EIAs and ELISAs

Similar to the western blot, enzyme immunoassays (EIAs) use antibodies to detect the presence of antigens. However, EIAs differ from western blots in that the assays are conducted in microtiter plates or in vivo rather than on an absorbent membrane. There are many different types of EIAs, but they all involve an antibody molecule whose constant region binds an enzyme, leaving the variable region free to bind its specific antigen. The addition of a substrate for the enzyme allows the antigen to be visualized or quantified (see the figure below).

In EIAs, the substrate for the enzyme is most often a chromogen, a colorless molecule that is converted into a colored end product. The most widely used enzymes are alkaline phosphatase and horseradish peroxidase for which appropriate substrates are readily available. In some EIAs, the substrate is a fluorogen, a nonfluorescent molecule that the enzyme converts into a fluorescent form. EIAs that utilize a fluorogen are called fluorescent enzyme immunoassays (FEIAs). Fluorescence can be detected by either a fluorescence microscope or a spectrophotometer.

Extended description

The figure numbers its three steps 1 to 3, left to right. (1) Antigen binds to surface: three red diamonds rest side by side on a tan surface. (2) Antibody-enzyme conjugate attaches to antigen: a green Y-shaped antibody, each carrying a purple oval enzyme at the free top end of its stem, opposite the two antigen-binding arms, binds to each diamond. (3) Substrate and enzyme interaction creates color change detection: beside each antibody’s enzyme a yellow circle (substrate) is joined by a curved gray arrow to a blue starburst (colored product).

Immunostaining

One powerful use of EIA is immunostaining, in which antibody-enzyme conjugates enhance microscopy. Immunohistochemistry (IHC) is used for examining whole tissues. As seen in the micrograph below, a section of tissue can be stained to visualize the various cell types. In this example, a mAb against CD8 was used to stain CD8 cells in a section of tonsil tissue. It is now possible to count the number of CD8 cells, determine their relative numbers versus the other cell types present, and determine the location of these cells within this tissue. Such data would be useful for studying diseases such as AIDS, in which the normal function of CD8 cells is crucial for slowing disease progression.

Immunocytochemistry (ICC) is another valuable form of immunostaining. While similar to IHC, in ICC, extracellular matrix material is stripped away, and the cell membrane is etched with alcohol to make it permeable to antibodies. This allows antibodies to pass through the cell membrane and bind to specific targets inside the cell. Organelles, cytoskeletal components, and other intracellular structures can be visualized in this way. While some ICC techniques use EIA, the enzyme can be replaced with a fluorescent molecule, making it a fluorescent immunoassay.

Check Your Understanding

Enzyme-linked Immunosorbent Assays (ELISAs)

The enzyme-linked immunosorbent assays (ELISAs) are widely used EIAs. In the direct ELISA, antigens are immobilized in the well of a microtiter plate. An antibody that is specific for a particular antigen and is conjugated to an enzyme is added to each well. If the antigen is present, then the antibody will bind. After washing to remove any unbound antibodies, a colorless substrate (chromogen) is added. The presence of the enzyme converts the substrate into a colored end product (see the figure above). While this technique is faster because it only requires the use of one antibody, it has the disadvantage that the signal from a direct ELISA is lower (lower sensitivity).

In a sandwich ELISA, the goal is to use antibodies to precisely quantify specific antigen present in a solution, such as antigen from a pathogen, a serum protein, or a hormone from the blood or urine to list just a few examples. The first step of a sandwich ELISA is to add the primary antibody to all the wells of a microtiter plate (see the figure below). The antibody sticks to the plastic by hydrophobic interactions. After an appropriate incubation time, any unbound antibody is washed away. Comparable washes are used between each of the subsequent steps to ensure that only specifically bound molecules remain attached to the plate. A blocking protein is then added (e.g., albumin or the milk protein casein) to bind the remaining nonspecific protein-binding sites in the well. Some of the wells will receive known amounts of antigen to allow the construction of a standard curve, and unknown antigen solutions are added to the other wells. The primary antibody captures the antigen and, following a wash, the secondary antibody is added, which is a polyclonal antibody that is conjugated to an enzyme. After a final wash, a colorless substrate (chromogen) is added, and the enzyme converts it into a colored end product. The color intensity of the sample caused by the end product is measured with a spectrophotometer. The amount of color produced (measured as absorbance) is directly proportional to the amount of enzyme, which in turn is directly proportional to the captured antigen. ELISAs are extremely sensitive, allowing antigen to be quantified in the nanogram ($10^{-9}$ g) per mL range.

