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Serology Assays for Antibody Detection

As clinical diagnostic and surveillance tools, serological assays such as the enzyme-linked immunosorbent assay (ELISA) and the lateral flow immunoassay (LFIA) have been extensively used in the detection and identification of infectious diseases. They achieve this aim through the use of highly specific recombinant antigens, or fusion proteins, to detect serum antibodies, which are used by the immune system to neutralize pathogenic bacteria, viruses or other microorganisms that cause disease. Serological assays have been successfully used in the detection of hepatitis viruses, the ebola virus, HIV and influenza.



Serology


Serology is the investigation of blood serum, the clear fluid that separates when blood clots. In research and clinical diagnostic studies, the term serology is synonymous with the identification of antibodies in the serum produced in response to a microbial infection. When a foreign microbe is introduced to an organism, the immune system initiates a number of mechanisms for its elimination, one of the most prominent being antibody production.

 

Immune system and the antibody response


Antibodies are produced by specialized white blood cells known as B lymphocytes or B cells. Expressed on the cell surface of B lymphocytes, B cell receptors (BCRs) bind to specific antigens against which it will initiate and antibody response. Upon activation by antigen binding, B lymphocytes proliferate and differentiate into B effector cells, which produce antibodies specific to the foreign antigen. Once antibodies with sufficient specificity to the antigen can be encoded, B effector cells secrete antibodies into the bloodstream. These antibodies then bind specifically with the foreign antigen for their elimination by the immune system.

 

Antibody structure and isotypes


Antibodies exist as Y-shaped units comprised of four polypeptide chains, two light chains and two heavy chains. Light chains are composed of a constant domain (e.g. kappa or lambda) which determines the class of the light chain, and one variable domain that is important for binding to the antigen. The heavy chain contains several constant domains and one variable domain for antigen binding. The composition of the heavy chain determines the overall isotype of each antibody.

Antibody isotypes: IgG, IgM, IgA, IgD and IgE

In mammals, specifically humans, antibodies are divided into five isotypes: IgG, IgM, IgA, IgD, IgE. The following table summarizes the properties and functions of each isotype.

 

Table 1. Antibody isotypes: IgG, IgM, IgA, IgD and IgE For Antibody structure and isotypes

Immunoglobulin
Isotype
Mol. Wt.
Structure
Function
IgGIgG1, IgG2a, IgG2b, IgG3, IgG4150 kDaMonomerIs the most abundant representing approximately 75 to 80% of human antibodies. Accounts for a majority of the antibacterial and antiviral activity in serum. They are produced mostly towards the end of a new infection and are most effective at eliminating known pathogens. In serological testing, the detection of IgG antibodies in serum is significant of a past infection.
IgMIgM900 kDaPentamerThe largest size antibodies, IgM are responsible for the agglutination of infected cells for elimination via phagocytosis. B lymphocytes produce IgM antibodies first in response to microbial infection. Although they have a lower affinity for antigens compared to IgG antibodies, the avidity of IgM antibodies is significantly higher as a result of its pentameric structure.
IgAIgA1, IgA2150-600 kDaMonomer/TetramerMost abundant in serum, saliva, mucus and intestinal fluid. They are dimeric antibodies capable of binding two antigens simultaneously to form large antigen-antibody complexes. IgA antibodies are responsible for the neutralization of viruses and the inhibition of microbial growth.
IgDIgD150 kDaMonomerTheir exact function largely remains unknown. IgD antibodies are expressed on the surface of B cells and are involved in pathogen binding.
IgEIgEMonomer190 kDaPlays a vital role in defending against parasitic infections and allergic reactions. IgE antibodies attach to tissue cells, especially mast cells, a special form of white blood cells. Mast cells are responsible for allergic symptoms such as reddening and hives on the skin or asthma-like breathing difficulties.



Significance of IgG and IgM determination in serology


Serological testing is used to detect an excess or deficiency in the two major classes of immunoglobulins, IgG and IgM. This provides vital information regarding the current status of an organism's immune systems and is used to help diagnose infectious diseases that affect the levels of one or more of these Ig classes. If an excessive amount of one or more of these Ig classes is present, further testing by immunofixation can be done to determine if the Ig comes from clones of an abnormal B lymphocytes (a characteristic of multiple myeloma).

The following table outlines the diagnostic interperation of possible scenarios that can be encountered when performing serological assays.

 

Interpretation of serological test results

IgG Test Result IgM Test Result Diagnostic significance
- - Indication of no infection or that the organism may be in the window period of infection.
- + Organism may be in the early stage of infection.
+ + Organism is in the active phase of infection.
+ - Organism may be in the late or recurrent stage of infection. Or may have had a past infection, but has recovered.



Types of serology assays


Serology assays, such as ELISA and LFIA, are the primary diagnostic method by which laboratories monitor infections with viruses and other various pathogens. While serologic tests are designed to be sensitive and specific, false-positives and false-negative results do occur. Therefore, it is strongly recommended to confirm new positive findings by both alternative diagnostic methods and testing additional subjects. Serological tests that have been rigorously validated for accuracy have the potential to transition from the laboratory setting to the point-of-care. Point-of-care-testing is a medical diagnostic test administered at or near the patient, such as a medical facility. (It is important to note that AAT Bioquest provides reagents intended for research use only.)

Enzyme-linked immunosorbent assays (ELISA)

The indirect ELISA method is a serological assay often used for the detection of primary antibodies directed for a specific antigen. In this method, the microbial-specific antigens are first immobilized onto the surface of a microtiter plate. Whole blood, plasma or serum samples are incubated in the well, to which primary antibodies may bind forming an immune complex with the antigens. Unbound primary antibodies are removed by washing, and the samples are then incubated with enzyme-labeled secondary antibodies directed against the host and target species of the primary antibody being used (Table 1). After washing away unbound secondary conjugates. a colorless substrate is introduced to the sample, which reacts with the enzyme conjugate and produces a measurable byproduct. Depending on the choice of substrate, this byproduct can be either colorimetric, chemiluminescent or fluorescent (Table 2). The magnitude of signal production is proportional to the amount of antigen in the sample. Use of a secondary detection reagents has significant advantages over direct ELISA, namely signal amplification, which arises from the specificity of the secondary antibody for a designated region on the primary antibody (e.g. Fc region). This permits multiple secondary antibodies to bind to a single primary antibody, amplifying the signal and improving sensitivity.

For in-depth info about ELISAs and secondary antibodies, click on:

 

Indirect ELISA
Figure 1. Indirect ELISA illustration.

 

Horseradish peroxidase (HRP)-labeled secondary antibody conjugates

Conjugate Host Isotype Species reactivity Enzyme label Unit size Cat No.
HRP Goat Anti-human IgG (H+L) Goat IgG Human HRP 200 µg 50262
HRP Goat Anti-human IgG (H+L) Goat IgG Human HRP 1 mg 50263
HRP Goat Anti-mouse IgG (H+L) Goat IgG Mouse HRP 200 µg 16728
HRP Goat Anti-rabbit IgG (H+L) Goat IgG Rabbit HRP 1 mg 16793


Substrates for detecting horseradish peroxidase (HRP)-labeled secondary antibody conjugates.

Substrate Detection Absorbance (nm) Ex (nm) Em (nm) Unit size Cat No.
ABTS Colorimetric 420 nm - - 1 L 11001
TMB Colorimetric 450 nm / Yellow
650 nm / Blue
- - 100 mL
1 L
11012
11003
Amplite™ Blue Fluorimetric - 324 nm 409 nm 25 mg 11005
Amplite™ ADHP Fluorimetric - 570 nm 583 nm 25 mg 11000
Amplite™ Red Fluorimetric - 570 nm 583 nm 1000 Assays 11011
Amplite™ IR Fluorimetric - 646 nm 667 nm 1 mg 11009
Luminol Chemiluminescent - - 410 nm 1 mg 11050



Lateral flow immunoassay (LFIA)

Lateral flow assays are paper-based diagnostic tests for the rapid detection and quantification of a target antigen. Because of its low development cost and ease of production, LIFAs have been extensively used in clinical laboratories for the qualitative and quantitative detection of specific antigens and antibodies. A variety of biological samples can be tested using LFIAs, including whole blood, plasma, serum, urine, saliva and sweat.

LIFA is based on four immunologic principles: particle agglutination (clumping), immunodot, immunofiltration and immunochromatography. First a liquid sample containing the target antigen is applied to the adsorbent sample pad at one end of the test strip. The sample pad, which contains buffer salts and surfactants, facilitates the binding of antigens in the sample to the 'capture' conjugates and on the membrane. The treated sample migrates through the conjugate release pad, which contains fluorescently labeled antibodies that are specific to the target antigen. The sample, now containing the conjugated antibody bound to the target antigen, migrates along the strip and into the detection zone, which is a porous neutrocellulose membrane comprised of specific biological components (e.g. antibodies or antigens) immobilized in lines. The significance of the detection zone is to react with the initial fluorescent antibody-antigen complex. Recognition of the sample antigen results in an appropriate response on the test line, while a response on the control line indicates the proper liquid flow through the strip. The read-out, represented by the lines appearing with different intensities, can be measured using a fluorescent strip reader.

Advantages of LIFA

  • High sensitivity and specificity
  • Simple one-step assay, no wash steps
  • Requires low sample volume for testing
  • Rapid results (5-30 minutes)
  • Opportunity for multiplex analysis using fluorescently-labeled antibodies
  • Compatible with whole blood, plasma, serum, urine , saliva and sweat

For in-depth info about primary antibodies and fluorescent secondary antibodies, click on: