DE2755559A1 - METHODS FOR CLEANING UP SERUM OR PLASMA - Google Patents

Description

DR. BERG DIl'L.-lNG. STAPF DIPL.-ING. SCHWABE DR. DR. SANDMAIR 275 55 5

PATENT LAWYERS

P.O. Box 860245 8000 Munich 86

Attorney File 28 679 ¹ ^ December 1977

THE WELLCOME FOUNDATION LIMITED LONDON, NWL / UK

Methods for purifying serum or plasma

The invention relates to a method for cleaning serum or plasma, in particular a method for cleaning of serum or plasma to be used for the detection of specific immunoglobulin M (IgM) antibodies.

The detection of specific IgM antibodies in the serum or Adult or neonatal plasma by immunofluorescence is a generally accepted sign of a active or recent infection; the accurate detection of specific IgM antibodies can be of the utmost importance when having infections like rubella, or toxoplasmosis

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/ Π R 1 L Bank accounts: Hypo-Bank Manchen 4410122850

fUO 1 «♦ _(BLZ 7O ₀₂₀₀₁₁ , _Swift code: HYPO DE MM

Bay «Vereinsbank Manchen 453100 (bank code 70020270) Postal check Manchen 65343-808 (bank code 70010080)

D / b. Telegrams: fl • (089) 988272 BERGSTAPFPXf 988273 TELEX: 988274 05 24 560 BERG d 983310

Suspected cytomegalovirus. Although immunofluorescence and other immunological tests in routine procedures proportionately Easy to do, they have two serious limitations: first, immunoglobulin can G (IgG) antibodies in non-fractionated serum samples mask the detection of specific IgM antibodies, namely by competitive inhibition with the IgM antibodies at the binding sites of the antigens used in the test. Second, a false positive IgM test can be made, if IgM antiimmunoglobulin ("RF") with specific IgG antibodies that have combined with the antigen used in the test react.

The absorption of serum in aggregated IgG can increase IgM anti-immunoglobulin activity and thus false positives IgM reactions are switched off (milling cutter, K.B., Shirodaria, P.V. and Stanford, CF. , Brit.Med.J. , 1971, III, 707); according to R. Gispen et al., Clin. Exp. Immunol., 1975, 22, 431, however, the results are not always satisfactory. Even a successful adsorption cannot reduce the possibility of a false negative IgM staining which result from competitive inhibition by specific IgG antibodies.

It has been shown that protein A, which is present in the cell wall of certain Staphylococcus aureus strains,

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both in purified form and in intact staphylococci with the Fc region of most, but not all ^{, subsets of human IgG connect "1} " (Ankerst, J. et al., J. Infect. Dis., 1974, 130, 268). Remaining IgG antibodies not adsorbed by Protein A can combine with the antigen used in the test; IgM "RF" can therefore react with the bound IgG and produce a false positive IgM result.

By fractionating the test sera using gel filtration or sucrose density gradient centrifugation, one can completely remove the IgG group of immunoglobulins from IgM and thus the effect of both competitive inhibition by specific IgG and false positives Switch off IgM reactions due to "RF" activity. However, these methods have the major disadvantage that they require expensive equipment and highly qualified personnel, and are very time consuming; this seriously limits the number of samples that can be processed by one person.

It has now been found that both the elimination of the "RF" effect and the competitive inhibition by Specific IgG can be achieved by taking serum or plasma samples with IgG, which is on particles of an inert substance is applied, brings into contact, and then a

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adding polypeptide known as protein A, which is obtained from staphylococci and which is bound to precipitant; this method allows the precise detection of specific IgM antibodies.

The invention relates to a method for the purification of serum or plasma, which is used for the detection of specific IgM antibodies is used in which the serum or plasma is brought into contact with IgG applied to inert particles will; This is followed by adsorption by staphylococcus protein A, which is either on the original bacteria or on precipitating agents bound is present.

The IgG is preferably of human origin; it can obtained from serum from patients suffering from myeloma or from normal human serum. The IgG fraction can be carried out by ion exchange chromatography, e.g. DEAE cellulose Column chromatography or some other known method. This faction can then be physically adsorbed by an inert carrier, which consists of a particulate material, e.g. latex, preserved red blood cells or polystyrene. Optionally, the immunoglobulin protein can be chemically attached to a Carrier polymer are coupled, with the aid of covalent bonds between the protein and the carrier. Suitable Carriers are cellulose, polyacrylamide gel balls, glass

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balls, agarose derivatives or cross-linked dextrans. Preferably, latex particles with a diameter of 0.2 up to 1.0 μm, preferably 0.6 μm, used as particles this size sediment more easily than smaller ones, but not too quickly and not before interacting with the serum took place. The concentration is 20 to 1000 mg IgG / g latex, preferably UO to 500 mg IgG / g latex.

A protein Α preparation can be made by using a suitable Staphylococcus aureus strain, e.g. Cowan I, grown in a suitable medium, and the protein by treatment with a suitable enzyme, e.g. lysostaphin, released from the bacteria. The insoluble material can then be centrifuged off, and a protein A preparation remains behind. This can then be mixed with a suitable precipitant consisting of a polymer can, e.g. a crosslinked polymer such as a crosslinked polysaccharide, or made of dextran, crosslinked with epichlorohydrin, or polyvinyl alcohol, crosslinked with epichlorohydrin, or other natural or synthetic polymers such as Agarose, cellulose or polyaminostyrene. The protein A can optionally also be added to other, non-polymeric precipitants, such as red blood cells, activated charcoal, activated glass balls or magnetic iron balls. That Protein A can be attached to the polymeric or non-polymeric substance

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be bound by methods commonly used to bind proteins to polymeric or non-polymeric substances or are known, so that a product is formed that contains 0.5 to 50 mg of bound protein A per gram of compacted particle mass, preferably contains 1 to 25 g of bound protein A per gram of compacted particulate mass.

Alternatively, and this method is preferred, the protein A is not purified and is on the parent bacteria available. A staphylococcal suspension of 5 to 20% by volume, preferably a 10% suspension, is then used.

The serum to be purified is obtained from a patient's blood sample which is allowed to clot; those after coagulation the remaining relatively clear liquid is the serum. Alternatively, plasma can also be cleaned; man it is obtained by adding an anticoagulant to the blood sample and centrifuging it, whereby the red blood cells are removed drop. The remaining relatively clear liquid is the plasma.

The IgG applied to inert particles can be provided for use in tests, e.g. in a glass or plastic container as well as protein A bound to a precipitant, together with appropriate instructions to carry out the cleaning. All for cleaning

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drive essential components can be in a box or Containers packed and offered. It is therefore emphasized that such a composition of the cleaning system from IgG bound to inert particles, Protein A bound to precipitant and the instructions, i.e. the offer a cleaning kit, represents a second object of the present invention.

Second, the invention relates to a set of reagents for the purification of serum or plasma in which specific IgM antibodies should be proven; it contains two containers, the first of which is bound to inert particles IgG preparation, the second containing a preparation of protein A obtained from staphylococci and that is bound to precipitants or the original bacteria. This also includes the corresponding instructions for use.

One or both components of the cleaning kit can be offered as a liquid or freeze-dried.

The protein Α preparation of the cleaning kit can be offered as a liquid in a buffer as Staphylococcal suspension with 5 to 20% by volume of bacteria, preferably as a 10% suspension. The suspension is offered in aliquots of 1 ml, 5 ml or 10 ml which, if desired, can also be freeze-dried. Optional

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the protein A can also be attached to a suitable polymeric or non-polymeric substance are bound, so that a preparation is obtained that contains 0.5 to 50 mg of bound protein A per Grams of compacted particulate mass, preferably 1 to 25 mg of bound protein A per gram of compacted particulate mass contains.

The one bound to a particulate material, e.g. latex IgG is preferably offered in aliquots of 1 to 10 ml of a suspension containing 20 to 1000 mg IgG / g latex, preferably tO contains up to 500 mg IgG / g latex. For example, the IgG can be bound to latex in such a way that a preparation of 2.5 mg IgG / ml of a 1% latex suspension is formed. To extend the shelf life of the cleaning kit components, preservatives can be added, e.g. a bacteriostat such as sodium azide, as well as other preservatives such as mannitol, sorbitol, or glucose.

The serum or plasma purified by the procedure described above and using the purification kit can be used in various immunological tests to detect specific antibodies against viruses, bacteria, parasites, Tissue antigens and other exogenous antigens are used, e.g. in tests in which immunofluorescence, hemagglutination, Hemagglutination inhibition, complement fixation, radioimmunoassay or enzyme-labeled antibody systems

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be used.

The components of the cleaning set can therefore also be used in reagent sets suitable for such immunological tests be incorporated.

The advantage of this purification process is that by using, for example, IgG bound to latex and protein A producing staphylococci with a relatively fast and
simple method both the false negative IgM detection due to competitive inhibition by specific IgG antibodies and the false positive IgM detection due to "RF" activity can be switched off. Furthermore, the method has the advantage that, in contrast to gel filtration and sucrose density gradient centrifugation, it can be carried out with small amounts of serum.

