These methods are dependant solely on the quality of the antiserum used
with migration of antigen via electrophoresis in RIE and diffusion in SRID,
both types of assay have similar levels of sensitivity and precision.
As seen from the results represented in Figures 2.6 (Chapter 2) and Figure 4.6, the composition of the m atrix has a distinct affect on the level
of PAPP-A detected. These effects have also been observed by Bjerrum
(1983) who found they were produced by the dissociation of sub-units, aggregation or denaturation of the antigen due to exposure to conditions
such as high tem perature, high salt, or extremes of pH. The
immunoprecipitate th at was produced being increased (or decreased) dependant upon which of these described factors was present. An increase in the size of precipitin ring (or rocket height) is probably due to a loss of antigenic determ inants, so th at migration through the gel is over a longer distance before the complexes are large enough to be retained w ithin the agarose network. A decrease can be due to partial unfolding of the protein or
loss of antigenicity by some of the molecules involved. However it has been
established th at using the RIE and SRID under constant conditions for all
the samples m easured then a level of PAPP-A could be determined when monitoring the purification scheme.
60 -i 50 - 40 - cn 30 - £ 20 J 10 0 5 15 20 25 30
P A PP-A C oncentration (jug/ml)
F ig u re 4.6
PAPP-A SRID illustrating the effect of salt concentration on the assay.
[Legend: ( ■: The samples were loaded in assay diluent containing male plasma, the plate was made in assay diluent containing a final concentration of 1 M salt, r2 = 0.997). (A: The samples were loaded in assay diluent at a final concentration of 1M salt (containing male plasma, Appendix 2), the plate was made in assay diluent containing a final concentration of 1M salt, r2 = 0.994). Other conditions were as outlined in Chapter 2, section 2.6.2. The PAPP-A concentration was calculated against the WHO reference pool, 78/610. ].
4.3.3 T he PA PP-A RIA
The PAPP-A assay described by Pinto-Furtado et al (1984) was less prone to effects of anti-coagulants on the assay and it also gave a linear correlation of values obtained when compared against an IRMA th at used the DAKO polyclonal and a monoclonal antibody directed against PAPP-A (Mowles et al. 1986) thus im parting to this assay a greater specificity for
PAPP-A. However this does not exclude the possibility th at the monoclonal
antibody is directed at an antigenic component other th an PAPP-A as it has 4 - 11
been shown th at it only binds to un-reduced PAPP-A (Chapter 3, Plate 3.1). PAPP-A has been shown to be linked to pro-MBP (Oxvig et al. 1993).
The advantage to this assay of affinity purifying the PAPP-A tracer on a heparin column was illustrated in Figure 2.9 (Chapter 2) with less than 23% of the tracer bound to excess antibody prior to chromatography. This compares favourably with a level of 30% observed by Pinto-Furtado et al. (1984). After affinity chromatography a greater than two fold increase in tracer bound in excess antibody was observed. The low level of tracer binding to PAPP-A antiserum could have been due to damage produced during the iodination procedure or to the presence of other proteinaceous m aterial in the iodination grade PAPP-A preparation (Chapter 6, Table 6.2).
-q 100 - 80 - 60 - 40 - 20 - 10 100 1000
PAPP-A Concentration (ng/ml)
10000 F ig u re 4.7A typical PAPP-A RIA calibration curve.
[Legend: Results were expressed as 1 Percent tracer bound: (Bound CPM - NSB)/(B0 - NSB) Vs PAPP-A standard, the curve was fitted using RIACALC as described in Chapter 2, section 2.6.4.5]
The PAPP-A RIA (Figure 4.7) had a detection limit as defined from its precision profile of 15 ng/ml and an upper limit of 500 ng/ml at a level of precision level as m easured by a coefficient of variance of 10% or less. This compared favourably to a detection limit of 10 - 15 ng/ml and an upper limit of 600 ng/ml th at was seen by Pinto-Furtado et ah (1984) using the described RIA procedure.
As seen from Table 4.2, the PAPP-A tracer undergoes changes with time th at reduce its ability to bind heparin. This reduction in binding is the factor th at limits the life of the tracer to about four weeks. As seen from Figure 2.9 (Chapter 2) it was essential to use the heparin bound fraction of PAPP- A tracer for the RIA. This assay was used to monitor development of a PAPP-A purification scheme.
T able 4.2 Percentage of PAPP-A tracer bound to a heparin affinity column. T im e A fter Io d in a tio n (days)
0 7 14 M
P e r c e n ta g e o f T otal
PA PP-A tr a c e r B o u n d 47% 25% 13% 10%
[Note: h The table represents the proportion of PAPP-A tracer bound Vs The age of PAPP- A tracer when separated using a heparin affinity column. The results are expressed as a percentage of total tracer applied that were bound to this affinity matrix under the conditions described in section 2.6.4.2 (Chapter 2).]
4.3.4 Q u a n tific a tio n by D en sito m etry
This method can be used once the PAPP-A has been enriched to a level where it can be visualised by protein staining and a discernible band could be distinguished from other serum/plasma components. It should be noted however th at the levels found using this method are for the monomeric
chain of PAPP-A (as seen under reducing conditions of SDS-PAGE).
A linear relationship was found to exist between the intensity of staining
and the protein concentration for CBB staining from 0 . 5 - 2 0 pg and 0.02 - 2.0 ng/mm2 for silver staining. (Merril, 1990).This compared favourably with the results th at were obtained in this thesis of a linear relationship between 1 - 8 pg/mm2 for CBB staining as was illustrated in Figure 4.8.
Therefore this method was used subsequently to calculate the recovery of
monomeric PAPP-A protein levels from SDS-PAGE gels (Chapter 5).
2.5
2
2.0
to _Q TD *5 0.5 0.0 A m o u n t Of M y o s i n / B a n d In M i c r o g r a m m e s figure 4.8A typical calibration curve of protein levels seen using video densitometry.
[Legend: The calculated area/absorbance, (Chapter 2, section 2.6.6) was plotted Vs the amount of myosin loaded onto an SDS-PAGE gel. The gel was stained with CBB, as described in Chapter 2, section 2.3.2.2d.]
To summarise, the PAPP-A assays were found to be affected by a variety of factors from:
• The prim ary quality of the individual assay components.
• The individual assay design with affects due to molecular characteristics of PAPP-A.
These factors have undoubtedly affected the interpretation of the various clinical studies th at were undertaken to see if PAPP-A could be used to monitor foetal well-being (Chapter 1, section 1.4.7). The assays developed and illustrated here were however sufficient for the purpose of monitoring a PAPP-A purification/enrichment procedure th at is outlined in C hapter 5.