GB2049185A

GB2049185A - Enzymeimmunoassay method and reagent tube

Info

Publication number: GB2049185A
Application number: GB8014006A
Authority: GB
Original assignee: Orion Yhtyma Oy
Current assignee: Orion Oyj
Priority date: 1979-04-30
Filing date: 1980-04-29
Publication date: 1980-12-17
Also published as: DE3016391C2; FI58839B; SE8003206L; FI58839C; DE3016391A1; GB2049185B; FR2455742A1

Abstract

An enzymeimmunoassay method is disclosed wherein a reagent tube is used having at least its interior surface coated with an antiserum or antigen. A sample to be investigated or a standard solution is introduced in the reagent tube together with antigen- or antibody- complex labelled with an enzyme which enzyme uses pyridine nucleotide as coenzyme. The solution is reacted with the coating, and thereafter the solution is removed and the reagent tube is connected to a second tube containing a substrate solution for the label enzyme. The substrate solution is reacted with the coating of the reagent tube and the compounds attached to it. The connected tubes are turned to transfer the solution to the second tube whereupon the reagent tube is removed and a quantitative fluorometric assay is performed on the combined materials in the second tube. A shaped reagent tube for carrying out the method is also disclosed.

Description

SPECIFICATION Enzymeimmunoassay method and reagent tube The present invention relates to an enzymeimmunoassay method, comprising introducing a sample to be investigated or a standard solution together with a label enzymeantigen- or enzyme-antibody-complex into a reagent tube having at least its interior surface coated wih an antiserum or antigen, reacting the solution with the coating, reacting the coating and the compounds attached to it with a substrate solution for the label enzyme, and quantitatively measuring the compound formed in the reaction.

Enzymeimmunoasay methods have been disclosed by Schuurs, A. H. and van Weeman, B.K. in Clinica Chimica Acta 81, 1-40, 1 977. Further methods have been disclosed in Finnish patents Nos. 52 154, 53 894, 54 033, and 54 034.

The object of the present invention is to produce a new method of performing enzymeimmunoassays which is more sensitive and more practical than the prior known methods.

This invention makes it possible to perform the measurements very easily and with great accuracy on both a micro and a macro scale.

The method according to the invention is characterized in that when the reaction of the sample to be investigated or the standard solution with the coating of the reagent tube is completed, the solution is removed from the reagent tube and the reagent tube is tightly connected with its mouth downwards to a second tube containing the substrate solution, the substrate solution is reacted with the coating of the reagent tube and the compounds attached to it, and upon completion of the reaction the connected tubes are turned to cause the solution to flow into the second tube, the reagent tube is removed, and the quantitative assay is carried out by fluorometry, whereby an enzyme utilizing pyridine nucleotide as coenzyme is used as the label enzyme.

The second tube is preferably a fluorometric cuvette, whereby the quantitative assay is carried out in the second tube.

According to a referred embodiment of the method according to the invention yeast glucose-5-phosphate dehydrogenase is used as the label enzyme.

To make the method more sensitive the fluorescence can be increased using known methods like enzymatic cycling (Lowry, O.H., Passonneau, J.V., Schulz, D.W. and Rock, M.K.: J. Biol. Chem. 236, 2746-2755, 1961, and Kato, T., Berger, S.J., Carter, J.A.

and Lowry, O.H.: Anal. Biochem. 53, 86-97, 1973) or by adding strong sodium hydroxide solution (Lowry, O.H., Roberts, N.R. and Kapphahn, J.l.: J. Biol. Chem. 224, 1047-1064, 1957).

The invention also relates to a reagent tube for carrying out enzyme-immunoassay method of the invention having at least its inner surface coated with an antiserum or antigen.

The reagent tube of the invention is characterized in that it has a conical lower portion ending in a sharp tip, and that at least one ring-shaped, resilient sealing element is provided at the outer circumference of the end of the reagent tube to be connected to the second tube. According to a preferred embodiment of the reagent tube an annular shoulder is provided at the outer circumference of the end thereof to be connected to the second tube, against which shoulder the end of the second tube is placed on connecting the tubes.