The following examples are intended to describe the invention in more detail.

example 1

Preparation of the reagents
a) Staphylococcal suspension

A protein A producing Staphylococcus aureus Cowan I
Strain was as described by G. Kronvall, J.Med.Microbiol. , 1973, 6,187, grown in nutrient medium. The culture was treated with formalin, washed in saline, and

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resuspended in phosphate-buffered saline (PBS) to form a 10% by volume bacterial suspension. The suspension was heated to 80 ° C., cooled rapidly, and further washed in phosphate buffered saline. The bacteria were resuspended in PBS containing 0.1% sodium azide and 2% mannitol to make a 10% by volume suspension. If desired, this can be freeze-dried in aliquots of 1 ml, 5 ml or 10 ml and ^{stored at 4 °} C. until use.

b) Latex IgG preparation

A 5% suspension of latex particles with a diameter of 0.6 μm was produced. An IgG solution (5 ml of a 5 mg / ml solution) which had been prepared from human serum by means of DEAE cellulose column chromatography, 2 ml of latex suspension and 3 ml of glycine saline solution buffer were mixed and heated to 56 ^° C. for 20 min heated. The mixture was centrifuged, washed with more buffer, centrifuged again, and finally made up to 10 ml with PBS, resulting in a latex IgG preparation with 2.5 mg IgG / ml of a 1% latex suspension.

Example 2

Cleansing the serum

5 ml of a blood sample from a patient suspected of having toxoplasmosis was allowed to clot.

The serum was pipetted off. - / 11

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180 μΐ PBS were added to a 20 μΐ portion of the serum, so that a 1:10 dilution was obtained. The 200 yl diluted serum were divided into two equal 100 μl portions divided, one of which was left untreated or not adsorbed. 100 yl of the 1% latex IgG preparation according to the example 1 were centrifuged and the supernatant removed. A 100 yl serving of the diluted serum was added to the Added latex pellet, stirred, and left to stand for 10 minutes at room temperature with occasional stirring for adsorption. 1 ml of the 10%, protein A-producing Staphylococcus aureus suspension according to Example 1 was centrifuged and resulted in 100 μΐ densely packed cells. The serum latex IgG mixture was added to the staphylococcal pellet, mixed well and stirred for 5 min allowed to stand at room temperature for adsorption.

The entire mixture, i.e. serum latex IgG and staphylococcal cells, was then centrifuged at about 3000 rpm for 10 minutes. The adsorbed serum was withdrawn and used for testing kept.

Example 3

Detection of specific toxoplasmosis IgM antibodies

Four multispot Toxoplasma antigen plates with 8 spots each were produced by placing a drop on each spot Antigen suspension was given and the excess was removed.

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The plates were dried and fixed in methanol for 10 min. Successive 1: 2 dilutions of the according to Example 2 prepared adsorbed serum and the unadsorbed serum prepared in PBS, such as dilutions from 1:10, 1:20, 1:40, 1:80, 1: 160, 1: 320, 1: 640, 1: 1280. The various dilutions of the adsorbed serum were applied to the antigen spots of two of the plates (No. 1 and 2), different dilutions of the non-adsorbed Serum was applied to the antigen spots of the two other plates (3 and 4).

The plates were placed in a humid chamber for 30 min at room temperature incubated, then the diluted serum was aspirated from the plates. Then the plates were then washed three times for 5 min in PBS.

Fluorochrome-labeled anti-human IgG from sheep and fluorochrome-unlabeled anti-human IgM from sheep (from Wellcome Reagents Ltd.) was diluted 1:20 in phosphate buffered saline. Excess phosphate buffered saline solution was removed from the antigen plates and fluorescent anti-human IgG was applied to plates 1 and 3, and on plates 2 and 4 fluorescent anti-human IgM applied. All plates were incubated in humid chambers for 30 min at room temperature, the conjugates were then sucked off and the plates three times for 5 min

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washed in phosphate buffered saline.

The plates were counterstained with 1: 5000 Evans Blue in PBS and rinsed in the saline solution. Subsequently were they are soaked up in buffered glycerol and examined under the microscope.

Results:

Plate 1 Plate 2 Plate 3

Plate 4

Serums

Absorbed absorbed

Not absorbed

Not absorbed

Conjugate titer

Anti-IgG Anti-IgM Anti-IgG

<1: 10 negative 1: 320 positive 1: 1280 positive

Anti-IgM 1: 320

positive but can be negative in some cases.

This indicates that the patient is infected with Toxoplasma suffers or has recently suffered from it.