In an indirect ELISA, we quantify antigen-specific antibody rather than antigen. We can use indirect ELISA to detect antibodies against many types of pathogens, including Borrelia burgdorferi (Lyme disease) and HIV. There are three important differences between indirect and direct ELISAs as shown in the figure below. Rather than using antibody to capture antigen, the indirect ELISA starts with attaching known antigen (e.g., peptides from HIV) to the bottom of the microtiter plate wells. After blocking the unbound sites on the plate, patient serum is added; if antibodies are present (primary antibody), they will bind the antigen. After washing away any unbound proteins, the secondary antibody with its conjugated enzyme is directed against the primary antibody (e.g., antihuman immunoglobulin). The secondary antibody allows us to quantify how much antigen-specific antibody is present in the patient’s serum by the intensity of the color produced from the conjugated enzyme-chromogen reaction.

As with several other tests for antibodies discussed in this chapter, there is always concern about cross-reactivity with antibodies directed against some other antigen, which can lead to false-positive results. Thus, we cannot definitively diagnose an HIV infection (or any other type of infection) based on a single indirect ELISA assay. We must confirm any suspected positive test, which is most often done using either an immunoblot that actually identifies the presence of specific peptides from the pathogen or a test to identify the nucleic acids associated with the pathogen, such as reverse transcriptase PCR (RT-PCR) or a nucleic acid antigen test.

Extended description

The figure numbers seven steps down two columns headed ‘positive sample’ and ’negative sample.’ (1) Primary antibody binds well: identical rows of yellow Y-shaped antibodies line the floor of both columns. (2) Blocking agent is added: a speckled layer covers the exposed surface between the antibodies in both columns. (3) Sample added: in the positive column a red particle (antigen) attaches to each antibody; in the negative column nothing attaches. (4) Unbound sample is washed away, shown by arrows; the positive column keeps its red particles and the negative column has none. (5) Antibody-enzyme conjugate is added: a second, purple-topped Y-shape attaches to each red particle in the positive column, while in the negative column the same purple-topped Y-shapes are drawn detached, unable to bind anything. (6) Unbound secondary antibody-enzyme conjugate is washed away: it remains attached in the positive column and disappears from the negative column. (7) Substrate is added: in the positive column a small circle beside each enzyme turns into a blue starburst (colored product); the negative column, having no enzyme left, shows no color change. Panel (b) is a photograph of a microtiter plate whose wells are shaded from clear at the edges to a deep teal-green toward the center in a checkerboard dilution pattern, with handwritten labels along its edges.

Extended description

The figure numbers seven steps down two columns headed ‘positive sample’ and ’negative sample.’ (1) Antigen is bound to well: identical rows of red diamonds line the floor of both columns. (2) Blocking agent is added: a speckled layer covers the surface between the diamonds in both columns. (3) Sample added: in the positive column a yellow Y-shaped primary antibody attaches to each diamond; in the negative column the same shapes are drawn unattached beside the diamonds. (4) Unbound sample is washed away: the positive column keeps its bound antibodies and the negative column loses them. (5) Antihuman enzyme-linked antibody is added: a second, purple-topped Y-shape attaches to each bound primary antibody in the positive column, while in the negative column only two of the purple-topped shapes are drawn, unattached. (6) Unbound antihuman antibody is washed away: it remains in the positive column and disappears from the negative column. (7) Substrate is added: in the positive column a small circle beside each secondary antibody turns into a blue starburst (colored product) — the artwork’s own label misprints this step’s reagent as ‘subtrate’ rather than ‘substrate’ — and the negative column shows no color change.

Check Your Understanding

Immunofiltration and Immunochromatographic Assays

For some situations, it may be necessary to detect or quantify antigens or antibodies that are present at very low concentration in solution. Immunofiltration techniques have been developed to make this possible. In immunofiltration, a large volume of fluid is passed through a porous membrane into an absorbent pad. An antigen attached to the porous membrane will capture antibody as it passes; alternatively, we can also attach an antibody to the membrane to capture antigen.

The method of immunofiltration has been adapted in the development of immunochromatographic assays, commonly known as lateral flow tests or strip tests. These tests are quick and easy to perform, making them popular for point-of-care use (i.e., in the doctor’s office) or in-home use. One example is the TORCH test that allows doctors to screen pregnant people or newborns for infection by an array of viruses and other pathogens (Toxoplasma, other viruses, rubella, cytomegalovirus, herpes simplex). In-home pregnancy tests are another widely used example of a lateral flow test (see the figure below). Immunofiltration tests are also popular in developing countries, because they are inexpensive and do not require constant refrigeration of the dried reagents. However, the technology is also built into some sophisticated laboratory equipment.

In lateral flow tests (see the figure below), fluids such as urine are applied to an absorbent pad on the test strip. The fluid flows by capillary action and moves through a stripe of beads with antibodies attached to their surfaces. The fluid in the sample actually hydrates the reagents, which are present in a dried state in the stripe. Antibody-coated beads made of latex or tiny gold particles will bind antigens in the test fluid. The antibody-antigen complexes then flow over a second stripe that has immobilized antibody against the antigen; this stripe will retain the beads that have bound antigen. A third control stripe binds any beads. A red color (from gold particles) or blue (from latex beads) developing at the test line indicates a positive test. If the color only develops at the control line, the test is negative.

Like ELISA techniques, lateral flow tests take advantage of antibody sandwiches, providing sensitivity and specificity. While not as quantitative as ELISA, these tests have the advantage of being fast, inexpensive, and not dependent on special equipment. Thus, they can be performed anywhere by anyone. There are some concerns about putting such powerful diagnostic tests into the hands of people who may not understand the tests’ limitations, such as the possibility of false-positive results. While home pregnancy tests have become widely accepted, at-home antibody-detection tests for diseases like HIV have raised some concerns in the medical community. Some have questioned whether self-administration of such tests should be allowed in the absence of medical personnel who can explain the test results and order appropriate confirmatory tests. However, with growing numbers of lateral flow tests becoming available, and the rapid development of lab-on-a-chip technology (described in the chapter introduction), home medical tests are likely to become even more commonplace in the future.

Extended description

Two labeled diagrams stack vertically, ‘Positive test’ above ‘Negative test.’ Each shows a tilted rectangular strip divided into three zones from left to right — a mix area, a test line, and a control line — with ‘flow’ arrows between them. Four numbered callouts above the positive test read: (1) Human chorionic gonadotropin (hCG) urine sample is applied to absorbent sample pad — the artwork’s own legend misprints this hormone’s name as ‘gonadortropin’ rather than ‘gonadotropin.’ (2) hCG antigen bonds with the anti-hCG antibody-colloidal gold conjugates. (3) hCG antigen bound to anti-hCG antibody-colloidal gold conjugate is captured by immobilized anti-hCG antibody. (4) Free hCG antibody-colloidal gold is captured by antibodies. In the positive-test row a starburst reading ‘color’ appears above both the test line and the control line. In the negative-test row, drawn without the numbered callouts, a starburst reading ‘color’ appears above the control line only. A legend beneath both rows keys four symbols: a green Y-shape for the hCG-first antibody, a small red diamond for human chorionic gonadotropin, a yellow circle bearing an orange Y for the hCG-second antibody-gold-nanoparticle conjugate (‘Au’), and a purple Y-shape for IgG.

Check Your Understanding

The table below compares some of the key mechanisms and examples of some of the EIAs discussed in this section as well as immunoblots, discussed in Detecting Antigen-Antibody Complexes.

