Antigen-antibody reactions are based on molecular interactions between antigenic epitopes and specific polyclonal or monoclonal antibodies. Precipitin procedures such as double immunodiffusion and counterimmunoelectrophoresis have been supplanted by easier and more rapid techniques (Fig. 1). The development of hybridoma technology allowed monoclonal antibodies to be produced against many clinically relevant microbial antigens, including surface proteins and exotoxins. Several antigen detection tests are potentially applicable to ocular infections (Table 1) and are most useful when infectious agents are difficult to culture.¹

TABLE 1. Immunologic Methods for Diagnosis of Ocular Chlamydial and Viral Infections

IMMUNOFLUORESCENCE

Fluorochrome-conjugated antibodies allowed to react with appropriate microbial antigens can be detected by an immunofluorescent method. Fluorescein is the most common conjugate, resulting in a bright apple-green color by fluorescent microscopy. Unlike other immunodiagnostic methods, immunofluorescence and immunoperoxidase staining are microscopic techniques that allow the presence of inflammatory cells to be noted in the background.

Direct immunofluorescence involves direct application of the fluoresceinated antibody to the specimen (Fig. 2). Indirect immunofluorescence entails mixing nonconjugated antibody with the specimen followed by application of a fluoresceinated antibody targeting the initial antibody (e.g., fluorescein-conjugated antihuman antibody). The indirect technique is usually more sensitive than the direct method. Distinguishing fluorescing apple-green particles from yellow, refractile debris relies on skilled observation.

Specimens are smeared onto a clean glass slide and fixed in cold methanol² or acetone. An immunofluorescent test is useful for diagnosing herpes simplex virus epithelial keratitis,^3–7,7a varicella-zoster virus epithelial keratitis,⁷ adenovirus conjunctivitis,⁸ and chlamydial conjunctivitis.^9,10 Immunofluorescence and immunoperoxidase can also demonstrate several bacteria, fungi, and protozoa^11,12,12a of ocular importance.

AGGLUTINATION REACTIONS

Particles coated with antibody can detect microbial antigens by producing agglutination. This immunochemical reaction causes visible clumping of the carrier material, whether erythrocytes, bacteria, synthetic beads, or phospholipid liposomes. Staphylococcus aureus is often used as a bacterial carrier in coagglutination reactions because its protein A naturally binds the F_c portion of most immunoglobulin subclasses, thereby leaving the immunoreactive end exposed. Latex and other inert materials (e.g., bentonite and collodion) are used in several commercial assays for evaluating common causes of meningitis and pharyngitis (e.g., Haemophilus influenzae, Streptococcus pneumoniae, group B streptococci, and Neisseria meningitidis) and can be applied to ocular infections.¹³ These assays detect soluble antigens as well as intact bacteria.

SOLID-PHASE IMMUNOASSAYS

Solid-phase assays are similar to agglutination reactions but use fixed polystyrene beads or wells that retain the microbial antigen. After the specimen is mixed with bound antibody, a tagged antibody is applied that further binds to the antigen, thus forming a sandwich of antigen between two antibodies. If the conjugate is a radioisotope (e.g., ³²P or ¹²⁵I), it is detected in a scintillation counter by radioimmunoassay. If the tag is an enzyme (such as horseradish peroxidase or alkaline phosphatase), the enzymatic substrate is added to yield a colored end product in an enzyme immunoassay, also called an enzyme-linked immunosorbent assay (ELISA). Chemiluminescent and other tags are under development. Several viral,^4,14 chlamydial,⁹ bacterial, fungal, and parasitic antigens can be detected with commercially available enzyme immunoassays.

