Response of Antigen Antibody Reaction

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BACKGROUND

The Antigen-Antibody reaction is an association between Antigen and Antibody. It is abbreviated as Ag-Ab reaction. The Ag- Ab reaction binds the antigen to antibodies to form antigen-antibody complexes. However the bonding of Antigen & Antibody is reversible in nature. Sometimes they produce immunity and sometimes they cause allergy & other incompatibilities.¹

So the basic objective of the test is to determine the response of antigen-antibody reaction.

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REQUIREMENTS

Chemicals:     Chloramphenicol Standards [Negative control, Concentrations 0.05, 0.15, 0.5, 1.5, 4.5 ng/ml]

        Wash Buffer [1X Wash Solution {Mix 1 ml of 20X wash solution with 19 ml distilled water}]

        HRP conjugate- Horseradish Peroxidase- 50 µl

        TMB Substrate [3,3′, 5,5′- Tetramethylbenzidine]- 100 µl

        Stop Buffer -100 µl (given in the ELISA KIT)

Apparatus:    96 well micro plate (antibody coated)

        Micropipette (100-1000 µl; 20-200 µl)

        Microtips (100-1000 µl; 20-200 µl)

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PROCEDURE

Before use the reagents are set to normal room temperature. Then from all standards (Chloramphenicol Standard) 100 µl is added in different wells from lower to higher concentrations order. 100 µl of sample is also added into different wells. 50 µl of HRP Conjugate is added to all the wells.² The wells are mixed for 30 seconds by gentle rotation. The plates are incubated for 30 minutes at room temperature in dark. After incubation wash the plate thrice with wash solution (250 µl). After washing pat dry the plate with tissue or towel by gently inverting it. Then 100 µl of TMB substrate is added. Again the wells are mixed for 30 seconds by gentle rotation. The plates are then incubated for 30 minutes in dark at room temperature. Then to stop enzyme reaction 100 µl stop buffer is added. Immediately read the absorbance of the standard and sample of the plate on a reader (Microplate reader) with 450 nm wavelength.³

Calculation

The mean relative absorbance percentage is obtained from each reference standard against its concentration in ng/ml (nanogram/millilitre).

Mean Relative absorbance (%) = (Absorbance Standard or Sample) / (Absorbance Zero Standard) × 100

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CONCLUSION

To determine the response of Ag-Ab reaction the absorbance of the plate wells is measured.

Figure 1: Standard and Sample Color After Adding Hrp Conjugate.

Figure 2: Standard and Sample Color After Adding Tmb Substrate.

Figure 3: Standard & Sample Color After Adding Stop Buffer.

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REFERENCES

1. Chaudhury MK, Good RJ, van Oss. Nature of the antigen-antibody interaction. Journal of Chromatography.1986:376: 111.
   
2. Martin TL, Mesulam MM, Mufson EJ. The light side if Horseradish peroxidise histochemistry. J Histochem. 1984:32(7):793.
   
3. Schmidt, Mazzella, Mathews; Methods in Molecular Biology, 2012: 507.