The reagent tube of the invention can be made of nylon, which is specially treated by a known method in order to attach antigen or antibody (Hornby, W.E. and Goldstein, L., in: Methods in Enzymology XLIV, K. Mosbach, eds., Academic Press, New York, 1976, p.

118). According to this treatment the tube is immersed in methanol which contains 18.8 % CaCI2 and 18.6 % H2O and is kept at 50-60"C by mixing with a magnetic stirrer.

By this treatment amorphous nylon is dissolved and the surface to be used is increased and its wetting ability becomes better. The dissolved nylon is washed away by incubating in water for several hours. After this the tube is immersed in 3.7 M HCI at 50-60"C whilst using a magnetic stirrer. After treatment the tube is washed until the pH is neutral.

The pretreated tube is activated by immersing it in 5 % glutaraldehyde in 0.5 M phosphate buffer, pH 5.0 for one hour at room temperature. Thereafter the tube is washed several times in acetate buffer, pH 5.0, and in phosphate buffer, pH 7.0. The activated tube is immersed in diluted antiserum in phosphate buffer and is incubated for three hours at room temperature. After incubation the tube is washed with 0.9 % NaCI solution, which contains wetting agent, 0,05 % TWEEN-20, and the tube is also washed thereafter with 0.133 M borate buffer, pH 8.0. The tube can be stored in the cold ether immersed in borate buffer which contains 0.02 % bovine serum albumin (BSA), or after treatment with BSAborate buffer emptying and freeze-drying.

Reagent tubes can be treated in large batches which guarantees quantitatively uniform coating of each tube. In the abovedescribed technique both the inside and the outside surfaces of the tube are affected.

However, the immunoreaction is applied only on the interior surface of the tube which will not be scratched or damaged during later handling.

The tube 1 has a cylindrical upper portion 2 and a conical lower portion 3. Because of this conical form it is possible to use small incuba tion volumes, e.g. from 10 yí and higher. The cylindrical upper portion 2 of the reagent tube 1 has on its outside an annular shoulder 4 and two annular but thinner projections 5, which are elastic and thus serve as sealing rings. It is obvious that the number of sealing rings may variate, but the tube must have at least one sealing ring. However, the shoulder 4 is not absolutely necessary, and it may, if desired, be left out.

Fig. 2 illustrates how the reagent tube 1 is coupled to a fiuorometric cuvette 6 by inserting into it. The reagent tube 1 is arrested with its shoulder 4 abutting against the top of the cuvette 6. The upper portion 2 of the reagent tube 1 is dimensioned to have an outer diameter slightly smaller than the inner diameter of the cuvette 6 but, on the other hand, the diameter of the sealing rings 5 is greater than the inner diameter of the cuvette thus providing a fluid-tight coupling of the two tubes to each other.

For the performance of the immunoassay the enzyme-labelled antigen or antibody and a standard or a sample to be investigated are pipetted into the reagent tube, e.g. in a total volume of 100 y The tube is closed an the immunoreaction is carried out during e.g. 14 h at 4"C. Thereafter the fluid is removed by suction and the tube is washed e.g. with 0.1 M TRIS-HCI buffer, pH 8.0. Then 1 ml of a substrate solution for the label enzyme is pipetted into the fluorometric cuvette and the reagent tube is coupled tightly to the cuvette.

The tubes are inversed and the enzymatical reaction is carried out by rotating or shaking the tubes at room temperature or at 37"C during 10-60 minutes. Thereafter the enzymatic reaction is stopped by inversion of the tubes so that the fluid flows into the cuvette.

The reagent tube is disconnected and the cuvette is placed in a fluorometer and the fluoroscence measured When the immunoassay is carried out in microscale the sample to be investigated or the standard and the enzyme-labelled antigen or antibody are pipetted into the reagent tube, e.g. in a total volume of 10 1. The tube is closed and incubated as described above.

