The immuassay handbook parte25


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The immuassay handbook parte25

  1. 1. 149© 2013 David G. Wild. Published by Elsevier Ltd. All rights reserved. Laboratory investigations of suspected (present or past) infections can be carried out using microbial methods, by detection of an antigen from the suspected organism, by detection of antibodies formed in the body as a response to the suspected organism, or by detection of DNA or RNA from the suspected organism. Each of these approaches has strengths and weaknesses. Microbial methods gener- ally take at least 24h to perform, and many organisms can- not be successfully cultured. Antigen levels are often too low to permit detection. Antibodies are not detectable for 1–4 weeks and are never detected in some subjects. Finally, DNA and RNA methods cannot establish a current infec- tion in many situations and are undetectable for some organisms because circulating levels are too low. When a complete investigation is required to establish the nature of an infection, all of these methods may be employed, but the cost of such a thorough study can be quite high. In many situations, it is possible to choose one approach that will answer the specific question under con- sideration. Measurement of antibodies is the method of choice for many investigations. For example, if the objective is to determine immunity against rubella, the use of anti- body methods is a clear choice because no virus will be detectable and the presence of antibody can confirm immu- nity based on actual infection or immunization. If a physi- cian suspects that an infection occurred some number of weeks or months in the past, as is often the case for syphilis or Lyme disease, the use of an antibody test is the most prac- tical approach to confirmation. Antibodies can also offer information that can distinguish recent from past infections. For example, the Epstein-Barr nuclear antigen 1 (EBNA-1) IgG antibody is not present in acute Epstein-Barr virus (EBV) infection, so the detection of viral capsid antigen (VCA) IgM and/or IgG antibody in the absence of EBNA-1 IgG antibody can be used to confirm active disease. Most immunoassays are used for the detection of defined entities, whether small molecules or proteins, rather than antibodies. Scientists who wish to measure antibodies need to understand the characteristics of antibodies that are rel- evant to assay development; these include affinity, avidity, and isotype. Further, the epitope detected by an antibody is not always clear; the epitope may differ between indi- viduals, at different stages of the disease, and also for dif- ferent strains of the same organism. The design of the immunoassay must take into account these features, and the diversity of antibodies has led to a wide range of assay designs for their detection. One advantage of antibody measurement is that concen- tration is often quite substantial, since the immune system is an effective manufacturing site. For very early detection, for example of HIV antibodies, quite sensitive methods may be required, but once antibody production has been established in the B cell, concentrations are quite high and only modest sensitivity is required. On the other hand, calibration of antibody measurement is notoriously difficult. Synthesis of antibodies is generally time-consuming and expensive, so human materials are often used for calibration. However, human antibodies may differ in their affinity, avidity, and epitope specificity, leading to considerable challenges. Recombinant antibodies are becoming available and may offer opportunities for improved calibration in the future. Assay Formats The common feature to almost all contemporary antibody measurements is the attachment of the target antigen to a solid phase. For rapid formats, latex strips are often employed. Bead-based assays are popular in clinical chemis- try settings. Single measurements are performed in many instruments from Roche, Siemens, Abbott, and other manu- facturers;multiplemeasurementsarepossibleusingLuminex instruments and related technology. Enzyme-linked immu- nosorbentassay(ELISA)measurementsarewidelyemployed for antibody detection, because they are easily prepared and can be run in almost all laboratories using inexpensive equip- ment. Planar arrays can extend the ELISA principles to per- mit measurement of multiple antibodies. See PRACTICAL GUIDE TO ELISA DEVELOPMENT. When extremely high specificity is required, such as in the blood bank setting, double-antigen sandwich assays may be employed. In this format, the antigen is immobi- lized on the solid phase at a low density. One arm of the antibody will bind to this target, leaving another recogni- tion site free. After a wash step, additional antigen bound to the labeled detection antibody is added, which greatly increases the likelihood that the antibody which was ini- tially captured has the correction recognition site. This technique has the disadvantage that different isotypes cannot be distinguished. Assay Development CAPTURE REAGENT SELECTION The capture reagent is the foundation on which the immu- noassay is built, and therefore, selection of the capture Detection of Antibodies Relevant to Infectious Disease Steven Binder ( Jennifer A. Isler ( C H A P T E R 2.8
  2. 2. 150 The Immunoassay Handbook reagent represents one of the most critical steps in devel- oping a specific assay to quantify the analyte of interest. The capture reagent should bind specifically to the targeted ana- lyte, and therefore, specificity is perhaps the most important consideration when selecting the capture reagent. For the detection of antibodies against infectious agents, the best capture reagent is an immunogenic portion of the infectious agent itself. It is recommended that experiments be per- formed to identify highly immunogenic regions of the infec- tious agent. This information can be predicted in silico using epitope modeling software but is most reliably obtained through epitope mapping studies. For example, antibodies generated in response to immunization (e.g., with a nonin- fectious form of the agent) can be tested for their reactivity to proteins of the infectious agent to determine which are most immunogenic. Immunogenic proteins can be further mapped using peptides or mutants to identify immunogenic epitopes. Species-specific differences should be considered in selecting the capture reagent. That is, if the assay is intended to detect antibodies to different strains of an infectious agent, the capture reagent should be well con- served across those strains. Alternatively, it may be desir- able to have a strain-specific capture reagent. One should also consider the heterogeneity of the antibody response (see below). It is recommended that an immunodominant antigen be selected to ensure that most if not all samples containing antibodies against the infectious agent are detected. Once potential capture reagents are identified, they can be screened for binding affinity and specificity. For this purpose, a positive control such as a polyclonal antibody preparation generated in the species of interest is recom- mended. The selected capture reagent should display high affinity and specific binding (as measured by low background and lack of matrix interference) in the desired matrix (e.g., human blood). Capture reagents that interact with proteins endogenous to the matrix will cause high background and may generate false-positive results. When immobilized on a solid phase, the capture reagent must retain the ability to bind the target analyte. Immobi- lization onto ELISA plates results from hydrophobic interactions between nonpolar portions of the capture reagent and the plastic matrix. In some cases, coating may mask or hide the region of the capture reagent required to bind the target antigen resulting in an inability to effec- tively capture the target antibody. If this happens, it is often helpful to label the capture reagent and link it to the plate via an interaction between the label and a high-affinity binding partner pre-bound to the plate. Commonly used examples include biotin-labeled reagents with avidin- coated plates and histidine-tagged reagents with nickel- coated plates. Because coating of the labeled reagents relies on binding of the label (not the capture protein per se) to the plate, the binding region(s) of the capture reagent are better positioned and more freely available to bind the target antibody. When labeling any reagent, it is impor- tant to understand the location and extent to which the reagent is labeled and to ensure that labeling does not alter the binding activity of the reagent. DETECTION REAGENT SELECTION The purpose of the detection reagent is to bind the tar- get antibody and allow a quantifiable signal to be mea- sured. Most commonly, an enzyme (e.g., horseradish peroxidase, HRP) is conjugated to the detection reagent and cleaves the appropriate substrate (e.g., tetramethyl- benzidine) to generate a chromogenic, chemilumines- cent or chemifluorescent signal proportional to the amount of target antibody bound. A wide range of enzyme-conjugated reagents is commercially available for this purpose; however, if it is not convenient to enzyme conjugate the desired detection reagent, a sec- ondary detection reagent can be used. For example, if a custom-generated mouse monoclonal antibody is used as the primary detection reagent, a commercially available HRP-conjugated goat anti-mouse antibody can be used as a secondary detection reagent to generate signal. The incubation temperature, buffer composition and pH, enzyme stability, and requirement for cofactors should all be considered when selecting an enzyme conjugate. As with all assay reagents, the concentration of the enzyme conjugate should be optimized for every assay. The required sensitivity is one of the biggest consider- ations in selecting a detection method, as summarized in Table 1. Chromogenic assays are less sensitive than chemi- fluorescent and chemiluminescent assays, but this is not necessarily a disadvantage. Highly sensitive assays have increased vulnerability to variations in signal when small perhaps unintentional changes are made in blockers or dilu- ents. Therefore, the appropriate detection method is often that which provides signal at or below the desired detection range of the assay. If the desired detection range of the assay is in the high picogram or microgram range, chromogenic assays are preferred, whereas chemifluorescent or chemilu- minescent assays are better suited to lower limits of detec- tion (i.e., in the low picogram or femtogram range). The Meso Scale Discovery (MSD) platform (www.mesoscale. com) uses electrochemiluminescence and is commonly used TABLE 1 Considerations for Selecting an ELISA Detection Method Detection Method Sensitivity Equipment Required Advantages Disadvantages Chromogenic ~5–200pg/well Absorbance plate reader Direct visualization; uses common equipment Decreased sensitivity Chemifluorescent ~1–5pg/well Fluorometer Highly sensitive High sensitivity can lead to variability Chemiluminescent ~1–5pg/well Luminometer Highly sensitive Signal fades quickly, can lead to variability across multiple runs
