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7.4.2. Distinguishing between non-linearity and antigen excess

Chapter 7

To check for antigen excess, Binding Site recommend performing an antigen excess check dilution in addition to the initial sample dilution, as described in the relevant product insert (Table 7.6). Such a protocol minimises reagent usage and ensures consistency.

Instrument Initial dilution
for κ and λ Freelite assays
Antigen excess check dilution
for κ and λ Freelite assays
Binding Site SPAPLUS1/10 1/1000*
Siemens BNII/ProSpec1/100 1/2000

Table 7.6. Antigen excess check dilutions on Binding Site SPAPLUS and Siemens instruments. * This is the overall dilution: 1/10 initial dilution + 1/100 manual dilution

Distinguishing between non-linearity and antigen excess is important because with non-linearity the lower result should be reported, whereas with antigen excess the higher result should be reported. The Binding Site recommends that when the result obtained at the antigen excess check dilution is more than 4-fold greater than the result obtained at the initial sample dilution, this sample should be considered to be in antigen excess. In this case the result obtained at the higher dilution should be reported. On the other hand, if the result from the antigen excess check dilution is less than 4-fold greater than that from the initial dilution, the sample should be considered non-linear and the value at the initial dilution should be reported. Examples of the use of this guidance to distinguish between antigen excess and non-linearity are shown in Table 7.7.

Dilution Result
(mg/L)
Sample 11/100 95.3
1/2000 202
Dilution results ratio* 2.1
Non-linear
DilutionResult
(mg/L)
Sample 21/100145
1/2000467
Dilution results ratio*3.2
Non-linear
DilutionResult
(mg/L)
Sample 31/10078.1
1/20004310
Dilution results ratio*55
Antigen excess

Table 7.7. Examples of κ FLC non-linearity and antigen excess on a Siemens BNII. *Results from antigen excess check dilution (1/2000) divided by those from the initial dilution (1/100). The results in bold should be reported.