Washing device

The invention concerns a washing device for treatment of rods attached to a plate with a solution. According to the invention the rods (6) are placed in ducts (4) through which the solution is passed. The invention can be used in particular in peptide syntheses and in immunological assay methods.

The invention concerns an apparatus and a method by means of which rods 
attached to a plate can be washed with liquids, such as washing buffers or 
organic solvents, or by means of which chemical or biochemical reagents 
can be allowed to act upon the rods. The invention is suitable for use, 
e.g., in peptide syntheses and immunological assay methods. 
It is known in prior art to synthesize peptides so that a first amino acid 
is covalently bound onto a solid face, whereupon a second amino acid is 
reacted with the first amino acid to form a peptide bond. This is 
continued until the desired peptide has been obtained. This process is 
described, e.g., in the paper by Merrifield, R., J. Am. Chem. Soc. 
85:2149-2154 (1963). 
Methods have been developed wherein a peptide is synthesized on heads of 
polyethylene rods attached to a plate. Thereby, certain steps in the 
synthesis, such as removal of the protection groups and washings, are 
carried out by washing the heads of the rods in basins that contain a 
reagent or a washing solution. 
The rods with the. peptides bound on them can be used directly, e.g., in 
immunological assay methods. In such cases as well, washing in a basin is 
used. The molecules bound on the peptides are also removed by washing the 
rods in basins. 
One drawback of the arrangement described above is high consumption of 
expensive reagents. 
Thus, the object of the present invention is to lower the consumption of 
these reagents and thereby to lower the costs. 
The object of the invention is achieved by the means stated in the patent 
claims. 
It is an essential feature of the invention that the rod to be washed is 
placed in a duct, and washing or reaction liquid is passed through the 
duct. The liquid is optimally passed in the ducts back and forth during 
the washing. 
According to one embodiment, the apparatus is connected with a valve 
arrangement by whose means mixing together of washing liquids or 
liquid-reaction fluids to be measured one after the other is prevented.