Thereafter the fluid is removed by suction and the reagent tube is washed, as described earlier. Then 1 5 Ibl of a substrate solution for the label enzyme is pipetted into the reagent tube and incubated gently shaking during 10-60 min atl37 C. Then 2 yl of 0.1 mol/l NaOH is pipetted into the reagent tube and the tube is incubated 10 minutes at 60"C in order to destroy the nicotine adenine dinucleotide phosphate (NADP+). Then 50 sl of the cycling reagent is added to the reagent tube in order to enhance the fluorescence and the tube is incubated 60 minutes at 37"C after which the fluorometric cuvette is pressed tightly on the top of the reagent tube.The coupled tubes are inversed and cooked 2 minutes to destroy the enzymes and stop the cycling reaction. The reagent tube is removed, 1 ml of indicator reagent is added to the cuvette and incubation is carried out for 20 minutes at room temperature. Finally the cuvette is placed in the fluorometer and the fluorescence measured.

The invention is illustrated by the following example.

Example Assay of testosterone in plasma.

20 yl of testosterone standard solution or sample extract is added to a reagent tube (Fig.

1) which is coated with testosterone-1 1 -succinyl-BSA-antiserum and containing a solution composed of 0.1 33 mol/l of borate buffer, pH 8.0, 0.02 % gelatine and then simultaneously or after some time the testosterone-3carboxymethyloxime-glucose-6-phosphate dehydrogenase (labelled testosterone) is added. The tubes are incubated over night at 4"C. Then the fluid is removed by suction and the tubes washed three times with 0.1 M TRIS-HCI buffer, pH 8.0 1 ml of the substrate solution for glucose-6-phosphate dehydrogenase is pipetted into the fluorometric cuvette.

The composition of this solution is 0.1 mol/l TRIS-HCI buffer, pH 8.0, 5 mmol/l MgCI2, 0.01 % BSA, 0.2 mmol/l NADP+, 1 mmol/l glucose-6-phosphate. The empty reagent tube is pressed tightly on to the fluorometric cuvette and the coupled tubes are inversed and the enzymatic reaction takes place at 37"C during 60 minutes with shaking (the reaction is linear at least 90 minutes). Thereafter the enzyme reaction is stopped by turning the tubes upside down so that the incubation solution flows into the cuvette, the immunoreagent tube is disconnected and the fluorescence measured with a fluorometer. With this method it is possible to determine testosterone concentrations in the region from 0.2-50 mmol/l without using fluorescence enhancement.

Claims

1. An enzymeimmunoassay method, comprising introducing a sample to be investigated or a standard solution together with a label enzymeantigen- or enzymeantibody-complex into a reagent tube having at least its interior surface coated with an antiserum or antigen, reacting the solution with the coating, reacting the coating and the compounds attached to it with a substrate solution for the label enzyme, and quantitatively measuring the compound formed in the reaction, characterized in that when the reaction of the sample to be investigated or the standard solution with the coating of the reagent tube is completed, the solution is removed from the reagent tube and the reagent tube is tightly connected with its mouth downwards to a second tube containing the substrate solution, the substrate solution is reacted with the coating of the reagent tube and the compounds attached to it, and upon completion of the reaction the connected tubes are turned to cause the solution to flow into the second tube, the reagent tube is removed, and the quantitative assay is carried out by fluorometry, whereby an enzyme utilizing pyridine nucleotide as co-enzyme is used as the label enzyme.

2. A method as in claim 1, characterized in that yeast glucose-5-phosphate dehydrogenase is used as the label enzyme.

3. A method as in claim 1 or 2, characterized in that the second tube is a fluorometric cuvette, and the quantitative assay is carried out in the second tube.

4. A method as in claim 3, characterized in that the fluorescence is amplified by enzymatic cycling or by adding sodium hydroxide.

5. A method as in claim 4, characterized in that the enzymatic cycling is carried out in the reagent tube.

6. A method as in claim 3, characterized in that sodium hydroxide is added to the solution in the second tube.

7. A reagent tube for carrying out enzymeimmunoassays, comprising a reagent tube having at least the interior surface thereof coated with an antiserum or antigen, characterized in that the reagent tube has a conical lower portion ending in a sharp tip, and that at least one ring-shaped, resilient sealing element is provided at the other circumference of the end of the reagent tube to be connected to the second tube.

8. Reagent tube as in claim 7, characterized in that an annular shoulder is provided at the outer circumference of the end thereof to be connected to the second tube.

9. A method in claim 1, substantially as described with reference to the accompanying drawing.

10. A method as in claim 1, substantially as described in the foregoing Example.

11. A reagent tube as in claim 7, substantially as described with reference to the accompanying drawing.