Immunoblots & Enzyme Immunoassays

Summary

• Enzyme immunoassays (EIA) are used to visualize and quantify antigens. They use an antibody conjugated to an enzyme to bind the antigen, and the enzyme converts a substrate into an observable end product. The substrate may be either a chromogen or a fluorogen.
• Immunostaining is an EIA technique for visualizing cells in a tissue (immunohistochemistry) or examining intracellular structures (immunocytochemistry).
• Direct ELISA is used to quantify an antigen in solution. The primary antibody captures the antigen, and the secondary antibody delivers an enzyme. Production of end product from the chromogenic substrate is directly proportional to the amount of captured antigen.
• Indirect ELISA is used to detect antibodies in patient serum by attaching antigen to the well of a microtiter plate, allowing the patient (primary) antibody to bind the antigen and an enzyme-conjugated secondary antibody to detect the primary antibody.
• Immunofiltration and immunochromatographic assays are used in lateral flow tests, which can be used to diagnose pregnancy and various diseases by detecting color-labeled antigen-antibody complexes in urine or other fluid samples.

Key terms

• enzyme immunoassays (EIAs) — type of assay wherein an enzyme is coupled to an antibody; addition of a chromogenic substrate for the antibody allows quantification or identification of the antigen bound by the antibody.
• fluorogen — nonfluorescent molecule that becomes fluorescent on enzyme or laser activation.
• fluorescent enzyme immunoassays (FEIAs) — EIA in which the substrate is a fluorogen that becomes fluorescent following reaction with the enzyme.
• immunostaining — use of EIA technology to deliver stain to particular cells in a tissue (immunohistochemistry) or specific targets within a cell (immunocytochemistry).
• Immunohistochemistry (IHC) — staining technique in which labeled antibodies are bound to specific cells in a tissue section.
• Immunocytochemistry (ICC) — staining technique in which cells are fixed and holes dissolved in the membrane to allow passage of labeled antibodies to bind specific intracellular targets.
• enzyme-linked immunosorbent assays (ELISAs) — specialized form of EIA in which either the primary antibody or the antigen is first attached to a solid surface such as the well of a microtiter plate.
• direct ELISA — enzyme-linked immunoabsorbent assay in which the antigens are immobilized in the well of a microtiter plate; only a single antibody is used in the test.
• sandwich ELISA — EIA in which the primary antibody is first attached to the wells of a microtiter plate, allowing it to capture antigen from an unknown solution to be quantified.
• primary antibody — in a sandwich ELISA, the antibody that is attached to wells of a microtiter plate to capture antigen from a solution, or in an indirect ELISA, the antigen-specific antibody present in a patient’s serum.
• secondary antibody — antibody to which an enzyme is attached for use in ELISA assays; in direct and sandwich ELISAs, it is specific for the antigen being quantified, whereas in indirect ELISA, it is specific for the primary antibody.
• indirect ELISA — EIA in which an antigen from a pathogen is first attached to the wells of a microtiter plate; the antigen then captures antibodies from patient serum to determine whether the patient currently has or previously had the disease.
• immunofiltration — technique in which antibody or antigen can be concentrated by passing fluids through porous membranes, and target molecules are captured as they pass.
• immunochromatographic assays — assay in which fluids are pulled through test strips by capillary action and antigen captured by mobile antibody-colored bead conjugates; a second, fixed antibody localizes the colored bead, allowing visualization.
• lateral flow tests — see immunochromatographic assays.

Practice

Explain the differences and similarities between EIA, FEIA, and ELISA

Describe the difference and similarities between immunohistochemistry and immunocytochemistry

Describe the different purposes of direct and indirect ELISA

This section is adapted from Microbiology, Section 20.4: EIAs and ELISAs by Nina Parker, Mark Schneegurt, Anh-Hue Thi Tu, Philip Lister, Brian M. Forster, and OpenStax, © OpenStax, licensed under CC BY-NC-SA 4.0. Access the original for free at openstax.org. Changes: all seven images re-encoded as WebP and rendered as mediafigures after image and PDF inspection, with kind="diagram" set on the EIA, ELISA, Indirect, Lateral, and Art Connection figures and kind="photo" on the IHC micrograph and the Torch pregnancy-test photo, overriding the media manifest’s JPEG-based photo guess for the five drawn figures; the EIA figure carries eager="true" as the page’s first figure; a longdesc added for the EIA, ELISA, Indirect, and Lateral figures, walking each numbered step in reading order; the Indirect ELISA figure’s own printed label misspells “substrate” as “subtrate” at its seventh step, transcribed as printed with the correct spelling named beside it in the longdesc and reported as a source defect; the Lateral flow figure’s own legend misspells “gonadotropin” as “gonadortropin,” transcribed as printed with the correct spelling named beside it in the longdesc and reported as a source defect; feature boxes rendered as callouts; the Clinical Focus Part 2 and Part 3 boxes’ “Jump to the previous/next Clinical Focus box” links are replaced with plain sentences naming that the case began in Section 20.1 (this run’s own naming rule, since the case runs past two sections) and where it continues, linking to Section 20.1 and, from Part 3, to Section 20.5 (both already on disk); the footnote in Part 3 is rendered as an inline parenthetical citation; the cross-reference to Section 20.1’s lab-on-a-chip figure is rendered as a link to the chapter introduction (on disk at authoring time); the cross-reference to Section 20.2 (m58901) is a link to that page; the “Immunoblots & Enzyme Immunoassays” table is a section-level recap spanning every EIA type this chapter covers rather than one objective’s own contrast, so it is transcribed as a Markdown table only, with no sortbins built for it, per this book’s chapter- or section-level recap table rule; the four source Multiple Choice items and the one Fill in the Blank item are adapted into Practice unchanged with their source keys; three of the section’s six body Check Your Understanding bullets are graded from this module’s own sentences rather than left in prose — “What do the direct and indirect ELISAs quantify?” is now a multiplechoice keyed by the indirect-ELISA paragraph’s own contrast sentence; “What physical process does the lateral flow method require to function?” is now a textin keyed by the paragraph’s own “capillary action” phrase; “Explain the purpose of the third strip in a lateral flow assay” is now a multiplechoice keyed by the Lateral figure’s own caption sentence, with distractors drawn from the module’s other lateral-flow steps — the source prints no key for any of the three; the other three body Check Your Understanding bullets remain self-checks with model answers assembled from this section’s own text, because each needs more than one sentence to answer honestly; the unkeyed Short Answer question about sandwich-ELISA epitopes remains a self-check whose model answer states what this section gives (the mechanics of the two-antibody sandwich) and stops, since the section identifies the secondary antibody as polyclonal but does not itself explain why that is advantageous; the unkeyed Short Answer question comparing the pregnancy strip to a direct or indirect ELISA is now a multiplechoice whose key is the paragraph’s own three reasons verbatim (“fast, inexpensive, and not dependent on special equipment”), with distractors negating each reason in turn; the unkeyed Critical Thinking Art Connection question is rendered as a mediafigure (its bare inline image has no source caption, so the caption above is author-written, describing only the unlabeled Y-shapes’ positions) immediately followed by a self-check whose model answer is assembled from the sandwich-ELISA paragraph’s own sentences; two filler textin items — a cloze from this section’s summary sentence about immunostaining, and a term-recall item for “Immunohistochemistry (IHC)” — fill the second objective group to the section floor; key terms compiled from the module’s fifteen defined terms and the book’s Glossary appendix, all fifteen taken directly from the appendix, except “immunostaining,” whose nearest appendix headwords (“immunostain,” “staining”) do not match the module’s own sense and whose meaning is instead drawn from this module’s defining sentence. A Check Your Understanding bullet in this section asks about “the secondary antibody in a direct ELISA,” though this section’s own text describes a secondary antibody only in the sandwich ELISA procedure; the bullet is transcribed and answered verbatim, drawing on the sandwich ELISA paragraph, as the module’s own wording, and flagged for the claim pass.