According to the invention, the plastic rods attached to the plate are 
treated with solutions. The rods are attached to the plate, e.g., in a 
matrix corresponding to an ordinary 8.times.12 microtitration plate. 
The washer comprises ducts, into which the heads of the rods are placed. At 
least at the locations of the rods, the ducts are somewhat wider than the 
thickness of the rods in order that the washing liquid could flow and 
by-pass the rods. The spacing, number, and length of the ducts naturally 
depend on the type of the plate that is used, on the number of plates, and 
on the way in which the plates are to be placed in the apparatus. 
The solutions are passed into the ducts preferably through a common 
distribution space. In a corresponding way, at the other end of the ducts, 
there is preferably a common space of removal. The liquid is measured into 
the distribution space, from which it is carried through the ducts into 
the space of removal. 
From the space of removal, if desired, the liquid may be recirculated into 
the distribution space. The solution is, however, optimally passed through 
the ducts back and forth between the distribution space and the space of 
removal until the solution has acted upon the rods for the desired period 
of time. 
To intensify the washing, facing the rods, the ducts may be provided with 
members that produce turbulence. 
If desired, ducts may be interconnected by means of transverse ducts. 
The operation of the apparatus is optimally controlled by means of a 
computer. 
The basin may be provided with a detector for monitoring the level of 
washing liquid, whereby the washing process can be controlled by means of 
the signals given by said monitoring detector. 
By means of the invention, the quantity of solution needed for washing can 
be reduced considerably as compared with a conventional washing in a 
basin. As a further advantage, a washing of maximal uniformity of all rods 
is obtained as the flow conditions for each of the rods are identical. 
Moreover, by means of increased flow rate and turbulence, the washing time 
can be made shorter or the result can be improved. 
The main parts of the apparatus shown in FIG. 1 are the base 1, the washing 
basin 2, and the reagent system 3. 
The washing basin comprises twelve washing ducts 4. Into the basin, five 
plates 5 can be placed for treatment at a time, each plate comprising 
8.times.12 rods 6. 
The ducts 4 are provided with a washing well 7 for each rod, the diameter 
of said well being somewhat larger than the diameter of the rod 6. Between 
the wells, the ducts are narrower than the rods. In this way the amount of 
solution required for filling the duct can be made little. The washing 
wells also produce turbulence, thereby improving the washing of the rods. 
At the initial ends of the ducts 4 there is a common distribution space 8, 
from which the solution flows into the ducts. On the other hand, at the 
final ends of the ducts there is a common space of removal 9, from which 
the spent liquid can be removed through the pipe 10. The solution is 
passed into the distribution space 8 from the reagent system through the 
inlet pipe 11. 
The washing basin can be closed by means of a lid 12. The edge of the lid 
is provided with a seal 13. In this way the washing space can be closed 
air-tightly and, if necessary, the washing can be carried out, e.g., in an 
inert gas phase. 
The washing basin is attached to the base by the intermediate of a motion 
mechanism 5. By means of the motion mechanism the inclination of the basin 
in the longitudinal direction of the ducts can be changed, whereby the 
solution is made to flow in the ducts back and forth. In the device shown 
in the figure the washing basin is hinged on the base at the initial end 
of the ducts, but of course it is also possible to use arrangements of 
other sorts to incline the ducts. 
To the exhaust pipe 10 of the basin, a vertical side branch 14 is 
connected, which is provided with a level sensor 15. During washing, the 
exhaust pipe is closed, whereby the liquid level in the side branch is at 
the same level as in the basin. In the first stage of washing, the final 
end of the basin is lowered, whereby the solution flows to the exhaust end 
and the level in the side branch rises. When the liquid surface has 
reached a certain level, the sensor gives a signal for reversing of the 
direction of inclination. In a corresponding way, when the liquid level is 
lowered to a certain level, the direction of inclination is reversed. 
After the desired time of washing or the desired number of inclinations 
the solution is removed out of the basin. 
Moreover, in the arrangement shown in FIG. 2, the guide rods 16 placed at 
the ends of the plates are seen, which fit into the recesses 17 provided 
in the basin. The guide rods support the plate so that the heads 18 of the 
rods 6 do not reach contact with the well bottoms. The guide rods also 
prevent movements of the plate during the inclinations. 
FIG. 3 shows a reagent system for use, e.g., in a peptide synthesis, 
wherein the reagents A, B and C can be passed by means of the pressure of 
an inert gas 19 (herein nitrogen) into the inlet pipe 11 of the washing 
basin. 
At the end of the inlet pipe 11 there is a valve 20, through which gas 
alone may be passed into the inlet pipe. Next, in the inlet pipe, there is 
a three-way valve 21, through which gas flows in the pipe, or a solution 
coming from the valve 26 flows in the pipe, or the reagent A is passed 
into the pipe. In a corresponding way, through the three-way valves 22 and 
23, gas can be passed from the preceding valve, or the reagent B or C, 
respectively. In a corresponding way, it is, of course, also possible to 
connect a higher number of reagent flasks to the system. If necessary, the 
whole inlet pipe can be cleared of reagents by means of gas. By means of 
this arrangement, it is possible to supply the reagent that is required at 
each particular time into the inlet pipe without mixing it at all with 
other reagents that might remain in the pipe system 
The gas pressure and so also the dosage rate of the reagents can be 
regulated by means of a pressure reduction valve 24. The arrangement 
further includes a closing valve 25. 
In a peptide synthesis it is possible to use amino acids protected with 
F-moc (9-fluorenylmethyloxycarbonyl) protection groups as a starting 
material. When the apparatus in accordance with the invention is used, the 
following washing procedure is carried out at the beginning of each 
synthesis cycle. 
1.times.130 ml of DMF (dimethylformamide), 5 min shaking 
4.times.130 ml of methanol, 2 min shaking 
1.times.130 ml of DMF, 5 min shaking 
1.times.90 ml of 20% piperidine in DMF, 30 min 
2.times.130 ml of DMF, 5 min shaking 
4.times.130 ml of methanol, 2 min shaking 
nitrogen drying, 15 min 
2.times.130 ml of DMF, 5 min shaking. 
After the synthesis is completed, the amino terminals can be acetylated, 
the following procedure being used: 
90 ml of DMF:acetic anhydride:diisopropylethylamine (v/v/v=50:5:1), 90 min 
shaking 
1.times.130 ml of DMF, 2 min shaking 
4.times.130 ml of methanol, 2 min shaking 
nitrogen drying, 15 min. 
For removal of the side chain protection groups the following procedure is 
employed: 
90 ml of TFA:phenol:ethanedithiol (v/v/v=95:2.5:2.5), 4 hours 
2.times.130 ml of 5% diisopropylethylamine/dichlormethane, 5 min shaking 
nitrogen drying, 15 min 
2.times.130 ml of distilled water, 5 min shaking. 
Finally the rods are kept in a methanol bath for 18 h. 
The peptide derived from the viral proteins can be used for the assay of 
corresponding anti-virus antibodies by means of EIA. Thereby, e.g., the 
following washing procedure is employed: 
90 ml of diluted antiserum, 4 hours shaking 
8.times.130 ml of washing buffer 
90 ml of alkaline phosphatase conjugated anti-Ig, 2 hours shaking 
8.times.130 ml of washing buffer, 5 min shaking. 
Finally the rods are subjected to the substrate in a microtitration plate, 
and the measurement is performed. 
When proteins bound to peptide are removed, the following procedure can be 
used: 
6.times.90 ml of disruption buffer, 10 min shaking 
4.times.130 ml of distilled water, 10 min shaking 
4.times.130 ml of methanol, 10 min shaking 
4.times.130 ml of distilled water, 10 min shaking.