Biomat has developed coated plates for Immunotoxicity ELISA tests

Applications & Techniques

Immunotoxocity Plates for ELISA tests

Different studies have indicated that the immune system of humans and animals is a potential target organ for adverse effects caused by drugs, environmental chemicals, and biotechnology derived products, and that damages to the system can be associated with morbidity and even mortality.

Immunotoxicology is the scientific discipline focused on the evaluation of potential adverse effects of xenobiotics (substances of natural or synthetic origin found but not produced in organisms) on the host immune mechanism that can lead to harmful changes in host responses.

The responses might manifest as:

  • an increased susceptibility to infectious diseases and tumorigenesis
  • the induction of hypersensitivity reactions
  • an increased incidence of autoimmune disease

Some regulatory agencies, e.g., Environmental Protection Agency (EPA), Food and Drug Administration (FDA), European Medicines Agency (EMEA), and the Ministry of Health, Labor and Welfare (MHLW) of Japan called for the evaluation of potential adverse effects on the immune system.

T-cell-dependent antibody response (TDAR) test has been identified in a regulatory context (International Conference on Harmonization) as a main functional test with good predictability in detecting potential immunotoxicity of some drugs.

By using exogenous antigens this technique can reflect the overall effect of a tested drug on the immune system and can predict changes in the immune function in addition to permitting an early prevision of the effects of the drugs on immunoregulation and immunotoxicity.

Biomat has developed Coated Plates for Immunotoxicity ELISA test.

ELISA (enzyme-linked immunosorbent assay) is an effective method to measure antibodies as it allows to collect samples serially compared to other methods and minimizing the quantities of reagents and antisera needed in the assay.

Biomat offers 96 Well Plates for Immunotoxicity coated with :

  • KLH (Keyhole Limpet Hemocyanin): can be either used as vaccine carrier protein acting as the hapten carrier part of the vaccine component or as a highly immunogenic antigen in order to assess the immune competence of an organism  and as a carrier of low molecular mass peptide and haptens, such as oligosaccharides, gangliosides or (glyco)peptides, designed to facilitate antibody production.
  • Tetanus Toxoid: commonly called lockjaw, is a serious bacterial disease that affects muscles and nerves. It is characterized by muscle stiffness that usually involves the jaw and neck that then progresses to involve other parts of the body. This disease is caused by neurotoxin from deep wound infection with Clostridium tetani. The use of this product can find application to set up assays where the level of anti-tetanus toxoid antibodies present in biological samples are measured spectrophotometrically.
  • DNP (Dinitrophenol) and TNP (Trinitrophenol): are haptens that can be attached to carrier proteins such as KLH (Keyhole Limpet Hemocyanin) or ovoalbumin; when one of these complexes is injected into appropriate animal models, produce an immune response which is measured as changes in the levels of anti DNP (or anti TNP) IgM and IgG. Thanks to these changes, the researchers can assess the impact of pharmacologic or genetic manipulations on the studied immune system.

For more info: Immunotoxicity ELISA coated surfaces

The preparation of the capture antigen that is coated on the plates is considered a very critical step. This point is where the Biomat 25+ years of experience in coating development brings unique value to the market.

USES

These Coated Plates for Immunotoxicity ELISA test are ideal for the setup of ELISA IgM and IgG assays to be used as biomarkers of immunotoxicity because they will measure levels IgM and IgG of anti KLH, anti DNP, anti TNP and anti-Tetanus toxoid in your samples. 

Anti-KLH, anti-tetanus toxoid, anti-DNP and anti-TNP IgM and IgG are routinely used as biomarkers of immunotoxicity.

In preclinical studies, animals are immunized with an antigen such as KLH while being dosed with a drug candidate. The levels of anti-KLH IgM and IgG are determined in serum or plasma collected at 5-7 or 14-21 days and compared with those in a control group that was not exposed to the drug. A decrease in anti-KLH levels in the treatment group provides evidence of immunosuppression.

Example protocol for mouse anti IgG KLH determination (the test has been carried out using a monoclonal mouse anti KLH )

  1. Add 100 µl of different concentrations of monoclonal mouse IgG anti KLH (BioLegend code 408502 at 0.5 mg/ml, from 0.0005 to 0.500 µg/ml, diluted in Sample Diluent code 400-1-100) to the wells of KLH coated plate and incubate for 60 minutes at room temperature
  2. Empty the wells and wash with Wash Buffer code 200-1-100 four times
  3. Add 100 µl /well of goat anti-mouse IgG -HRP (Jackson ImmunoResearch code 115-035-003, diluted 1:25,000 in Diluent for HRP conjugate code 400-2-100) and incubate for 60 minutes at room temperature
  4. Empty the wells and wash with Wash Buffer code 200-1-100 four times
  5. Add 100 µl /well of TMB substrate solution code 500-1-100) and incubate 15 minutes at room temperature
  6. Stop the substrate reaction by adding 100 µl /well of Stop Solution for substrate code 600-1-100 and read the optical density values at 450 nm

Figure 1: the figure shows the mO.D. directly proportional to the concentration of mouse IgG anti KLH bound to KLH coated microplate.

Figure 2: the figure gives an idea of the dilution factor to apply to the serum/plasma of the immunized mouse under evaluation; where k means a dilution of 1:1,000.


For more information:

Immunotoxicity ELISA coated surfaces

Immunotoxicity Plates

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Biomat Immunotoxicity Plates for ELISA test