  3. 3. 151CHAPTER 2.8 Detection of Antibodies Relevant to Infectious Disease to develop assays with a high degree of sensitivity and dynamic range. MSD assays employ a ruthenium-labeled detection antibody which, upon electrochemical stimula- tion via the cathode-coated MSD plate, emits light propor- tional to the amount of antibody in the sample. The light signal is amplified by multiple excitation cycles for increased sensitivity, and background levels are low, which typically results in excellent signal-to-noise ratios. BLOCKING BUFFERS Because the binding capacity of an ELISA plate well is typically in excess of the amount of capture reagent used to coat the well, a blocking solution can be used. It consists of an irrelevant, immunologically nonreactive protein that coats the remaining surface of the well to reduce nonspe- cific binding of proteins in the test sample or assay reagents. Detergents have also proven successful to minimize non- specific protein binding. Commonly used blockers include albumin, casein, milk, Tween®, Triton®, and dextran sul- fate. The effectiveness of the blocker varies depending on a number of components of the assay, perhaps the most important of which is the matrix in which the analyte will be measured. A clear indicator of nonspecific binding is the back- ground signal generated in the absence of target antibody (i.e., the analyte to be measured). High background levels can substantially reduce the signal-to-noise ratio of the assay and thereby limit the assay’s detection range. In addition to blocking the plate immediately following coat- ing, incorporation of irrelevant proteins or detergents in subsequent wash steps can also help to minimize back- ground signal. Optimal blocking conditions, including the blocking solution, its concentration of irrelevant protein or detergent, and blocking incubation time and tempera- ture, are best determined experimentally. This can be done by varying the blocking conditions listed and evaluating the effect on background signal and signal-to-noise. HETEROGENEITY OF THE ANTIBODY TO BE MEASURED The immune response to an infectious agent is almost always polyclonal. The population of target antibody is often quite diverse and reflective of the number of immu- nogenic constituents of the infectious agent. To ensure that positive samples are not missed in the assay (false neg- atives), it is important that the assay design permits the detection of the full range of antibodies present in all indi- viduals. This can be accomplished by pairing immuno- dominant proteins or epitopes for capture with generic reagents (e.g., antibody against species-specific IgG) for detection. ISOTYPE SPECIFICITY Antibody isotyping allows one to identify the serological class of the target antibody. This is commonly performed to better characterize the antibody and provide helpful infor- mation related to the antibody response (e.g., timing of response and antibody structure). In the context of an ELISA assay, the isotype of the target antibody can be determined using a capture or detection antibody that is specific for an antibody isotype (e.g., IgG and IgM). These isotype-specific antibodies are commercially available and are offered as kits specifically for the purpose of determining the isotype of the antibody of interest. Other methods, including agarose gel diffusion and isotyping “strip” kits, can also be used for isotyping. Just as the isotype specificity of an ELISA reagent can be useful for isotyping the target antibody, one may want to avoid isotype-specific reagents during assay development if the isotype of the target anti- body is not known. SPECIAL CONSIDERATIONS FOR VACCINE IMMUNITY STUDIES The ability of a vaccine to elicit an antibody response against the desired antigen(s) is effectively measured using a quan- titative immunoassay in which the end result can be reported as an antibody titer. Commercial kits are available to quantitate antibody titers against common vaccine antigens; however, rare or experimental vaccines require the develop- ment of novel immunoassays. Because the antibody response to a vaccine will differ depending on the individual, it is not practical to generate a calibration curve from which absolute concentrations can be extrapolated. Therefore, quantifica- tion of antibody titers is commonly performed using serial dilutions of the test sample where the titer is reported as the highest sample dilution that produces a signal reliably above background. The quantity of antibody is not the only important factor in understanding the immune response to a vaccine; an isotype-specific detection antibody (as described above) is commonly incorporated to track how the antibody response matures over time. Assay Validation QUANTITATION RANGE The quantitation range of an immunoassay should be suitable for the expected antibody concentrations to be measured in the unknown sample given acceptable levels of dilution of that sample. A quantitation range is defined by an upper limit and lower limit of quantitation. Those limits, together with a recommended minimum of four antibody concentrations in