Appendix XV L. Immunonephelometry for Vaccine Component Assay
GENERAL PRINCIPLE
The general principle of nephelometry is described in general chapter 2.2.1. Clarity and degree of opalescence of liquids. Immunonephelometry measures turbidity that is mainly due to immune complexes formed by the antigen-antibody reaction. Although immunonephelometry is applicable for the quantification of both antibodies and antigens, this general chapter describes the quantification of antigens as vaccine components.
APPARATUS
The nephelometer used is described in general chapter 2.2.1. Clarity and degree of opalescence of liquids. However the measurement may be made at a non-zero angle different from 90°.
METHODS
In general, the progress of the reaction measured by light scattering can be described in 3 steps.
Initially, a baseline level of light scattering due to the reaction medium is detected. After the 1st reagent (antigen) is added, an increase in the signal is observed followed by a plateau. When the 2nd reagent (antibody) is added, a 2nd increase of the signal is observed with a 2nd plateau followed by a final increase in the intensity of the signal, which continues until a 3rd plateau is reached. The measurement zone starts from the addition of the 2nd reagent until the higher intensity of light scattering (the 3rd plateau) is reached depending on the concentrations of the component to be assayed.
The following methods can be used to quantify the immune complexes formed during this type of reaction.
End-point nephelometry
The light scattering is measured after the immune complex has formed, i.e. after about 60 min. The value of the end-point is in this case directly proportional to the content of the component to be assayed when an excess of antibodies is used. A blank is necessary in order to subtract the value of nonspecific scattering due to the reaction mixture and to the reaction/measurement cell.
RATE nephelometry
Rate nephelometry is based on the rate of immune complex formation (rate of increase in light scattering), which is proportional to the concentration of the component to be assayed.
There are 2 types of rate nephelometry.
Fixed-time rate nephelometry
A 1st measurement of light scattering is carried out a few seconds after the last reagent has been added. A 2nd measurement is carried out after a fixed time interval established during development of the method. The difference between the 2 values is proportional to the quantity of the component to be assayed.
Rate nephelometry using the peak of the first derivative
The formation of the immune complex is described by the curve of the first derivative of the variation in light scattering with time (dΘD/dt). Two successive peaks are observed due to the addition of the 1st and then the 2nd reagent.
The height of the 3rd peak of the first derivative is proportional to the quantity of component to be assayed in the reaction medium. Successive measurements are carried out a few seconds after the 2nd reagent has been added.
In this case, the light scattered by the reaction medium, the component and the reagent does not affect the initial value of the derived signal.
OPERATING CONDITIONS
Reagents and reference standards
The reagents, diluents and samples to be examined must be clear and free from any suspended particles. To this end, a preliminary filtration or centrifugation step may be considered.
The antisera or antibodies are selected based on their specificity, their precipitating capacity and their avidity for the antigen. Indeed, high avidity results in clearly detectable signals being generated rapidly, which thus improves the precision of the results.
In addition, it is important to define the range of antigen concentrations to be assayed with an excess of antibodies and to verify the absence of a zone effect in this range.
It is recommended to ensure that the profiles of response will be similar to that of an appropriate reference standard.
Pre-treatment of the samples to be examined
An appropriate pre-treatment could be envisaged to eliminate an adjuvant or another interfering substance.
Assay
Ensure that all the reagents are at a suitable temperature to optimise the reaction.
To the samples diluted with a suitable medium in the reaction measurement cell, add a fixed quantity of antibodies. Carry out several determinations of the dilution being examined.
In some cases, the progress of the reaction may be improved by introducing additional substances into the reaction medium (e.g. polyethylene glycol).
The reference standards and samples must be treated in the same manner.
Performance of a blank test
To ensure that the assays are specific, only the scattering due to immune complexes must be measured. Other particles, the matrix of the sample, the reagents or the reaction measurement cell may interfere. In this case and if the end-point method is used, it is necessary to carry out a blank test to avoid taking non-specific scattering into account.
Calibration
The calibration curve is obtained by preparing a set of dilutions of a reference standard. The concentrations are chosen in order to encompass the working range.
Methods of calculation
The parallel line method (see general chapter 5.3) or a calibration curve is used for the calculations.
VALIDITY OF THE TESTS
Validity of the calibration Curves
Only calibration curves that comply with the defined validity criteria may be used.
The following parameters are examples to be used to define validity criteria:
Validity of the results
The following criteria are examples of validity criteria of the samples to be assayed: