A. Antibody Activity Threshold IndexCompare Samples to 1 U/ml Calibrator or Internal Control = Positive/Negative Cut-off.
Example:
Results The sensitivity of the assay to detect anti-ACWY IgG, from either natural infection or vaccination, is controlled so that the 1 U/ml Calibrator represents a threshold OD for most true positives in human serum diluted to 1:100 or greater. Visual inspection of the data in the above graph shows the following:
Calibrators – dilution curve of anti-ACWY antiserum, derived from ACWY vaccination, shows the OD range of the assay; high value indicates optimal sensitivity of the assay.
1 U/ml: a 'Cut-off' line has been drawn to indicate a threshold distinguishing between Positive/Negative. This is not a clear-cut threshold, rather a low OD area that could represent either low positives or high background negatives.
Positive Control – antiserum with reactivity to Meningococcal Group A; value range is on the label. This Control can be used to assess reproducibility and/or normalize between-assay variation.
Internal Control – a true positive from a normal individual that represents the lab's experience in distinguishing low positive from negative samples. This should be run in each assay to supplement the 1 U/ml Calibrator for Positive/Negative discrimination purposes.
Samples A,B,C,D – 3 samples (A, B, C) are negative: below the threshold; 1 sample (D) is positive: clearly above the threshold.
The 1 U/ml Calibrator can be used to calculate a Threshold Index that numerically discriminates Positive/Negative:
Divide each Sample net OD by the 1 U/ml Calibrator net OD. Values above 1.0 are a measure of Positive Antibody Activity; below 1.0 are Negative for antibody.
B. Positive Index Experimental sample values may be expressed relative to the values of Control or Non-immune samples, by calculation of a Positive Index. One typical method is as follows:
1. Calculate the net OD mean + 2 SD of the Control/Non-immune samples = Positive Index.
2. Divide each sample net OD by the Positive Index. Values above 1.0 are a measure of Positive Antibody Activity; below 1.0 are Negative for antibody.
A sample value would be Positive if significantly above the value of the pre-immune serum sample or a suitably determined non-immune panel or pool of samples, tested at the same sample dilution.
This calculation also quantifies the positive Antibody Activity level, assigning a higher value to samples with higher Antibody Activity, and vice versa.
Example:
Results Experimental Samples are represented as follows:
C – Calibrator
I – Internal Control; lab's threshold positive serum
E – Experimental sample
Antibody Titer & Specificity Dilutions of antibodies with specificity for the different Group antigens were run in the assay to demonstrate the efficacy of the multiple antigen (ACWY) coated plate. Titers were calculated as inverse of the dilution that produced a 1.0 OD in the assay.
Results Rb x-Gr A: rabbit immunized with the Group A segment of the Menveo vaccine by Sanofi Pasteur. Titer: 47.5 k
Rb x-Gr CWY: rabbit immunized with the Group CWY segment of the Menveo vaccine by Sanofi Pasteur. Titer: 255 k
Ms x-Gr A: mouse monoclonal antibody specific to Group A antigen; a candidate WHO standard. Titer: 13.6 k
Hu x-Gr A/C: a human serum pool reactive with Groups A and C provided by the CDC as a Reference Pool. Titer: 40 k
Quality ControlThe calibrators and controls must perform as stated in the manual. High blanks or Negative controls (A450 >0.5) is usually due to inefficient plate washing or too much sample or high amount of antibody-HRP conjugate. Please read the manual carefully before using the kit.
COA
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