TITLE: Protocol of Spotted Fever Rickettsia IgG
AUTHORS: narankhajid

[DESCRIPTION]
All serum samples were tested for Rickettsia IgG using a commercial ELISA kit (NovaTec Immunodiagnostica GmbH, Dietzenbach, Germany) according to the manufacturer’s instructions(S9).

[STEPS]
SECTION: Protocol of Spotted Fever Rickettsia IgG
1. 
SECTION: Protocol of Spotted Fever Rickettsia IgG
1.1. Dispense 100µl controls and diluted samples into their respective wells. Leave well A1 for substrate blank. 
SECTION: Protocol of Spotted Fever Rickettsia IgG
1.2. Cover wells with the foil supplied in the kit. 
SECTION: Protocol of Spotted Fever Rickettsia IgG
1.3. Incubate for 1 hour at 22-25°C.
SECTION: Protocol of Spotted Fever Rickettsia IgG
1.4. When incubation has been completed, remove the foil, aspirate the content of the wells and wash each well four (4) times with 300 µl of Washing Solution. Avoid overflows from the reaction wells. The soak time between each wash cycle should be >5 sec. At the end carefully remove remaining fluid by tapping strips on tissue paper prior to the next step! 
SECTION: Protocol of Spotted Fever Rickettsia IgG
1.5. Note: Washing is critical! Insufficient washing results in poor precision and falsely elevated absorbance values. 
SECTION: Protocol of Spotted Fever Rickettsia IgG
1.6. Dispense 100 µl IgG HRP conjugate into all wells except for the blank well (e.g. A1). Cover with foil.
SECTION: Protocol of Spotted Fever Rickettsia IgG
1.7. Incubate for 30 min at room temperature in the dark. Do not expose to direct sunlight.
SECTION: Protocol of Spotted Fever Rickettsia IgG
1.8. Repeat step 4. 
SECTION: Protocol of Spotted Fever Rickettsia IgG
1.9. Dispense 100 µl TMB Substrate Solution into all wells 
SECTION: Protocol of Spotted Fever Rickettsia IgG
1.10. Incubate for exactly 15 min at room temperature in the dark. 
SECTION: Protocol of Spotted Fever Rickettsia IgG
1.11. 10.Dispense 100 µl stop solution into all wells in the same order and at the same rate as for the TMB Substrate Solution. 
SECTION: Protocol of Spotted Fever Rickettsia IgG
1.12. Any blue colour developed during the incubation turns into yellow. 
SECTION: Protocol of Spotted Fever Rickettsia IgG
1.13. Note:Highly positive patient samples can cause dark precipitates of the chromogen!These precipitates have an influence when reading the optical density. Predilution of the sample with physiological sodium chloride solution, for example 1+1, is recommended. Then dilute the sample 1+100 with dilution buffer and multiply the results in NTU by
SECTION: Protocol of Spotted Fever Rickettsia IgG
1.14. 11.Measure the absorbance of the specimen at 450/620 nm within 30 min after addition of the Stop Solution.
SECTION: Measurement
2. Adjust the ELISA Microwell Plate Reader to zerousing the substrate blank in well A1.

If - due to technical reasons - the ELISA reader cannot be adjusted to zero using the substrate blank in well A1, subtract the absorbance value of well A1 from all other absorbance values measured in order to obtain reliable results! 

Measure the absorbance of all wells at 450 nmand record the absorbance values for each control and patient sample in the distribution and identification plan. 

Dual wavelength reading using 620 nm as reference wavelength is recommended. 

Where applicable calculate the mean absorbancevaluesof all duplicates. 
SECTION: Run Validation Criteria
3. 
SECTION: Run Validation Criteria
3.1. ·Negative Control < 0.2 absorbance units 
SECTION: Run Validation Criteria
3.2. ·Positive Control > 0.8 absorbance units 
SECTION: Run Validation Criteria
3.3. ·Cutoff Calibrator 0.25-0.55 absorbance units 
SECTION: Run Validation Criteria
3.4. ·Cutoff Calibrator / Negative Control ratio > 1.5
SECTION: Run Validation Criteria
3.5. ·Positive Control / Cutoff Calibrator ratio > 2.0 


If these criteria are not met, the test is not valid and must be repeated.
SECTION: Calculation of Results
4. The cut-off is the mean absorbance value of the Cut-off control determinations. 

Example: Absorbance value Cut-off control 0.54 + absorbance value Cut-off control 0.52 =1.06 / 2 = 0.53 
Cut-off = 0.53 
SECTION: Interpretation of Results
5. Samples are considered POSITIVE if the absorbance value is higher than 10% over the cut-off. 
Samples with an absorbance value of 10% above or below the cut-off should not be considered as clearly positive or negative
SECTION: Interpretation of Results
6. Øgrey zone 
It is recommended to repeat the test again 2 - 4 weeks later with a fresh sample. If results in the second test are again in the grey zone the sample has to be considered NEGATIVE. 

Samples are considered NEGATIVE if the absorbance value is lower than 10% below the cut-off. 
SECTION: Results in Nova Tec Units
7.   

SECTION: Results in Nova Tec Units
8. Example:    (1.786 x 10)/(0.53 )= 34  NTU (NovaTec Units)
Cut-off:    		10 		NTU
 Grey zone:  		9-11 		NTU
 Negative:   		<9 		NTU
 Positive:   		>11              	NTU