40 Antibodies
A nasal swab sample holds thousands of kinds of molecule besides the viral protein that a COVID-19 antigen test is built to detect. The test line must turn red when that protein is present and stay blank otherwise. Gold collects at the line when a target molecule links the gold-labeled detection antibody to the capture antibody, so the strip responds to any molecule that both antibodies bind.
An antibody that also bound a protein present in every nasal sample would lack analytical specificity (diagnostic performance). Every test would read positive, so \(TN = 0\) and the clinical specificity, \(TN/(TN+FP)\), would be zero. A lateral flow test therefore needs an antibody that binds the target with negligible binding to other molecules in the sample, has a measured binding strength, and is made to within a stated tolerance from lot to lot (Koczula and Gallotta 2016).
The Y-shaped molecule
An antibody, or immunoglobulin, is a Y-shaped protein. A protein is a chain of amino acids, small molecules linked end to end, folded into a definite shape. An antibody is built from four such chains: two identical heavy chains and two identical light chains. Disulfide bonds, which link sulfur atoms on two amino acids, hold the chains together (Clark et al. 2018). Each chain folds into compact units called domains, a variable domain at its tip and one or more constant domains behind it (Janeway et al. 2001b). IgG, one class of antibody, has this structure (Figure 40.1).
The two arms are the Fab regions, which bind antigen, and the stem is the Fc region; a flexible hinge joins them (Janeway et al. 2001b). The variable domains differ in amino-acid sequence from one antibody to the next, while antibodies of the same class and species have the same constant domains (Clark et al. 2018). At the tip of each arm, the variable domains of one heavy and one light chain together form the antigen-binding site (Janeway et al. 2001b). The variable tips therefore determine which target an antibody binds: binding depends on how well the site and the target match in shape and chemistry (Clark et al. 2018). The two arms are identical, so one IgG carries two copies of the same binding site.
The sandwich and the control line
The part of the antigen that an antibody binds is its epitope, and most protein antigens carry several different epitopes (Parker et al. 2016, sec. 20.1). A sandwich test holds the antigen between two antibodies (Koczula and Gallotta 2016). Both can attach to one antigen at once if they bind different epitopes. Two antibodies whose epitopes are the same or overlap block each other, and no sandwich forms.
The control line usually carries an antibody that binds antibodies from another species (Koczula and Gallotta 2016), such as an anti-mouse antibody when the detection antibody comes from a mouse. Because mouse antibodies of one class have the same constant domains, an anti-mouse antibody binds mouse antibodies of that class whatever their target. The control line therefore captures the detection antibody and its gold whenever the sample flows, with or without target.
Where antibodies come from
Antibodies are made by B cells, white blood cells that each make antibodies with a single kind of binding site (Parker et al. 2016). A person carries at least \(10^{11}\) different antibodies (Janeway et al. 2001a). During an immune response, the B cells whose antibodies bind the foreign molecule most tightly are selected to multiply, so binding strength tends to rise as the response continues (Janeway et al. 2001a).
Monoclonal and polyclonal antibodies
Antibodies for a test are raised in an animal: a mouse, rabbit, or goat is injected with the target protein. Many of its B cells respond, each to its own epitope, and each multiplies into a clone: cells descended from one parent cell, all identical. The animal’s serum, the liquid part of blood after clotting (Betts et al. 2022), therefore holds a mixture of antibodies against different epitopes, called a polyclonal antibody (Parker et al. 2016, sec. 20.1). A monoclonal antibody comes from a single clone: every molecule has the same sequence and binds the same epitope (Table 40.1). In 1975 Köhler and Milstein fused B cells from an immunized mouse with tumor cells that keep dividing in culture. The fused cell, a hybridoma, keeps dividing in culture like its tumor parent and makes the one antibody of its B-cell parent (Köhler and Milstein 1975; Parker et al. 2016, sec. 20.1).
| Monoclonal | Polyclonal | |
|---|---|---|
| Source | one B-cell clone, grown as a hybridoma | many B-cell clones in an immunized animal |
| Epitopes recognized | one | several |
| Batch to batch | uniform once the hybridoma exists | composition varies |
Monoclonal antibodies, mostly from mouse hybridomas, are preferred for lateral flow tests because one defined antibody can be produced in large quantities (Koczula and Gallotta 2016).
Ranking antibodies by affinity
An antibody’s binding site holds its epitope with a strength called its affinity (Clark et al. 2018). Affinity is quantified by the dissociation constant \(K_D\): the concentration of unbound antigen at which, at equilibrium, half of the antibody’s binding sites hold antigen. Its unit is moles per liter (M), and antibody values are given in nanomolar or picomolar, where \(1~\text{nM} = 10^{-9}\) M and \(1~\text{pM} = 10^{-12}\) M (Brown et al. 2020). The rate constant \(k_\text{on}\) measures how fast antibody and antigen bind and \(k_\text{off}\) how fast the complex dissociates (both defined in binding). Their ratio is \(K_D = k_\text{off}/k_\text{on}\).
Among antibodies that bind a suitable site without cross-reacting, a lower \(K_D\) means the antigen is held more tightly at equilibrium, since less antigen is needed to fill half the sites. The analytical sensitivity of a lateral flow assay is limited in part by the \(K_D\) of its antibody–antigen pair (Koczula and Gallotta 2016); the time to a visible line also depends on \(k_\text{on}\) (binding). A polyclonal antibody mixes binding strengths and has no single \(K_D\).
Example: antibody pairs and a polyclonal lot
A protein carries four epitopes, numbered 1 to 4, with a monoclonal antibody against each. Either antibody of a pair can serve as the capture antibody, so there are \(4 \times 3 = 12\) ordered (capture, detection) pairs. If epitopes 2 and 3 overlap, the pairs (2, 3) and (3, 2) fail, leaving 10. A test line printed with 2.0 µg of a polyclonal antibody of which 2.5% binds the target (illustrative values) carries \(0.025 \times 2.0~\mu\text{g} = 0.050~\mu\text{g} = 50\) ng of target-binding antibody.