Example H

Detection of specific anti-nuclear IgM antibodies. The procedure of Example 3 was followed, however
anti-nuclear antigen was used instead of toxoplasma antigen. The following results were obtained:

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79 · Titer negative Conjugate <1:10 negative Anti-IgG <1:10 positive Anti-IgM 640 160 wrong
positive Anti-IgG 1: 80-1: Anti-IgM

Serums

Absorbed
Absorbed

Not absorbed

Not absorbed

This shows a false positive result due to "RF" with non-adsorbed serum.

Example 5

Preparation from IgG, coupled to aminoethyl cellulose. 0.5 g of aminoethyl cellulose were mixed with 10 ml (0.5 mol / 1) Sodium hydroxide solution and then washed out thoroughly with distilled water until the pH was about 7.0. the Aminoethyl cellulose was suspended in 12 ml (0.05 mol / 1) of phosphate buffer at pH 7.0, and 1 ml of an aqueous buffer was added Solution of 25% glutaraldehyde given. After stirring for 2 hours at room temperature, the processed cellulose became separated from the liquid phase by suction with a Buchner funnel and five times with phosphate buffer (at least 500 ml) washed to remove residual glutaraldehyde. The cellulose "cake" was in phosphate buffer resuspended, 1.5 mg solution of human IgG was added and the volume was increased to 10 ml with buffer.

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filled. The cellulose IgG preparation was at room temperature Rotated for 2 h and then on a Buchner funnel with phosphate buffer containing 1 mol / 1 sodium chloride, washed to remove non-covalently bound antibody, then it was made 1% in PBS or 10% suspension, i.e. 50 ml or 5 ml, respectively.

Example 6

IgG coupled with microcrystalline cellulose. Microcrystalline cellulose (about 1 g Sigma Cell 20) was washed in a Buchner funnel with at least 250 ml of distilled water. 1 g of the dried washed Cellulose was weighed into a bottle. 10 mg of sodium periodate was dissolved in 3 ml of distilled water; 3 ml of this solution were added to 1 g of cellulose and rotated overnight at room temperature. The sample was centrifuged, the supernatant removed and the activated cellulose suspended in 10% ethylene glycol to terminate the reaction; Then she was in a Buchner funnel with ethylene glycol, distilled water and finally with 0.1 mol / l carbonate / bicarbonate buffer with pH 9.5. IgG (3 mg in 10 ml carbonate / bicarbonate buffer) was added to the prepared cellulose (Ig) and put on overnight Room temperature rotates. The cellulose IgG preparation was then centrifuged and the supernatant removed. It was

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de a sodium borohydride solution (10 ml of 1 mg / ml) prepared, added to the cellulose sediment and rotated for 2 h at room temperature; it was then centrifuged and
washed thoroughly in phosphate buffered saline,
finally suspended in PBS to form a 1% or 10% suspension, ie 100 ml or 10 ml.

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Claims (11)

Patent claims: 1. Method for the purification of serum or plasma used for the detection of specific immunoglobulin M (IgM) antibodies is used, characterized in that that the serum or plasma with immunoglobulin G (IgG) applied to an inert particle material in Is brought into contact, which is followed by adsorption by staphylococcal protein A, which is either on the original bacteria or is present bound to precipitant. 2. The method according to claim 1, characterized in that the inert particulate material Latex, preserved red blood cells, polystyrene, cellulose, polyacrylamide gel beads, glass beads, agarose derivatives or cross-linked dextrans. 3. The method according to claim 2, characterized in that the inert particulate material latex with a particle diameter of 0.2 to 1.0 µm. 4. The method according to claim 3, characterized in that the diameter of the latex particles 0.6 pm. - / 18 809825/0014 2 / b b b δ 5. The method according to any one of claims 2 to U, characterized in that the IgG concentration yields between 20 and 1000 mg IgG / g latex. 6. The method according to claim 5, characterized that the IgG concentration is between UO and 500 mg IgG / g latex. 7. The method according to any one of the preceding claims, characterized in that / ?, the Protein A is present on the original bacteria or used as a 5 to 20% by volume staphylococcal suspension will. 8. The method according to claim 7, characterized in that that the suspension is a 10% by volume suspension. 9. The method according to any one of claims 1 to 6, characterized in that the precipitant, to which the protein A is bound, a cross-linked polysaccharide, dextran cross-linked with epichlorohydrin, polyvinyl alcohol cross-linked with epichlorohydrin, agarose, cellulose, polyaminostyrene, red blood cells, activated carbon, activated Glass balls or magnetic iron balls is. - / 11 «098? s / new I. I bbbb J 10. The method according to any one of claims 1 to 6 or 9, characterized in that the Concentration of protein A bound to precipitant 0.5 to 50 mg protein A per gram of compacted particle mass amounts to. 11. The method according to claim 10, characterized in that the concentration of the bound Protein A is 1 to 25 mg of protein A per gram of compacted particulate mass.