El valle del Colca-Chivay

Inject serum into naive

recipients

t

Day 7

Immunise recipients

CFA/OVA

Day 28

Assess Systemic Immune Responses

Figure 9.3 A schematic representation of the experimental protocol for the affinity absorption of OVA from the serum of OVA fed BALB/c donor mice and the subsequent introduction of the OVA depleted serum and the affinity absorbed, biologically processed OVA into groups of adult recipient mice.

5. Total eluted material from N°4, above. The eluant collected from both column runs of the serum from OVA fed BALB/c mice was concentrated to the pre-column volume of 7ml as previously described.

6. Unbound material obtained when serum from OVA fed BALB/c donor mice was passed through the RaBSA affinity column. The unbound material was collected and concentrated to the pre column volume as described previously.

Small aliquots of all samples were retained for measurement of immunoreactive OVA by sandwich ELISA and SDS-PAGB separation followed by silver staining. Recipient mice were immunised and challenged as previously described and systemic DTH and antibody responses to OVA were assayed.

Results

1. Sandwich ELISA and SDS PAGE analysis

Serum obtained from mice fed with either OVA or saline were passed down either the MaOVA affinity column or the RaBSA column and the concentrations of immunoreactive OVA in the various preparations obtained are given in Ikble 9.2. In the case of saline fed donor mice OVA was not detected in either the bound or unbound material from both the MaOVA and the RaBSA columns. Concentration of the fractions from the affinity columns to a suitable volume for injection into recipient mice, using an "Amicon" concentration cell, necessitated the measurement of the amount o f OVA contained in the filtrate through the YMIO membrane (molecular weight cut off of 10 kDa) to make sure their was no loss of OVA in this concentration step. Measurement of these fractions demonstrated no detectable OVA. Serum from OVA fed donors showed no reduction in immunoreactive OVA after passage through the RaBSA column (1967ng/ml before and 2060 ng/ml after) whereas, the MaOVA column removed most of the immunoreactive OVA from the serum of OVA fed donors.

Table 9.2 Concentrations of immunoreactive OVA in the serum of BALB/c mice following various manipulations.

Serum Column Fraction Cone"

l.OVA fed mice MaOVA pre treatment 2326ng/ml

2.OVA fed mice MaOVA l^Unbound* 1458ng/ml

3.OVA fed mice MaOVA l°Eluant* 4144ng/ml

4.OVA fed mice MaOVA 2°unbound* 1067ng/ml

5.OVA fed mice MaOVA 2°Eluant* 516ng/ml

6. OVA fed mice RaBSA pre treatment 1967ng/ml

7.OVA fed mice RaBSA Unbound 2060ng/ml

8.OVA fed mice RaBSA Eluant <2ng/ml

9.Saline fed mice MaOVA Unbound <2ng/ml

10.Saline fed mice RaBSA Unbound < 2ng/ml

* 1 ^Unbound= Unbound material from first passage of serum from OVA fed mice *l°Eluant=Acid elution of bound material from first passage of serum from OVA fed mice

*2°Unbound=Unbound material from passage of 1° unbound material

*2°Eluant=Acid elution of bound material from passage of 1° Unbound material

SDS-PAGE followed by silver staining confirmed the results obtained in the sand­ wich ELISA and only eluants obtained following the passage of serum from OVA fed d o n o rs th ro u g h the MaOVA colum n show ed e v id e n ce o f any b ands corresponding to native OVA, i.e. a doublet at approximately 46 and 43 kDa. No other bands were observed in these samples.

U n vivo determ ination of the biological activity of the bound and unbound m aterial from the RaBSA and MaOVA columns.

Recipients of serum from OVA fed donor mice showed significant suppression of their DTH responses when compared to recipients of MaOVA passaged serum from saline fed mice (p < 0.0005; Figure 9.4) or recipients of RaBSA passaged serum from saline fed donors (p < 0.0005; Figure 9.4). Recipients of MaOVA passaged serum from OVA fed donors, i.e. from which immunoreactive OVA had been removed, showed no significant suppression when compared to recipients of serum from saline fed donors which had been identically treated (p=N S; Figure 9.4). In contrast, recipients of the eluant obtained after MaOVA passage of serum from OVA fed donors showed significant suppression of their systemic DTH responses ( p < 0.0005: Figure 9.4). Recipients of RaBSA passaged serum from OVA fed donors showed significant suppression of their systemic DTH responses when compared to recipients of RaBSA passaged serum from saline fed donors ( p < 0.0005: Figure 9.4).

The systemic antibody response did not differ significantly between the 6 recipient groups (Figure 9.4).

Comment Interestingly serum from OVA fed mice before passage through the MaOVA column appeared to contain less im m unoreactive OVA than the first column eluant (2326ng/ml vs 4144ng/ml). This discrepancy may reflect poor quanti­ fication at the upper end of the standard curve in this particular run of the sandwich ELISA. Alternatively the continued handling and final concentration of the eluant material may also have contributed to the difference. Passage of serum from OVA fed donors through the RaBSA column had no effect on the total levels o f immunoreactive OVA in the unbound material (1967ng/ml before passage and 2060ng/ml after passage through the column).

F ig u re 9 .4 E ffect o f rem oving im m unoreactive OVA from serum and subsequent i.p. injection of this removed OVA in BALB/c recipients. Groups of recipients (n = 8) were injected i.p. with (1) serum from saline fed mice after passage through a MaOVA affinity column, (2) serum from saline fed mice after passage through a RaBSA affinity column, (3) serum from OVA fed mice, (4) unbound material obtained after passage of serum from OVA fed mice through a MaOVA affinity column, (5) bound material obtained from passage of serum from OVA fed mice through a MaOVA affinity column and, (6) unbound material obtained from passage of serum from OVA fed mice through a RaBSA affinity column.

*: comparison with group 1, **: comparison with group 2, ***: comparison with group 4

D o n o r s e r u m t r e a t m e n t ( g r o u p s )

group 1 I I Unbound nncterial after passage of serum from

saline fed mice through the M«OVA column group 2

group 3 group 4 group 5

group 6

Unbound material after passage of serum from saline fed mice through the Rex BSA column OVA fed

Unbound material after passage of serum from OVA fed mice through the McxOVA column

Bound material after passage of serum from OVA fed mice through the M ex OVA column

Unbound material after passage of serum from OVA fed mice through the RaBSA column

mm

System ic DTH responses