Precipitation and agglutination are the visible expression of the aggregation of antigens and antibodies through the formation of a framework in which antigen particles or molecules alternate with antibody molecules (Fig. 10-1). Precipitation is the term for the aggregation of soluble test antigens. Precipitation is the combination of soluble antigen with soluble antibody to produce a visible insoluble complex. Agglutination is the process whereby specific antigens (e.g., red blood cells) aggregate to form larger visible clumps when the corresponding specific antibody is present in the serum.

Figure 10-1 Agglutination patterns.
A, Slide agglutination of bacteria with known antisera or known bacteria. *Left,* Positive reaction; *right,* negative reaction. B, Tube agglutination. *Left,* Positive reaction; *right,* negative reaction. (From Barrett JT: Textbook of immunology, ed 5, St Louis, 1988, Mosby.)

Artificial carrier particles may be needed to indicate visibly that an antigen-antibody reaction has taken place; examples include latex particles and colloidal charcoal. Cells unrelated to the antigen, such as erythrocytes coated with antigen in a constant amount, can be used as biological carriers. Whole bacterial cells can contain an antigen that will bind with antibodies produced in response to that antigen when it is introduced into the host (Table 10-1).

Table 10-1

Examples of Carriers

Type (Reagent)	Type of Assay	Principle	Result
Latex particles	C-reactive protein (CRP)	A suspension of polystyrene latex particles of uniform size is coated with the IgG fraction of an antihuman CRP-specific serum.	If CRP is present in the serum, an antigen-antibody reaction takes place. This reaction causes a change in the uniform appearance of the latex suspension and a clear agglutination results.
Stabilized sheep erythrocytes sensitized with rabbit gamma globulin suspended in buffer solution	Rheumatoid factor (RF)	RF acts like antibodies against gamma globulin that acts as the antigen.	If gamma globulin is attached to a particular carrier (e.g., RBCs or latex particles), the reaction of RF with gamma globulin becomes a visible agglutination.

The quality of test results depends on the following technical factors:

• Time of incubation with the antibody source (e.g., patient serum)

• Amount and avidity of an antigen conjugated to the carrier

• Conditions of the test environment (e.g., pH, protein concentration)

Agglutination tests are easy to perform and, in some cases, are the most sensitive tests currently available. It is important to note that quality results are dependent on the proper training of the person performing the assay and adherence to strict quality control regulations (e.g., positive and negative control sera). Agglutination-type tests have a wide range of applications in the clinical diagnosis of noninfectious immune disorders and infectious disease.

In latex agglutination procedures (Box 10-1), antibody molecules can be bound to the surface of latex beads. Many antibody molecules can be bound to each latex particle, increasing the potential number of exposed antigen-binding sites. If an antigen is present in a test specimen such as C-reactive protein, the antigen will bind to the combining sites of the antibody exposed on the surface of the latex beads, forming visible cross-linked aggregates of latex beads and antigen (Fig. 10-2). In some procedures (e.g., pregnancy testing, rubella antibody testing), latex particles can be coated with antigen. In the presence of serum antibodies, these particles agglutinate into large visible clumps.

Box 10-1 Immunologic Assays Performed by Latex Particle Agglutination

• C-reactive protein

• Immunoglobulin G rheumatoid factors

• Immunoglobulin M rheumatoid factors

• Rubella antibody

Procedures based on latex agglutination must be performed under standardized conditions. The amount of antigen-antibody binding is influenced by factors such as pH, osmolarity, and ionic concentration of the solution. A variety of conditions can produce false-positive or false-negative reactions in agglutination testing (see Table 10-4).

Coagglutination and liposome-enhanced testing are variations of latex agglutination (Fig. 10-3). Coagglutination uses antibodies bound to a particle to enhance the visibility of agglutination. It is a highly specific method but may not be as sensitive as latex agglutination for detecting small quantities of antigen.

Figure 10-3 Diagram of liposome-enhanced latex agglutination reactions. (Adapted from Neo-Planotest Ducoclox slide test, Organon Teknika, Durham, NC.)

Pregnancy Testing

The principle of antigen and antibody interaction has been applied to pregnancy testing since the first agglutination tests were developed in the 1960s. These assays have replaced animal testing.

Human Chorionic Gonadotropin

Pregnancy tests are designed to detect minute amounts of human chorionic gonadotropin (hCG), a glycoprotein hormone secreted by the trophoblast of the developing embryo that rapidly increases in the urine or serum during the early stages of pregnancy.

This glycoprotein hormone consists of two noncovalently linked subunits, alpha (α) and beta (β). The α unit is identical to that found in luteinizing hormone (LH), follicle-stimulating hormone (FSH), and thyroid-stimulating hormone (TSH). The β subunit has a unique carboxy-terminal region. Using antibodies made against the β subunit will cut down on cross-reactivity with the other three hormones. Accordingly, many pregnancy test kits contain monoclonal antibody (MAb) directed against the β subunit to increase the specificity of the reaction.

For the first 6 to 8 weeks after conception, hCG helps maintain the corpus luteum and stimulate the production of progesterone. As a general rule, the level of hCG should double every 2 to 3 days. Pregnant women usually attain serum concentrations of 10 to 50 mIU/mL of hCG in the week after conception. If a test is negative at this stage, the test should be repeated within a week. Peak levels are reached approximately 2 to 3 months after the last menstrual period (LMP).

Agglutination Inhibition

The determination of in vitro agglutination inhibition depends on the incubation of the patient’s specimen with anti-hCG, followed by the addition of latex particles coated with hCG. If hCG is present, it neutralizes the antibody; thus, no agglutination of latex particles is seen. If no hCG is present, agglutination occurs between the anti-hCG and hCG-coated latex particles.

Pregnancy Latex Slide Agglutination

Alternate Procedural Protocols

Latex agglutination slide tests have been replaced in many situations (e.g., home testing; see Chapter 9) by one-step chromatographic color-labeled immunoassays for the qualitative detection of hCG in urine (e.g., Clearview hCG II and Clearview hCG Easy, Wampole Laboratories, Princeton, NJ). Another variation is a one-step chromatographic color-labeled immunoassay for use with urine or serum (e.g., Wampole PreVue hCG Stick or Cassette, Status hCG).

Flocculation Tests

Flocculation tests for antibody detection are based on the interaction of soluble antigen with antibody, which results in the formation of a precipitate of fine particles. These particles are macroscopically or microscopically visible only because the precipitated product is forced to remain in a confined space.

Flocculation testing can be used in syphilis serologic testing (see Chapter 18). These tests are the classic Venereal Disease Research Laboratories (VDRL) and rapid plasma reagin (RPR) tests. In the VDRL test, an antibody-like protein, reagin, binds to the test antigen, cardiolipin-lecithin–coated cholesterol particles, and produces the particles that flocculate. In the RPR test, the antigen, cardiolipin-lecithin–coated cholesterol with choline chloride, also contains charcoal particles that allow for macroscopically visible flocculation.