Background in DIAPOPS

  1. Introduction
  2. Contamination with old PCR products
  3. Unsuccessful washing after addition of Streptavidin-enzyme conjugate
  4. Old substrate
  5. Unspecifically binding of Streptavidin-alkaline phosphatase conjugate
  6. Lack of blocking in the incubation step in the hybridization detection of the solid phase product
  7. Conclusion

1. Introduction
In all diagnostic assays, including the DIAPOPS analysis ( DIAPOPS introduction), it is very important to clearly distinguish between positive and negative samples. In order to differentiate a true positive sample from a negative sample, it is important to know the level of signal from wells without added template DNA. These blank or blind samples give the background signal, which should be low and uniform. They should be treated like all other samples, and should go through all steps in the DIAPOPS procedure ( DIAPOPS Procedure). One or more blank samples should be included in every DIAPOPS analysis to ensure the background level is normal.

A number of different problems can give rise to an increased level of background signals in DIAPOPS. These reasons are explained in detail below.

2. Contamination with old PCR products
If the PCR mix is contaminated with old PCR products ( Carry-over preventions), there will be raised readings from every sample tested, including blanks. The PCR product concentration amplified from these contaminating products should theoretically be the same in every sample. However, different levels of product concentration may be observed in otherwise identical wells. This will be measured as a raised background level in DIAPOPS, frequently with variable signals. If the increased background level is caused by a contamination with old PCR products, the true positive signals is expected to be above the background signal depending on the level of contamination.

Contamination with old PCR products can be checked by running the liquid phase products on an agarose gel ( Liquid phase PCR product analysis). The contamination can be seen as faint bands of the correct size and normally with a slight variation in product concentration. However, the limit of detection with DIAPOPS often is better than the agarose gel ( Data from a comparison between DIAPOPS and liquid phase product concentration), and a very low level contamination may give an increased background signal in DIAPOPS, which is not detectable on the agarose gel. If there is an increased background signal, and all other theories have been tested and rejected, this very low level contamination is probably the cause, and a change of all solutions for the PCR mix is the best way of resolving the problem ( The solution to a contamination).

3. Unsuccessful washing after addition of Streptavidin-enzyme conjugate
The washing of NucleoLink Strips is discussed in this section. In this discussion it is assumed that all other washes have been successful, so the problem will only originate from this specific step ( DIAPOPS Procedure). If the NucleoLink wells are not washed properly ( The term: Wash three times ) after addition of Streptavidin-enzyme conjugate (in short called "conjugate"), some of this conjugate will remain in the wells. This will yield very high background signals, most likely higher than the correct signal. Only very high signals in all wells will be measured, and true positive samples will not be recognized. This problem may be solved by closely following the described washing procedure ( DIAPOPS Procedure), and ensuring that each washing cycle is performed optimally ( The term: Wash three times ). If a thorough washing does not solve the problem, it may be caused by abnormal adsorption of the conjugate to the surface of the NucleoLink Strips ( A case story of a background problem) or unspecifically binding to the solid phase primer ( Unspecific binding to the solid phase primer of the streptavidin conjugate).

4. Old substrate
The substrate for the enzyme may deteriorate on standing, or if stored under incorrect conditions where autohydrolysis may occur. This can also happen if the substrate solution has not been stored in the dark. This may yield an increase in background signal, because the substrate is already fluorescent or colored before addition to the NucleoLink wells. As a consequence of this problem, all levels of background can be seen from slightly raised reading to signals at a level where the true positive samples are totally concealed. A reading of the substrate alone in an empty well will reveal if this is the problem.

The correct storage conditions differ with various substrates. As a consequence, the rate of autohydrolysis will also vary from one substrate to the other. The storage capability of the diluted 4-MUP substrate ( 1 mM 4-MUP dissolved in 1 M diethanolamine (pH 9.8), 1 mM MgCl<font size=1>2</font> (1000 ml)) is over 6 months, if stored at -20ºC ( Storage of the 4-MUP dilution).

5. Non-specific binding of Streptavidin-alkaline phosphatase conjugate
The streptavidin-alkaline phosphatase conjugate (in short called "conjugate") has been observed to adsorb to the surface of the NucleoLink Strips even after blocking of the surface ( A case story of a background problem). Furthermore, it has been observed, that the solid phase primer can bind the conjugate non-specifically ( Unspecific binding to the solid phase primer of the streptavidin conjugate). The binding caused by the solid phase primer is not sequence specific, and the problem may be solved by purchasing a new synthesis of a primer with the same sequence ( A new solid phase primer with the same sequence).

6. Lack of blocking in the incubation step in the hybridization detection of the solid phase product.
In the hybridization detection of the solid phase product in the DIAPOPS procedure ( DIAPOPS Procedure), Blocking Reagent (BR) ( 5 x SSC, 0.1% Tween 20, and 0.5% Blocking reagent (BR) (500 ml)) is added to all incubation buffers. If BR is not added, the background signals will increase, because the streptavidin-enzyme conjugate will adsorb to the surface ( Blocking with BR).

As blocking reagents to prevent this adsorption, BSA (bovine serum albumin) ( DIAPOPS buffer with 10 mg/ml BSA (100 ml)) and BR ( Blocking with BR) were tested, and a concentration of 0.5% BR proved to be the best choice ( Blocking with BR). Signals from positive samples with a high template concentration will most likely be seen above the background level if no blocking agent is used, but samples with low template concentration may not yield signals above the background.

7. Conclusion
Background problems will normally appear only if thorough washing procedures are not followed, or if blocking reagent is not used in the incubation buffers during detection of the solid phase product. A special, PCR related, problem to DIAPOPS is carryover of old PCR products ( Introduction), which will raise the background level. Care should be taken to avoid this problem.

In very infrequent cases, the Streptavidin-enzyme conjugate can bind to the solid phase primer ( Unspecific binding to the solid phase primer of the streptavidin conjugate). This can be solved by using a new synthesis of the same solid phase primer sequence ( A new solid phase primer with the same sequence). Furthermore, in one single case the streptavidin-alkaline phosphatase conjugate has been observed to adsorb to the surface of the NucleoLink Strips even after blocking of the surface ( Introduction). The solution to this was to purchase a conjugate from a different manufacturer.

If the raised background level is caused by any problem other than carry over contamination of old PCR products ( Carry-over preventions), a new detection by hybridization can be performed without making a new amplification ( Rehybridization to the solid phase PCR product).