between, are used to generate a calibration curve in which the known antibody concentra- tions of a control sample are plotted against the signal gen- erated upon analysis of that control sample. The calibration curve is then used to determine the concentration of anti- body in unknown samples based upon the signal generated during analysis. A validated quantitation range that requires control samples with known concentration of antibody demon- strates acceptable precision and accuracy. In addition to the antibody concentrations of the calibration curve points (i.e., the calibrators), quality control samples of at least high, medium, and low concentrations (relative to the upper and lower limits of quantitation) must also demon- strate acceptable accuracy and precision. The accuracy of the method describes the closeness of the test results obtained by the method to the known concentration in the
  4. 4. 152 The Immunoassay Handbook control sample and is typically reported as the relative error. The assay precision describes the closeness of repeat measurements of antibody concentration when assays are performed multiple times on the same plate (intra-assay) or across plates (inter-assay). Current regulatory guide- lines (e.g., from the U.S. Food and Drug Administration (FDA) and the European Medicines Agency (EMA)) sug- gest that a validated assay to quantitate antibody demonstrate accuracy ±20% of the known antibody con- centration and precision of ≤20% coefficient of variance. Additional information related to target validation param- eters can be found in the current regulatory guidance and industry white papers. DILUTIONAL LINEARITY As mentioned above, the quantitation range of an immu- noassay should be suitable for the expected antibody con- centrations to be measured at acceptable dilutions of the test sample. When antibodies are present in test samples at concentrations above the quantitation range of the assay, they are commonly diluted in order to bring the antibody concentration into the quantification range. To demon- strate the degree to which a sample can be diluted and still yield accurate back-calculated concentrations of the target antibody, a dilutional linearity assessment should be per- formed. This is typically done by spiking a high concentra- tion of antibody into the test sample matrix and preparing serial dilutions of that sample so that the resulting concen- trations fall within the quantitation range of the assay. Dilutional linearity is considered acceptable when 80% of the observed test results that fall between the upper and lower limit of quantitation yield back-calculated antibody concentrations ±20% of the known concentration (after multiplication by the dilution factor). The highest dilution of antibody that meets the above criteria is defined as the limit of dilution. It is important to note cases in which a dilution of the target antibody that should theoretically generate a signal above the upper limit of quantification actually falls within the range of quantification. This may indicate a potential prozone effect (also referred to as high-dose hook effect) in the assay. This can occur when the level of anti- body is in excess relative to the assay reagents thereby satu- rating the binding capacity of the capture and detection reagent and inhibiting signal generation. One must pro- ceed with caution when analyzing test samples using an assay with a suspected prozone effect. To avoid potential underestimation of antibody concentrations at high levels, it is recommended that test samples be analyzed at multi- ple dilutions to demonstrate dilutional linearity (described as parallelism when referring to test samples). SELECTIVITY AND SPECIFICITY Selectivity is the degree to which the assay measures the target antibody in the presence of other materials expected to be present in test sample matrix (e.g., plasma). Similarly, specificity is a measure of the assay’s ability to bind the antibody of interest, specifically in the presence of struc- turallysimilarcompoundsorotherpotentialcross-reactants expected to be present in test samples. Appropriate selection of capture and detection reagents is critical to ensure that the assay does not generate high levels of signal in the absence of target antibody. To assess selectivity, indepen- dent lots of blank test sample matrix should be spiked with the target antibody, and the antibody concentration measured to calculate the percent recovery for each sam- ple. The selectivity of the method is considered accept- able if the recovery of the antibody is ±20% of the known antibody concentration in at least 80% of the samples tested. Similarly, specificity can be evaluated by spiking antibody into sample matrix that is additionally spiked withanexpectedconcentrationofapotentialcross-reactant. Specificity is acceptable if the recovery of the antibody in the presence of the potential cross-reactant concen- trations is within 80–120% relative error when com- pared to the known concentration of spiked antibody. In addition, samples containing only the potential cross- reactant should yield results below the lower limit of quantification. STABILITY In any validated assay, it is important to verify that the analyte is stable over the storage conditions of the test samples, namely storage time and temperature. Antibodies are relatively stable when stored frozen for a long period of time (e.g., 1 year); however, less than ideal storage con- ditions and repeat freeze–thaw cycles can lead to degrada- tion within the test sample. Stability is typically evaluated by preparing multiple levels of quality controls (i.e., anti- body spiked in the appropriate matrix) to mimic a test sample. The time and temperatures tested should reflect the possible storage conditions of the sample and should also incorporate potential errors that may occur during sample handling (e.g., samples left on the benchtop). A common experimental design includes short-term assess- ment at ambient and refrigerated temperatures, long-term assessment under frozen conditions, and repeat freeze– thaw cycles. Stability is generally considered acceptable when the measured concentrations of at least two thirds of the stability samples and 50% or more of each concentra- tion level are within the accuracy and precision criteria established for the method. Instrumentation For simple strip tests, visual inspection is often employed, and no instrumentation is required. Strip tests are inex- pensive and can be employed outside of traditional labora- tories and in countries where equipment is limited. Because antibodies are generally stable when dried, blood spots can be collected in remote settings and transferred to a testing center for elution, further increasing the opportunities for testing in developing countries. ELISA equipment is widely available and may support colorimetric, fluorometric, or chemiluminescent detec- tion. For increased throughput, automation of sampling, pipetting, and washing is possible with robotic equipment that may have the capacity to handle as few as 2 or as many as 16 microtiter plates simultaneously. Because concen- trations of antibodies are generally not low, 30–60min
  5. 5. 153CHAPTER 2.8 Detection of Antibodies Relevant to Infectious Disease incubations are generally possible, and total assay time for an ELISA test may be 2–3h. Multiplex bead assays have become popular since the introduction of this technology in 1997. There are many situations where multiple antibodies should be measured at the same time to aid in patient management. For example, measurement of mumps, measles, rubella, and varicella (MMRV) antibodies is commonly requested to establish immunity to childhood infections. The Luminex 200 sys- tem, described in another chapter, is widely used for this purpose. Antigen arrays can also be used for measurement of multiple antibodies. Applications The list of infections that are currently identified by serol- ogy is quite long. The paragraphs below summarize some of the more significant uses as well as several emerging applications. BLOOD BANK TESTING Potential blood donors must be screened for transmissible diseases to ensure the safety of the blood supply. HIV, hepatitis, and syphilis testing are universally performed on blood donors; screening for other infections such as Human T-cell lymphotropic virus (HTLV), malaria, and Chagas disease is performed in regions where these patho- gens are encountered. HIV testing is perhaps the most demanding of immuno- assays currently in use, because of the high requirements for sensitivity and specificity. In a laboratory setting, cur- rent fourth generation tests must exceed 99.5% sensitivity and 99.7% specificity; requirements are slightly less for rapid tests designed for use in a community setting. In order to meet the specificity challenge, some manufactur- ers use immunometric (sandwich) formats, where one arm of the HIV antibody recognizes a target fixed to a solid surface, and the other arm of the antibody recognizes an antigen attached to a label or enzyme, ultimately leading to detection by fluorescence or chemiluminescence. Both IgG and IgM antibodies must be detected, if present. Fur- ther, all results no matter what method is used initially for screening, must be confirmed. For some time, western blot methods were used for this purpose; but today, the sensi- tivity of the screening test may exceed the sensitivity of the western blot test, so supplementary testing using a nucleic acid amplification test (NAAT), e.g., based on polymerase chain reaction (PCR), is required. Finally, HIV is a virus possessing many serotypes, and antibody recognition must be broad enough to recognize these serotypes, as well as HIV-2, a different strain with significant sequence modifi- cations. Figure 1 illustrates an example of how laboratories screen and confirm for HIV antibodies. CONFIRMATION OF ACUTE DISEASE STATUS Many diseases can be confirmed in the acute phase using antibody detection. Infectious mononucleosis, a common infection in children and teens, has been detected for many decades using simple tests for “heterophile antibody”, a simple strip test that looks for antibodies in human serum that react with sheep or horse blood extracts. Today, a more sensitive test for IgM antibodies for the VCA antigen from Epstein–Barr virus (EBV) is preferred, but this test is only performed in full service laboratories. Additional information about the time since infection can be deter- mined using IgG EBV VCA as well as IgG EBV early anti- gen (EA-D). Finally, the absence of IgG EBNA rules out late infection or re-infection, since this antibody typically appears 3 months after the initial exposure. Figure 2 sum- marizes the appearance over time of these antibodies. Many physicians are not trained to interpret these results, and further advances in technology may change how tests are used. For example, antibodies to EBNA were deter- mined until 2000 using indirect immunofluorescence. This method detects many EBNA molecules in the virus, some “early” and some “late”. However, all modern meth- ods detect antibodies to the recombinant EBNA-1 pro- tein, against which antibodies are formed quite late in the process, improving the utility of this determination. PATHOGENS THAT CAN AFFECT PREGNANCY Another major use of antibody testing is the screening of pregnant women for diseases that may be transmitted to their offspring. This testing differs from blood bank test- ing in that only active, untreated disease is of interest. Toxoplasma, rubella, cytomegalovirus (CMV), and syphi- lis are commonly requested. Herpes simplex virus (HSV) testing is also performed in some countries. Screening may include both IgG and IgM antibodies, since a recent infec- tion may sometimes be detected only by IgM antibodies. VACCINATION EFFICACY The testing of hospital employees for measles, mumps, rubella and varicella zoster virus was previously mentioned, and multiplex methods to monitor diphtheria and tetanus have also been reported. In addition, the development of new vaccines generally requires antibody testing to establish whether the proposed formulation is effective in eliciting a response and has reduced the need for more cumbersome neutralization methods. The recent introduc- tion of vaccines for human papillomavirus and pneumococ- cus has been greatly aided by rapid antibody screening. TRANSPLANTATION AND IMMUNOSUPPRESSION MONITORING Many of the same tests that are used for monitoring acute infection, disease during pregnancy, and the efficacy of vaccination are also of value for evaluation of both the donor and the recipient prior to transplantation. For example, organs from donors with EBV or CMV infec- tions can cause serious disease with recipients who have not been previously infected. Antibody screens are inex- pensive and can help identify potential issues. Follow-up testing often requires NAAT to confirm the presence of active infection and in some cases establish viral load. Immunosuppression of a patient receiving the organ to
  6. 6. 154 The Immunoassay Handbook FIGURE 1 An example of a current laboratory algorithm for HIV testing. Presented at the third CDC-APHL HIV Diagnostics Conference, March 24–26, 2010, Orlando, Florida. (The color version of this figure may be viewed at FIGURE 2 Appearance over time of antibodies resulting from an EBV infection. Adapted from J.M. Seigneurin, Apport du laboratoire dans l’infection à virus Epstein-Barr (Laboratory diagnosis of Epstein-Barr virus infections). Immuno-analyse & Biologie spécialisée 17: 33–39, 2002. (The color version of this figure may be viewed at
  7. 7. 155CHAPTER 2.8 Detection of Antibodies Relevant to Infectious Disease reduce the risk of rejection may allow a sequestered virus (CMV, EBV, or HSV) to flare and put both patient and organ at risk. EPIDEMIOLOGY The identification of new infectious diseases, such as hanta virus, often leads to a need to determine the prevalence of exposure in the local human population. Also, testing of animal populations can be helpful to determine the species that are likely to be carriers of the disease. These studies can be supplemented by reverse transcriptase polymerase chain reaction (RT-PCR) analysis to identify animals that were chronically shedding the virus—for hantavirus, this turned out to be the deer mouse. Also, many patients may have been exposed to an infection without expressing symptoms; using serology, the true incidence of the dis- ease may be more accurately determined. During the 1999 West Nile virus outbreak in New York City, it was dem- onstrated that only 1% of those infected had reported symptoms. ANIMAL TESTING Animals used for research are regularly tested for evidence of previous or current infection. Since many animals must be tested in mouse or rat colonies, multiplex methods that can simultaneously screen for multiple infections have become popular. Similarly, domestic animals are often screened for infectious diseases in veterinary settings. BIOWARFARE Antibody detection is not likely to be the first choice in the event of a biowarfare attack if an acute agent like anthrax is used. However, many other scenarios have been consid- ered where widespread antibody testing might be necessary to determine the prevalence of an infection and also to help rule out infection in anxious individuals who are concerned about past exposure. Further Reading Andreotti, P.E., Ludwig, G.V., Peruski, A.H., Tuite, J.J., Morse, S.S. and Peruski, Jr., L.F. Immunoassay of infectious agents. Biotechniques 35, 850–859 (2003). Bissonette, L. and Bergeron, M.G. Diagnosing infections—current and anticipated technologies for point-of-care diagnostics and home-based testing. Clin. Microbiol. Infect. 16, 1044–1133 (2010). Deshponde, S.S. Enzyme Immunoassays: From Concept to Product Development. (Kluwer Academic Publishers, Norwell MA, 1996). Lim, D.V., Simpson, J.M., Kearns, E.A. and Kramer, M.F. Current and developing technologies for monitoring agents of bioterrorism and biowarfare. Clin. Micro. Rev. 18, 583–607 (2005). Mairhofer, J., Roppert, K. and Ertl, P. Microfluidic systems for pathogen sensing: a review. Sensors 9, 4804–4823 (2009). Uttamchandani, M., Neo, J.L., Ong, B.N. and Moochhala, S. Applications of micro- arrays in pathogen detection and biodefence. Trends Biotechnol. 27, 53–61 (2009).