Immunoassay- A Powerful Analytical Technique

Immunoassay is a biochemical test that measures the concentration of an analyte in a biological liquid using the reaction of an antibody or antibodies to its antigen. The assay takes advantage of the specific binding of an antibody, which are special proteins made in the body in response to invading microorganisms or other foreign substances, to its antigen. The qualities of immunoassay that makes it one of the powerful analytical techniques are its specificity, sensitivity and versatility.

This technique can measure both the presence of antigen (direct) or antibodies (indirect) in a given specimen. For instance, when seeking to detect the presence of an infection the concentration of antibody specific to that particular pathogen is measured. For measuring hormones such as LH, FSH and Insulin, the hormone acts as the antigen.

Detecting the quantity of antibody or antigen can be achieved by labeling either the antigen or antibody. The label may consist of an enzyme: Enzyme immunoassay (ELISA), radioisotopes such as I-125: Radioimmunoassay (RIA), magnetic labels: Magnetic immunoassay (MIA) or fluorescence. Other techniques include agglutination, nephelometry, turbidimetry and Western Blot. Calbiotech offers the former two-immunoassay kits (ELISA & RIA) with a superior quality. The majority of the Calbiotech kits are in ELISA format.









Figure 1. Simplified scheme of Calbiotech ELISA kits.

In addition to colorimetric (ELISA) assays, Calbiotech also offers chemiluminescent (lumELISA) assays, which employs special type of substrate, known as luminol that emits chemically produced lights. For better sensitivity, dynamic range, linearity and shorter assay time, Chemiluminescent kits are the options.

Commonly the designs of a Calbiotech ELISA kit fall within one of the following types. To keep the assays easy to understand, the brief explanations are accompanied with pictorially representations.

1. Sandwich assay Type

• Antibodies immobilized on the polystyrene micro-titer plate (capture antibody) will bind the target analyte present in samples.
• The second antibody (detection antibody), tagged to an enzyme, will recognize a different epitope on the analyte and binds the target on a different position.
• The unbound analyte and/or detection antibodies will be washed out
• Substrate (TMB) is added. The remaining bound enzyme catalyzes the oxidation reaction of TMB to produce color (Blue).
• The enzymatic reaction is stopped by adding diluted acid to yield a yellow color.
• The yellow color can be read spectrophotometrically at 450nm.


Figure 2. Direct immunometric assay (ELISA) detecting antigen.

The Calbiotech chemiluminescent (lumELISA) version of the typical sandwich assay follows similar protocol to the regular ELISA kits except that luminol substrate is used and the enzymatic reaction of the substrate is not stopped by a diluted acid solution. Once the luminol substrate is added, the photons should be counted immediately using luminometers.


Figure 3. Direct immuno-metric assay (lumELISA) detecting antigen

The typical dose-response curve looks like the following:


Figure 4. Dose-response curve showing direct relationship between signal developed and analyte concentration.

Assays are also designed to measure antibodies, produced by the body against a certain pathogens, in biological samples. In this type of assays, the antibodies are considered as the analyte. Because the analyte is surrounded on two sides, the assay procedure can be categorized as sandwich assay.

In this assay type:

• The pathogen itself is immobilized on the polystyrene micro-titer plate and the target antibody (if present) will bind itself to the antigen
• After un-reacted analyte is washed out , a secondary antibody tagged to the an enzyme is added and binds to the target antibody
• The unbound enzyme tagged secondary antibody is washed out
• Substrate is added and the remaining bound enzyme will catalyze its oxidation reaction to produce color (Blue)
• The oxidation reaction is stopped by adding a diluted acid to yield yellow color.
• The yellow color can be read spectrophotometrically at 450nm.


Figure5. Indirect immunometric assay (ELISA) detecting antibody

In all of the above immunometric assays, the intensity of the color developed is directly proportional to the concentration of the analyte.

Generally sandwich assay designs are suitable if the target molecules are large enough (proteins and antibodies) to bind two separate antibodies at the same time. Competitive assay designs are for small target molecules.

2. Competitive Assay Type

• Capture antibody is immobilized on the polystyrene micro-titer plate
• Both antigens (the analyte and the conjugated antigen) will compete to the same site in the capture antibody
• The plate is washed out to remove unbound free and conjugated antigens.
• Substrate(TMB) is added and the remaining enzyme catalyzes its oxidation reaction to produce color (blue)
• The oxidation reaction is stopped by adding diluted acid to yield yellow color
• The yellow color can be read spectrophotometrically at 450nm


Figure 6. Competitive assay detecting antigen.

In competitive assays, the color developed is inversely proportional to the analyte concentration in the biological sample.


