1 subtype of influenza A virus
The world’s attention has been on the outbreak of so-called “Swine flu”, recently declared a pandemic by World Health Organization – the first in 40 years. The Swiss pharmaceutical company Novartis AG recently announced that it had successfully manufactured a vaccine, making it likely that there will be supplies ready by the fall – in time for the start of the new flu season.
May 2009
This enzyme, once the target of a popular antibody-based test for prostate cancer eventually replaced by PSA, is now part of a modern immunotherapy approach to treating prostate cancer. Can you guess what it is?
Prostatic acid phosphatase (PAP)
This enzyme was the original tumor marker for prostate adenocarcinoma. Although enzymatic activity continued to be the method of choice, immunoassays for PAP were once commercially available. Both types of assays for PAP have all but been replaced by prostate-specific antigen (PSA). A new approach to therapy of prostate cancer is the use of autologous antigen-presenting cells pulsed ex vivo with a recombinant fusion protein consisting of granulocyte-monocyte colony stimulating factor and PAP. This new commercially available product (Provenge) is the first immunotherapy regimen to receive approval from the Food and Drug Administration.
April 2009
At last month’s AAAAI meeting, a study showed that administration of very small amounts of this protein over time helped children with severe allergy. Can you guess what it is?
Cupin (Peanut allergen)
The “cupins” are a superfamily of proteins with a beta-barrel core domain (shown in red). They include two types of proteins (vicilins and legumins) which represent the common allergens in peanuts. Investigators at Duke University recently reported that children enrolled in an open-label trial of oral immunotherapy using such peanut proteins have shown significantly decreased levels of IgE specific anti-peanut antibody.
March 2009
This month’s Clinical Chemistry includes a study looking at antibodies against larger aggregates of this hormone, secreted exclusively by fat cells. Can you guess what it is?
Adiponectin
Pictured is the simplest form of this hormone made by adipose cells, consisting of three peptide monomers. Adiponectin is a “good” cytokine (analogous to HDL as the “good” lipoprotein particle) because it appears to counteract the effects of pro-inflammatory cytokines found in patients with insulin resistance and the “metabolic syndrome”. Measuring adiponectin may have a future role in cardiovascular risk modification and the report by Liu et al in the March Clinical Chemistry seemed to support the use of commercially available ELISAs. However, more work must be done to analyze the way in which more complicated aggregates of the protein (including “high molecular weight” adiponectin) may affect these assays.
February 2009
Lack of antibody production against the organism growing around the beta-hemolytic Staphylococcus on this plate has resulted in the potential return of a disease long thought conquered. Can you guess what it is?
Hemophilus influenzae
Five children were sickened in Minnesota last month by H. influenzae, type B (Hib) and one died. Three of the children (including the one who died) had not received any vaccinations, raising concerns about the recent tendency for parents to decline vaccination. There is also currently a shortage of Hib vaccine. One of the characteristics of Hib is its requirement for certain nutrients released from blood agar by other bacteria, hence its growth around the “staph streak” on this plate.
January 2009
This molecule is the target of an antibody that causes a common thyroid disorder but measurement of its activity in the clinical laboratory is controversial, as shown by a report in the January issue of Clinical Chemistry. Can you guess what it is?
TSH receptor
Graves disease is caused by an autoantibody to the receptor on thyroid cells for thyrotropin (or thyroid stimulating hormone, TSH). When the antibody binds to the receptor, it mimics the action of TSH, producing increased production and release of thyroid hormone. Anti-TSH receptor may be detected by the ability of antibodies in the patient’s serum to block the ability of labeled TSH bind to bind to receptors in vitro (so-called “blocking” antibodies) or by the ability of antibodies in the patient’s serum to stimulate cultured thyroid cells (so-called “stimulating” antibodies). Catherine Massart and her colleagues looked at several commercially available assays for blocking antibody in the January issue of Clinical Chemistry (55:183-186, 2009) and they found poor precision and significant variability.
December 2008
The microorganism in this lung biopsy has provoked an exaggerated immune response which may be diagnosed by finding high levels of both IgE and IgG antibodies. Can you guess what it is?
Aspergillus
Allergic bronchopulmonary aspergillosis (ABPA) refers to a condition characterized by an unusual immunologic reaction to inhaled antigens of Aspergillus species (often in patients with asthma) that results in bronchial wall damage and bronchiectasis. The diagnosis is a clinical one but laboratory testing may offer support for ruling it in or excluding it. Helpful laboratory evidence includes a high level of IgE anti-Aspergillus antibody; a high level of total IgE; and the presence of IgG anti-Aspergillus antibody, detected by either immunodiffusion or immunoassay.
November 2008
The diagnostic significance of antibodies to this molecule has recently been questioned and this controversy is discussed in the Fall Immunotes CDID newsletter. Can you guess what it is?
Cardiolipin
Check out the on-line Fall 2008 CDID newsletter to see why some experts believe that antibodies against other phospholipids may be more important in the diagnosis and management of anti-phospholipid antibody syndrome.
October 2008
The molecule at the bottom, which results from the action of tissue transglutaminase, may be a better target for an assay that measures antibodies in celiac disease. Can you guess what it is?
Deamidated Gliadin Peptide
Celiac disease is an autoimmune disorder of the small intestine in which lymphocytes damage the absorptive villi, causing blunting and malabsorption. The pathogenesis involves presentation of peptides derived from gliadin, a protein found in wheat and other grains. Deamidation of the peptides helps this process to be more effective. Although certain major histocompatibility complex (MHC) proteins are required to properly present these dangerous peptides, other genetic (and possibly environmental) factors are also responsible. Autoantibodies develop against the enzyme which catalyzes the deamidation reaction (tissue transglutaminase) and these are considered very sensitive and specific for the diagnosis. Previously, antibodies to gliadin have not been considered specific as these may develop in patients with malabsorption due to other causes. However, recent studies have shown that antibodies specific for the deamidated peptides may be useful in celiac disease.
September 2008
This month’s issue of Clinical Chemistry features a reprint of a “classic” report describing an immunoassay for the molecule depicted in red in this cartoon. Can you guess what it is?
Troponin T
Dr. Hugo Katus described the discovery of cardiac-specific troponin T, which anchors the troponin complex to tropomyosin (the cord-like structure pictured in blue). During depolarization, calcium binds to troponin C and troponin I (the yellow structure) moves out of the way, allowing actin (orange) to interact with myosin (pink). This generates contraction of the muscle fiber. The original troponin T assay provided results in 90 minutes; modern advances have allowed manufacturers to create assays for cardiac-specific troponin T (and troponin I) that reduce the turnaround time to 20 minutes or less.
August 2008
Amgen recently announced the success of a clinical trial of denosumab, a monoclonal antibody directed against the molecule which helps produce the multi-nucleated osteoclast pictured. Can you guess what it is?
RANK ligand
New bone formation requires initial bone resorption (similar to digging out loose plaster before respackling dry-wall board). When the balance between resorption and formation is disturbed, osteoporosis results. Current drugs like bisphosphonates try to poison the osteoclast to prevent excessive bone resorption. Amgen’s new monoclonal antibody inhibits the molecule (RANK ligand) which promotes osteoclast development and activates osteoclasts. It binds to the “receptor activator of nuclear factor kappa B”.
July 2008
Alpha-1-Protease Inhibitor
The CDID award for best abstract at this year’s AACC meeting will be presented to a group who looked at a new way to measure this molecule complexed to a common tumor marker. Can you guess what it is?
Prostate-specific antigen (PSA) is a serine protease inactivated by serine protease inhibitors. Most of the focus on complexed (or, alternatively, free) PSA has been on its binding to anti-chymotrypsin. Recently, investigators at University of Helsinki looked at PSA bound to alpha-1-protease inhibitor (also known as alpha-1-antitrypsin). They employed proximity ligation assay (immunoassay using oligonucleotides and PCR). At the 2008 Issues in Immunodiagnostics symposium, an award was presented to lead author Lei Zhu.