IMMUNODETECTION OF OCULAR INFECTIONS

Rapid diagnostic tests have high predictive value (Table 2) for adenovirus conjunctivitis,^8,15,16 herpes simplex virus dendritic epithelial keratitis,^4,7,12,17 and chlamydial conjunctivitis.^9,18,19

TABLE 2. Performance of Immunodiagnostic TestsCompared to Culture for Ocular Infections

TUBERCULOSIS

The tuberculin skin test (Mantoux test) uses purified protein derivative (PPD). After subcutaneous injection of 0.1 mL PPD, the extent of induration is used to assess the T-cell responsiveness of the individual to tuberculin antigen. The more inflammation, the higher the chance of being infected. Lymphocytic swelling greater than 15-mm diameter is indicative of tuberculosis. A cut point of 10 mm is used for patients at higher risk of tuberculosis, and a reading of 5 mm may be sufficient for those at very high risk.²¹ Companion anergic testing (e.g., candidin, tetanus toxoid, and/or mumps skin tests) for evaluating false-negative PPD testing is no longer widely recommended.

HISTOPLASMOSIS

The histoplasmin skin test has been used in the diagnosis of presumed ocular histopasmosis.²² However, potential reactivation after skin testing,²³ false-positive testing, uncertain diagnostic correlation, and reduced availability²⁴ limit the general use of this method in ophthalmology.

OTHER INFECTIONS

Skin tests have been applied in the diagnosis of ocular leishmaniasis,²⁵ blastomycosis, cat-scratch disease, and tularemia. Alternative tests are now available for these conditions.

LOCAL ANTIBODIES

The presence of antibodies in the ocular fluids may indicate local production, sequestration, or diffusion from the blood. Tears can be used for detecting specific immunoglobulins during infections of the ocular surface.²⁶ A high titer of specific immunoglobulin A indicates active infection.²⁷

Intraocular cellular immunity is partly under local control.²⁸ Specific antibodies in the tear film can be detected in the intraocular fluids. Aqueous humor and vitreous have been used to evaluate the humoral immune response to bacterial,²⁹ viral,^30,31 and parasitic^32,33 infections within the eye. Some have used this method to evaluate patients with uveitis.^34,35

The demonstration of local antibody production can be assessed by measuring the ratio of specific antibody relative to the total amount of immunoglobulin. Albumin concentrations in the eye and serum may be substituted for the total immunoglobulin levels. The antibody coefficient (C), also known as the Goldmann-Witmer, Dernouchamps, or Desmonts coefficient, is calculated by the following formula:

C = (inverse titer of specific antibody in ocular fluid) × (total immunoglobulin level in serum)(inverse titer of specific antibody in serum) × (total immunoglobulin level in ocular fluid)

This ratio estimates the intraocular antibody level relative to serum by accounting for diffusion of similar macromolecules across the blood–eye barrier. The higher the antibody coefficient, the more likely there is local production of specific antibody rather than just leakage across a disrupted blood–ocular barrier. Coefficient values greater than 4 are strongly suggestive of ocular infection.³⁶

CIRCULATING ANTIBODIES

Serum is commonly used for the serologic diagnosis of infectious diseases. In ophthalmology, serology is most often used to detect prior exposure and, less often, to confirm the diagnosis of a primary infection. Acute infections are diagnosed by a fourfold or greater rise in antibody titer. Identifying seroconversion requires at least two different samples separated by a sufficient time interval.

Because antibodies persist for years, a single positive test does not indicate when exposure took place. For prevalent conditions, such as chlamydial and herpes simplex virus infections, serologic testing is generally limited to epidemiologic surveys. Because many healthy people have had prior exposure to these agents, a positive antibody test during reinfection or reactivation of a latent infection must be interpreted with caution in the diagnosis of ocular disease. A true negative test, however, helps to exclude a particular etiology from the differential diagnosis.

SEROLOGY OF OCULAR INFECTIONS

Serologic testing is most useful for diagnosing diseases caused by organisms that are difficult to identify by other means (Table 3). The detection of specific immunoglobulin (Ig) M) or a rising titer of specific IgG can help to confirm viral conjunctivitis due to adenovirus, enterovirus 70, or herpes simplex virus. Examples of ocular infections diagnosed by serological testing are syphilis, Lyme disease, toxoplasmosis, toxocariasis, Epstein-Barr virus (EBV) infection, human T-cell lymphotropic retrovirus type 1 (HTLV-1) disease, and acquired immune deficiency syndrome (AIDS). Molecular mimicry in HLA-B27 associated anterior uveitis with bacterial antigens can be examined with serologic tests.³⁷

TABLE 3. Serology of Ocular Infections

Adenovirus

Paired sera can be used in detecting a rising titer of specific anti-adenovirus antibodies during adenovirus conjunctivitis. Discrepancies and cross-reactions make serologic interpretation difficult.³⁸

Herpesviruses

Paired sera show diagnostic rising antibody titers during primary infection with herpes simplex virus (HSV). Circulating IgG, but not IgM, persists for life in most people. Seroepidemiologic studies show that more than 80% of adults have detectable HSV-1 IgG. Nondiagnostic fluctuation in serum IgG may occur during recurrent ocular herpes.³⁹ Secretory IgA can be detected in the tear film during recurrent HSV keratitis.^40–42 IgG can be detected in the aqueous humor during HSV uveitis or retinitis.

In EBV infection, the antibody profile can help distinguish recent from remote infection. Heterophil antibody and antibody to early antigen (EA) are present only during acute infection. Immunoglobulin G to viral capsid antigen (VCA) begins early and persists for years. Antibody to viral nuclear antigen (EBNA) develops later and then persists.

Human Retroviruses

Human T-cell lymphotrophic virus type 1 (HTLV-1) is a cause of adult T-cell leukemia/lymphoma and a chronic myelopathy (tropical spastic paraparesis). This retrovirus has been seroepidemiologically associated with intraocular inflammation in endemic areas such as Japan, the Caribbean, and central Africa. Serum is screened for seropositivity by agglutination or ELISA and then sent for Western blot confirmation.

Screening for human immunodeficiency virus (HIV-1) infection is commonly done by an enzyme immunoassay to detect anti-HIV antibodies. Positive results are verified by a Western blot procedure that uses viral proteins separated by gel electrophoresis so that two or more antibodies to HIV proteins (p24 and p31) and glycoproteins (gp41 and gp120/gp160) can be detected by an enzyme-labeled antihuman immunoglobulin reaction.

Spirochetes

The diagnosis and management of syphilis rely on nontreponemal and treponemal antibody tests. Nontreponemal tests, based on the original Wassermann complement-fixation method, detect antibodies to phospholipids. These anticardiolipin antibodies are measured by the Venereal Disease Research Laboratory (VDRL) and rapid plasma regain (RPR) tests and give a quantitative titer that is followed to assess treatment efficacy. A positive nontreponemal test can occur during other conditions such as systemic lupus erythematosus. Specific antibodies to Treponema pallidum are measured by an indirect immunofluorescent test (FTA-ABS) or a hemagglutination assay (MHA-TP). A reactive treponemal test is present during active syphilis and after adequate treatment.

Specific antibodies to Borrelia burgdorferi can be measured in Lyme borreliosis and confirmed by Western blot analysis. IgM and IgG antibodies can be measured in serum and cerebrospinal fluid.⁴³.

Bartonella henselae

Specific IgM, IgG, and IgA antibodies can be detected by enzyme immunoassay for diagnosing cat-scratch disease.

Toxoplasma gondii

Finding specific antibodies at any titer by indirect immunofluorescence or enzyme immunoassay supports the clinical diagnosis of ocular toxoplasmosis. A serologic pattern of toxoplasmic infection in the distant past consists of IgG and no IgM or IgA. The presence of high-titered IgG and detectable IgM and/or IgA (by either ELISA, indirect immunofluorescent assay [IFA], or immunosorbent agglutination) indicates recently acquired infection. Different methods for detecting IgG, such as the differential agglutination test and the IgG avidity test, also help to differentiate chronic and acute infections.⁴⁴

Toxocara canis

A positive ELISA test for antibodies, regardless of titer for Toxocara canis, aids in the laboratory evaluation of ocular toxocariasis. A specific serologic test for Toxocara cati is also available. A high intraocular antibody level provides additional laboratory confirmation.

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