Antibodies are Y-shaped biomolecules made in the B lymphocytes or B cells (specialized white blood) cells during an adaptive immune response. The B cells release the antibodies in their millions into the bloodstream and lymphatic systems, where they bind to and destroy corresponding antigens (foreign substances like disease-causing pathogens).
Antibodies’ primary characteristics include specificity ( they attach to specific antigens), immunological memory Maintain a record of previously-encountered pathogens) and immune tolerance (preserve healthy cells).
Consequently, antibody detection in blood samples aids in applications like clinical research, diagnostics, therapeutics, forensics, and serological safety. Below are five techniques used to detect antibodies in blood samples.
Enzyme-linked Immunosorbent Assay (ELISA) Test
Enzyme-linked immunosorbent assays (ELISA) tests primarily detect proteins like antibodies and antigens in biological samples like blood. They build on the biomolecules’ specificity characteristics to detect the presence and concentration/ quantity of a protein analyte; the analyte could be an antigen or an antibody.
ELISA kits are plate-based test kits utilizing 96- and 384- multi-well plates; a 384-well plate facilitates testing multiple blood samples simultaneously, saving time. Also, depending on the protein analyte, ELISA kit plates may feature immunological, non-treated, and cell culture surfaces. However, all ELISA test kits, regardless of the surface, all ELISA test kits adhere to the ELISA principle.
So, What is the ELISA principle? The principle of ELISA builds on antigen-antibody interaction to capture the target analyte.
ELISA protocols for manufacturing an immunological test kit are coating, incubation, and detection.
Coating entails applying an immobilized antigen on the ELISA kit well plates. Depending on the ELISA test type, the wells may feature a capture antibody that immobilizes the antigen.
After immobilizing the target antigen, ELISA kit manufacturers incubate the plate wells. Incubation entails creating an antibody-antigen complex. Depending on the ELISA kit type, the manufacturers incubate the wells several times, breaking each incubation routine with a washing stage.
The incubated molecules feature conjugated enzymes, hence the name enzyme-linked immunosorbent assay.
Lastly, target protein detection is the goal of ELISA test kits, so the kits feature substrates like biotin to facilitate colourimetric detection. The substrates generate water-soluble coloured by-products to facilitate detection.
Despite the ELISA principle being the core reference in how the kits detect antibodies in the blood, the protocols vary slightly according to the ELISA types. The four ELISA test types are direct, indirect, competitive, and sandwich. Each type has its protocol variation, pros, and cons.
Western Blot/ Immunoblotting
Western blotting or immunoblotting is among the most popular biomolecule deception techniques to identify target molecules from a complex mixture. Like the ELISA technique, the Western blot leverages antibody specificity to detect antibodies in a blood sample.
The technique identifies antibodies and determines their sizes or molecular weight and quantity in the complex mixture. Below is an overview of the Western blot protocol to better understand its efficiency in detecting antibodies from blood samples.
While ELISA kits utilize well plates, the Western blot technique utilizes gel electrophoresis as its detection medium. Gel electrophoresis is a macromolecule separation technique that passes an electric current through a special gel to separate macromolecules like proteins by their molecular weight.
The electric current propels the macromolecules through the goal at different speeds based on their molecular weight, forming protein bands. So, macromolecule sorting by size is the first stage in the Western lot protocol.
The second is transferring the sorted protein bands onto a blotting membrane or solid support. The blotting membrane undergoes blocking during the transfer to prevent non-specific binding to the membrane. Non-specific binding compromises the detection process because specific bands will not show.
The Western blotting protocol ends with visualization using staining techniques to identify the target antibody. Staining in Western blotting entails using secondary antibodies.
Enzyme-linked Immunospot (ELISPOT)
An Enzyme-linked Immunospot (ELISPOT) assay is an immunoassay technique employing a similar analyte detection protocol to the ELISA protocol described above. However, the primary difference between the two antibody detection techniques is that while ELISA assays detect antibody presence and quantity, ELISPOT assays detect cellular immune responses.
So, how do ELISPOT kits work? The ELISPOT protocol substitutes the target antigen immobilized in the ELISA coating stage with cytokine-specific monoclonal antibodies (mAbs).
Second, the incubation agents in ELISPOT are the cells under observation ( primarily cytokines because they produce antibodies. However, ELISPOT utilizes similar enzyme conjugates to ELISA assays (biotin and streptavidin) to detect the immune response in the substrates.
ELISPOT assays detect the volume of target antibodies produced by cytokines and are ideal for supplementing ELISA assays for comprehensive blood antibody content readings.
Latex Agglutination/ Latex Fixation Test (LAT)
The late agglutination test (LAT) leverages the antigen-antibody locking mechanism to detect specific antibodies in body fluid samples, including blood. The technique is among the most prevalent disease-detection methods in diagnostics or disease detection.
LAT antibody detection entails coating latex balls with a target antigen and dipping them in a blood sample. The antigen-coated balls clump together (agglutinate) if the corresponding antibody is in the blood sample.
Microparticle Enzyme Immunoassay
The Microparticle Enzyme Immunoassay (MEIA) also relies on the antibody-antigen complex under the ELISA principle to detect antibodies in blood samples. However, the primary difference between MEIA and ELISA assays is that the former features microspheres that function as solid-phases supports over a suspension rather than welled plates.
Moreover, one study comparing the two immunoassay techniques established that MEIA is faster and more accurate than ELISA. Therefore MEIA assays are prevalent in therapeutic monitoring where speedy results are necessary.
Conclusion:
Each blood antibody technique has advantages and disadvantages based on sensitivity, accuracy, speed, and volume, among other parameters. Therefore, each assay has its ideal application in research and medicine based on suitability and cost.