June 2008
If you discover this bug on your arm this month, your doctor will order a laboratory test looking for antibodies against a particularly devious spirochete. Can you guess what it is?
Borrelia burgdorferi
Borreliosis (“Lyme disease”) is an infectious disease caused by a spirochete bacterium transferred via the bite of the deer tick (shown is Ixodes dammini, the tick responsible for infections in the eastern part of the U.S.). Identifying antibodies against B. burgdorferi is an important part of the diagnosis and this serology test is an especially challenging one. The test only positive in a minority of patients soon after exposure and then, when either neurological or joint symptoms begin, positive results need to be confirmed by Western blot. Wear long pants while hiking this summer!
May 2008
This antigen will be coming your way this month and we hope that you don’t have IgE antibodies against it. Can you guess what it is?
Oak Tree Pollen
The vaguely triangular shape and the “warty” granular surface identifies this as the pollen of the oak family of trees. Oak trees are present in most of the U.S. and they shed enormous amounts of pollen, making them an important cause of seasonal allergy. If you know that you are not allergic to tree pollen, watch out for grass (June and July) and weed pollen (September and October) later this year. If you are interested in allergic disease, check out the report of the 2008 meeting of the AAAAI (the major organization in the U.S. of allergists) in the new issue of our on-line CDID newsletter “Immunotes” sometime this month.
March 2008
Investigators look for antibodies to this enzyme in patients with inflammatory bowel disease in this month’s issue of Clinical Chemistry. Can you guess what it is?
Alpha-enolase
A number of antibodies (both autoantibodies and antibodies to foreign antigens) are associated with inflammatory bowel disease (ulcerative colitis and Crohn’s disease). Two commonly described ones include the autoantibody that produces an “atypical” perinuclear ANCA (anti-neutrophil cytoplasmic antibody) pattern and the antibody against the yeast Saccharomyces cerevisiae (ASCA). Vermeulen et al followed up on recent findings that these patients had antibodies to alpha-enolase (a small enzyme involved in the glycolytic pathway) but their presence in healthy controls and patients with other gastrointestinal disorders precludes use of these antibodies as a diagnostic test (see Clin Chem 2008; 54:534-541.)
February 2008
Certain versions of this relatively common autoantibody can produce this appearance when serum is chilled. Can you guess what it is?
Cryoprecipitable Rheumatoid Factor
Many patients infected with hepatitis C virus develop an abnormal immune response that includes the presence of rheumatoid factor (autoantibody against IgG) with a peculiar physicochemical property. When the temperature is lowered, it denatures and comes out of solution. The denaturation is reversible (it instantly goes back into solution once the temperature is raised) and concentration-dependent (at higher concentrations, denaturation occurs at higher temperatures). In some patients, the rheumatoid factor response may even become monoclonal.
January 2008
Like Janus, the two-faced Roman god, a particular anti-nuclear antibody can produce this two-faced appearance, with some cells positive and others negative. Can you guess what it is?
Proliferating Cell Nuclear Antigen (PCNA)
Autoantibodies to PCNA exhibit the speckled immunofluorescence pattern only on some Hep2 cells because the antigen expression occurs during late G1 and early S phase of the cell cycle just before DNA synthesis. Autoantibodies to PCNA are detected in the sera of 3–5% of patients with SLE. Anti-PCNA antibody has not been detected in other autoimmune diseases and is thought to be specific for SLE.
December 2007
IgE antibodies against this mold may be responsible for serious allergic disease which is exacerbated during winter months because of overheating of air-tight homes. Can you guess what it is? (Answer next month)
Stachybotrys
Recently, attention has been given to the possibility that fungal growth in indoor environments (especially with water damage) can produce serious disorders in the building’s occupants. The symptoms of so-call “sick building syndrome” vary, but respiratory disorders attributed to allergic reactions predominate. When air samples have been tested, in addition to common indoor fungi such as Penicillium, Cladosporium and Aspergillus, a high percentage of Stachybotrys has been detected. This fungus likes to grow on cellulose and finds warm moist building material particularly attractive. It is not clear to what degree specific IgE-mediated reactivity against fungal antigens contributes as the fungi may also produce mycotoxins capable of causing the same symptoms.
November 2007
Antibodies against the protein shown in yellow (not the more famous proteins shown in red and blue) will see increased use beginning this month. Can you guess what it is?
Influenza Nucleoprotein
Every year the winter months bring cold and snow and respiratory tract infections. Most are viral in nature and most are due to the common cold virus. Influenza A and B viruses cause annual outbreaks of more serious respiratory tract disease, usually associated with systemic symptoms. Commercial “immunochromatographic” enzyme immunoassays, commonly employed to screen nasal washings for influenza A and B virus even though their sensitivity is not exceedingly high, use antibodies specific for nucleoprotein antigens, not the surface antigens hemagglutinin (H; shown in red) and neuraminidase (N; shown in blue). These latter antigens are the primary target of naturally occurring antibodies. Because these may change due to point mutations (antigen “drift”) or, more dramatically, recombination with animal viruses (antigen “shift”), antibodies from prior infection may not be protective this year. We hope you got your flu vaccine!
October 2007
According to a recent study, antibodies to this HLA-related glycoprotein may contribute to allograft loss even when the recipient is well-matched for HLA. Can you guess what it is?
MICA (MHC Class I-related Chain A)
HLA Class I (HLA-A & HLA-B) and Class II (HLA-DR) antigens are the most important to match between donor and recipient but so-called “minor” antigens are also important, especially if they are expressed on the surface of endothelial cells. This is because antibodies to such antigens can cause vascular rejection. Most of the “minor” antigens are red blood cell antigens (such as the ABO system) but a recent study found that some proteins coded within the Major-Histocompatibility-Complex Class I gene complex that resemble Class I proteins may also be important. These proteins do not function as presenters of peptide antigens to lymphocytes. Rather, they are induced by stress and may signal to a natural killer cell that the cell is in trouble. Note that the structure is similar to Class I MHC but that the “light chain” beta-2-microglobulin is missing.
September 2007
Antibodies involved in the most common drug allergy are directed against this structure, not the drug itself. Can you guess what it is?
Penicilloyl Hapten
IgE antibodies to penicillins are not directed against the parent compound, but against haptens that form when the drug binds to protein. There are several sites for this binding to occur. The most common (forming the "major" allergenic determinant) is the carbonyl carbon within the beta-lactam ring (as shown in this diagram). Other sites (forming "minor" allergenic determinants) include the carbonyl carbon and the sulfur atom of the outer thiazolidine ring.
August 2007
Pseudomonas Aeruginosa
The 2007 CDID Best Abstract Award went to authors of a study that showed that measuring IgG antibodies to products of these organisms could help predict a serious consequence of cystic fibrosis. Can you guess what they are?
Cystic fibrosis produces chronic lung changes that result in colonization with this gram-negative rod. Early antibiotic treatment can prevent this. The winners of this year's CDID award for best abstract presented a study at the annual AACC meeting in July showing how levels of IgG antibodies against Psuedomonas antigens could identify this complication, allowing for early intervention. See Leal T et al, Clin Chem 53(S6):A210, 2007. Thanks to Dr. N. Banaei of Stanford University for the image of the mucoid Pseudomonas.
July 2007
We are moving beyond autoantibody targets, beginning with this month's quiz. The laboratory use of antibodies to the green epitopes in this cartoon will be discussed during this month's "Issues in Immunodiagnostics" session at the AACC meeting. Can you guess what they are?
Free Light Chains
The cartoon is the immunoglobulin molecule, with two heavy chains and two light chains (all linked by disulfide bridges). Epitopes on the light chains such as the ones colored green are hidden when the light chains are attached to the heavy chains. When the light chains are free, however, these antibodies are able to detect these proteins. Sensitive nephelometric assays for such free light chains in serum are able to detect imbalances between the two types of light chains (kappa and lambda) that occur in myeloma. Although originally used primarily to monitor disease in patients whose myelomas produced light chains only, these assays are also finding a place in the management of patients with myelomas that produce intact monoclonal immunoglobulins as well.