Figure 7. Dose-response curve showing indirect relationship between the signal generated and the analyte concentration.

To learn more about our technologies, download a package insert, MSDS or to place an order for immediate delivery, please visit our website at www.calbiotech.com or CLICK HERE!

New Product Focus: Lysozyme ELISA

New Product Focus
Lysozyme ELISA for the detection of human lysozyme in serum, plasma or stool.

The Calbiotech Lysozyme Assay Kit (Cat.# LS162C) provides researchers with a sensitive assay to measure levels of lysozyme in human samples. This direct sandwich ELISA assay can detect lysozyme from 50ng/ml down to 0.75 ng/mL.

Lysozyme is one of the anti-microbial agents present in spleen, lung, kidney, white blood cells, plasma, saliva, and tears and is also found in human milk. Lysozyme has 130 amino acids and its natural substrate is the bacterial cell wall peptidoglycan. Since synthesized by granulocytes and macrophages, lysozyme can act as a useful marker for myelomonocytic cells. Increased levels of lysozyme in urine and serum are diagnostic indicators for acute monocytic leukemia and acute myelomonycytic leukemia. Elevated lysozyme levels were found in synovial fluids of the inflammatory arthritides and osteoarthritis. Salivary lysozyme, a marker for oral infection and hyperglycemia, might display a significant relationship with hypertension, an early stage of cardiovascular disease.

Increased serum lysozyme activity is also present in tuberculosis, sarcoidosis, megaloblastic anemias, acute bacterial infections, ulcerative colitis and Crohns disease. Elevated lysozyme levels in urine and serum occur during severe renal insufficiency, renal transplant rejection, urinary tract infections, glomerulonephritis and nephrosis.

Principal of the Assay
The human Lysozyme ELISA kit is designed for detection of Lysozyme in human plasma, serum, or stool. This assay employs a quantitative sandwich enzyme immunoassay technique that measures Lysozyme in 75 minutes. A monoclonal antibody specific for Lysozyme has been pre-coated onto a microplate.

Lysozyme in standards and samples is sandwiched by the immobilized antibody and an HRP-conjugate monoclonal detection antibody specific for Lysozyme. All unbound material is then washed away and a peroxidase enzyme substrate is
added. The color development is stopped and the intensity of the color is measured.

Assay Specifications
Run time: 75min
Dynamic range: 0.75 – 50ng/ml
Sample type: Serum, Plasma, Stool
Sample volume: 25ul

This new product is easy to use, more accurate, and faster than its competitors and it comes with the great value and unmatched quality that are built in to all Calbiotech kits. To download the package insert, MSDS or to place an order for immediate delivery, please visit our website at www.calbiotech.com or CLICK HERE!

Preventing Contamination: Tweak your technique.

Calbiotech Immunoassay kits are easy to use, stable and reliable kits. Each kit comes with a package insert that is short and so vividly written that even a new ELISA user (technician) can comfortably perform the assay with a great accuracy and success. Nevertheless, like any other analytical measurement methods, ELISA is also susceptible to some contamination if we do not handle the reagents and/or instruments properly.

Today we will look at some common contamination which can occur during simple assay performance and ways to prevent them.

Cross-Contamination and Prevention

Sample-to-sample (carry-over)

In an assay involving multiple samples, remnants of sample A, after delivery, can mix with the subsequent sample B. This cross-contamination between samples may cause a pseudo positive test result.

Prevention:

• Change the tip after each sample.

Pipette-to-sample

Contaminated tips and/or a contaminated pipette can contaminate samples.

Prevention:

• Change the tip after each sample.
• Only use new or sterilized pipette tips.
• If you think the source of contamination is the pipette, clean or autoclave the parts of the pipette which are in contact with the sample.

Sample-to-pipette

Contamination can occur if sample or any other fluid is allowed to enter the cone of the pipette.

Prevention:

• Keeping the pipette vertical during pipetting can prevent liquids from running into the pipette body. Avoid inclining your pipette excessively.
• Always store the pipette vertically.
• Release the plunger slowly to prevent samples from jumping into the cone of the pipette.

Well-to-well

During assay performance, the washing step in the assay procedure is very critical. It is very possible that wells can cross-talk between each other. Make sure you consider the following points:

Prevention:

• Do not over fill the wells with wash buffer during the washing step.
• Clean your washing machine system (or bottle, if manual washing is used) regularly.
• Use clean distilled or de-ionized water while diluting the concentrate wash buffer provided in Calbiotech ELISA kits.

We hope you found our quick note on preventing cross contamination useful. Please share anything you think we missed in the comment section of this blog.

For technical support with running any of our assays, please contact support@calbiotech.com and we will get back to you immediately.