GB2138132A - Storage-stable antibody-linked erythrocytes - Google Patents

Abstract

Antibody-linked and optionally enzyme-treated erythrocytes which have been treated with an alpha , omega -aliphatic dialdehyde of the general formula OHC-(CH2)n-CHO, in which n is a whole number of from 1 to 5.

Description

SPECIFICATION Storage-stable antibody-linked erythrocytes The present invention is concerned with new and improved carriersforantigens and antibodies which are storage-stable and with the use thereof for carrying out diagnostic and analytical procedures.

Diagnostic and analytical procedures are known for the measurement and determination of microbial and other biological materials, including antigens, and of antibodies reactive therewith. Such procedures should be specific, very sensitive and simpie to carryout. Furthermore, such procedures must be capable of being carried out in a very short period of time because, under certain circumstances, rapid diagnosis can be of great importance.

Labelling either antigen or antibody with radioiostopes is the basis of many very sensitive assays but such procedures are not very desirable because of the necessity of having to deal with radio-active materials, which can be dangerous and which, in any case, requires the use of special precautions. Sensitive procedures have also been developed using enzymes linked to antibody.

Adsorbing or linking antigen or antibody on to particles affords simple, less sensitive and often only semiquantitative procedures. With erythrocytes as carriersforeitherantigen or antibodies,the reactions are more sensitive and, with specially enzyme-treated carriererythrocytes,the reactions approach the sensi tivityoftheenzyme-linked orELlSAtests. In the reverse passive haemagglutination test (RPH) and in the related Mr PAH, Mrs PAH and SPACE reactions, the enzyme-treated linked erythrocytes may be considered to be the label for antibody, as is the isotope or enzyme in the various radio-immunoassays (RiA) and ELISAtest, respectively.

Enzyme-treated, antibody-linked erythrocytes have proved to be extremely sensitive to RPH tests, the specific antibody-linked carrier red cells simply being added to serial dilustions of the sample to be assayed for antigen. The assay end-point is the lasttube orwell showing aggregation of the antibody-linked erythrocytes. The procedure can be used, for example, as a measure of IgE and gAin human serum (see Scott, Thornley, Coombs & Bradwell, Int. Arch. Allergy, 64, 222/1981). Furthermore, the assay is of comparable sensitivity to RIA, measuring down to below 0.5 ngiml.

In addition, the procedure can be used to detect and thus diagnose respiratorysyncytial virus in nasopharyngeal secretions of infants (see Crange, Stott, Nagington & oombs,J. med. Virology, 8, 15311981). It has also been used to measure rota virus and investigations with herpes virus are in progress.

The RPH is also a final stage in tests for antibody {Mr PAH and Mrs PAH reactions) and these assay proce- dures have proved very successful in measuring bacterial antibodies in bruceilosis (see Coombs, Edebo, Feinstein & Gurner, Immunology, 34, 1037/ 1978) and in salmonellosis (see Edebo, Nilsson, Lindberg, Svenungsson & Coombs, J. Path. microbiol.

Scand. Sect. B, 89,341/1981); in measuring reaginic or IgE antibodies to clinically important allergens (see Scott, Thornley & Coombs, Int. Arch. Allergy, 64, 230/1981 ) and in measuring antibodies of all classes to antigens insoluble in physiological fluids but of considerable clinical interest, for example gliadin in coeliac disease and collagen in rheumatoid arthritis (see Kieffer, Frazier, Daniels, Ciclitera & Coombs, J.

Immunol. Methods, 42,129/1981 and Coombs, Gurner, Oldham, Barnes & Kieffer, Int. Arch. Allergy, 1982 (in the press)).

Antibody-linked erythrocytes and especially anti body-linked enzyme-treated erythrocytes or 'carrier red ceil reagent' are very much more sensitive in antigen detection than other particles, such as latex, where antibody is simply adsorbed.

However, at present, these sensitive carrier erythrocyte reagents have to be prepared afresh everytwoto three weeks because the erythrocytes slowly disintegrate and lyse after this period. Admittedly it has recently been found that they may be successfully cryo-preserved but this cryo-preservation procedure is a costly and time-consuming process. Consequently, carrier erythrocyte reagents with a long shelf life would have considerable value.

We have now found that treating antibody-linked and optionally enzyme treated erythrocytes with certain oc, w-aliphatic dialdehydes preserves the erythrocytes from lysis, while only very slightly reducing their antigen-specific reactivity.

Thus, according to the present invention, there are provided antibody-linked and optionally enzymetreated ervthrocyteswhich have been treated with an oX eo-aliphatic dialdehyde of the general formula OHC-(CH2)n-CHO, in which n is a whole number of from 1 to 5 and is preferably 3.

Thepresentinvention also provides a processfor the treatment and stabilisation of antibody-linked and optionally enzyme-treated erythrocytes, wherein antibody-linked and optionally enzyme-treated erythrocytes are treated with a low concentration of an oc, -aliphatic dialdehyde of the general formula OHt- (CH2)nCHO, in which n is a whole number of from 1 to 5 and is preferably 3.

The cx,w-aliphatic dialdehydes used according to the present invention are of low molecular weight, good results having been obtained with the use of, for example, glutaraldehyde.

Furthermore, we have ascertained that it is impor tantonlyto use low concentration of the dialdehyde, good results having been obtained in the concentration range of from 0.01 to 0.05% by weight and preferablyoffrom 0.01 to 0.015% byweight.

When using enzyme-treated erythrocytes, there may be employed any of the methods already known for this purpose. Obviously the enzymetreatment should only be for such a period oftimethatthe eryth rocytes remain intact, i.e. the enzyme treatment must be stopped before the erythrocytes have been lysed. By way of example, satisfactory results are obtained by treating erythrocyteswith a 0.25% by weight solution oftrypsin, chymotrypsin, papein or urinimidase at ambienttemperature for 20 to 30 minutes.

According to the present invention, the methods known for coupling immunoglobulinsto optionally enzyme-treated erythrocytes can be used, good results having been obtained with chromicchloride.

Thetypes of antisera which can be used according to these erythrocyte-antibody linked procedures include IgG and IgM monoclonal antibodies and heterogenous antisera to virus, such as herpes, hepatitis B, rota virus and respiratorysyncytial virus; antibodies specific for schistosomes and malaria parasites and antibodies specificforserum proteins, such as human immunoglobulin, complement components and acute phase proteins, such as C-reactive protein and a- foetal protein. Ce Is coupled with antibodies specific for immunoglobulin can be used to detect antibodies in the sera of patients specific for bacteria such as Brucella, Salmonella and other pathogens.

The stabilised eryth rocytes according to the present invention can,forexample, be used for: 1) RPH tests (a) giving rapid diagnosis of infective diseases by detecting microbial products in body fluids; for example the diagnosis of meningococcal and Mycobacterium tuberculosis infections; (b) in assay proceduresfor plasma proteins,for example immunoglobulins, complement components and acute phase proteins and the like, and to detect deficiencies; (c) in assay procedures in the manufacture of, for example, interferon; and 2) Mr PAH and Mrs PAH tests to measure thetitre and class of reactive antibodies in a large variety of systems.

Furthermore, it appears that the stabilised erythrocytes according to the present invention are of potential usefulness in direct antiglobulin rosetting reactions (DARR) as well as in indirect antiglobulin rosetting reactions (IARR).

The stabilised erythrocytes according to the present invention are very simple to use: apart from the actual reagents, onlysimpleequipment is required, such as microtitre plates, pasteur pipettes, loops for serial dilution tests and the iike. Thus, tests can be carried out in the field under extremely primitive conditions, so that, in addition to being extremely useful for rapid tests by physicians and in sophisticated clinical laboratories, they can also becarried out in places where equipment is virtually non-existent.

Thus, the present invention also provides a test kit comprising, in itssimplestform,two bottles of erythrocytes stabilised according to the present invention, one being coupled with the specific antibody and the other (control) being coupled with immunoglobulin ofthe same species as was used to raise the antisera.As an additional control,the kit can also comprise a preserved sample ofthe material to be tested for as a positive control.

The following Example is givenforthe purpose of illustrating the present invention: Example.

IgG coupling to trypsin-treated erythrocytes using chromic chloride.

Reagents Phosphate buffered saline (PBS) (comprising 8.0 g./litre NaCI, 0.2g./litre KCI, 1.15 g./litre anhydrous Na2HPO4and 2.0 g./litre anhydrous KH2PO4) NaCI 9 gms/litre (0.9%) Trypsin NaCI 8.0 g./litre KCI 0.4g./litre Na2HPO4(Anhyd) 0.12g./litre Glucose 1.0 g./litre Phenol red 0.01 litre Trypsin (Difco Grade 1 :250) 2.5 g./litre Stir magnetically and then filterthrough a membrane filter. Store at 20"C. This represents 0.25% trypsin (w/v).

Adjust the pH to 7.0 with N/10 NaOH before use.

Anti4rypsin Trypsin inhibitorfrom soybean (Sigma-Trypsin inhibitor No. T.9003) 1% (w/v) in PBS (keptfrozen).

Make 0.025% solution in PBS on day of use (1/40 of stock1%).

Chromic chloride.

CrCl3 2.25M (599.5 g./litre in distilled water) = 60%.

Working stock = 1/60 of above in 1 % NaCI; adjust pH to 5.0 with 0.2M NaOH.This 1% solution diluted to approximately 0.02% (1/501/45 dilution of 1 % stock) 0.02% on day of coupling.

All above stored at +4"C.

IgG lgG to be coupled must be dialysed against 0.9% NaCI solution, spun and adjusted to 2 mg/ml. to 0.5 mg/ml., depending upon the strength ofthe antisera.

Erythrocytes.

The erythrocytes are well washed (5 or 6 times) in a large volume of (at least 20 ml/2 ml packed cells) PBS before the trypsin treatment, the procedure being similarfor erythrocytes from ox, sheep, donkey and humans. Afterwashing, make a 10% suspension in PBS (v/v).

Trypsin treatment.

Mix equal volumes of pre-warmed (37 C.) 10% erythrocytes and 0.25% trypsin (pH 7.0) and leave the mixturefor20minutes ina37"C.waterbath,inverting occasionally. After20 minutes, spin and wash twice in PBS at 950G/5 minutes.

Anti-trypsin.

Resuspend deposited erythrocytes in an equal volume of 0.25% trypsin inhibitor, mix and leave for 10 minutes at ambienttemperature.Top up the containerwith NaCI solution, spin andwash twice in 0.9% NaCI solution at950G/5 minutes. Remove the lastwash and leave the packedtrypsin-treated erythrocytes ready for coupling.

Coupling protein to trypsin-treated erythrocytes.

Using plastic disposable (LP3)tubes: IgG 2 mg/ml in NaCI 50,at.

packed trypsin-treated erythrocytes 50 ILl.

chromicchloride 0.02% 100 ply Add the chromicchloride slowly, whilst mixing on a Rotamix. Mixfor30 seconds, cap the tubes and rotate slowlyfor 60 minutes at ambienttemperature. Fill tube with PBS, spin and wash threetimes in PBS at 400 G/5 mins. Finally, suspend in 5 ml. of PBS to give a 1% suspension of erythrocytes.

Set up 2 drops of aboveto checkfor autoagglutination. Also use an anti-immunoglobulin over a range of conentrations to check coupling of immunoglobulinto erthrocytes and a standard concentration of antigen to confirm antibody activity has remained.

This is most simply achieved in a standard reverse passive haemagglutination titration.

Glut;rraldehyde-treatmentafterprotein coupling using chromic chloride.

Glutaraldehyde 1% solution in NaCI from purified E.M. grade 25% stocksolution,further diluted 1/10 in NaCI just before use.

Procedure.

lna 10mi.centrifugetube,add5ml.of 1% antibody-coupled erythrocytes. On a Rotamix add 0.5 ml. of 0.1 % glutaraldehyde slowly to the eryth rocytes, mixfor30 seconds, then rotate over and over on Matburn rotatorfor 60 minutes at ambient temperatu re. Then spin and wash three times in PBS (MSE No. 2for5 minutes). Finally, resuspendto 1% in 0.02% sodium azide solution in PBS or other preservative and store until required for use, for example fortesting human serum.

Claims (17)

1. Antibody-linked erythrocyteswhich have been treated with an a,oa-aliphatic dialdehyde of the general formula OHC(CH2)nCHO, in which nisa whole number offrom 1 to 5.

2. Antibody-linked erythrocytes according to claim 1, which have been treated with the os,o)- aliphatic dialdehyde in a concentration of from 0.01 to 0.05% by weight

3. Antibody-linked erythrocytes according to claim 2, which have been treated with the o:,ci)- aliphatic dialdehyde in a concentration of from 0.01 to 0.15% by weight.

4. Antibody-linked erythrocytes according to any ofthe preceding claims, which have been treated with glutaraldehyde (n = 3).

5. Antibody-linked erythrocytes according to any ofthe preceding claims, which have also been enzyme treated.

6. Antibody-linked erythrocytes accordingto claim 5, wherein the enzyme treatment has been carried out with trypsin, chymotrypsin, papein or urinimidase.

7. Antibody-linked erythrocytes according to claim 1, substantially as hereinbefore described and exemplified.

8. Aprocessforthe preparation of antibody linked erythrocytes according to claim 1, wherein antibody-linked eryth rocytes are treated with a low concentration of an a,co-aliphatic dialdehyde of the general formula OHC(CH2)nCHO, in which n is a whole number offrom 2to 5.

9. A process according to claim 8, wherein the dialdehyde is used in a concentration offrom 0.01 to 0.05% by weight.

10. A process according to claim 9, wherein the dialdehyde is used in a concentration of from 0.01 to 0.015% byweight.

11. A process according to any of claims 8 to 10, wherein the dialdehyde used is glutaraldehyde.

12. A process according to any of claims 8 to 11, wherein, prior to treatment with the dialdehyde, the erythrocytes are treated with an enzyme.

13. A process according to claim 12, wherein the enzymetreatment is carrieå outwith a 0.25% by weight solution of the enzyme atambienttempera- turefor20to30minutes.

14. A process according to claim 12 or 13,wherein the enzyme used istrypsin, chymotrypsin, papein or urinimidase.

15. A process according to claim 8forthe preparation of antibody-linked erythrocytes, substantially as hereinbefore described and exemplified.

16. Antibody-linked erythrocytes,wheneverpre- pared by the process according to any of claims 8to 15.

17. test kitforcarrying out assays, comprising erythrocytes according to any of claims 1 to 7 and 16, coupled with a specific antibody and, separately therefrom, erythrocytes coupled with immunoglobu lin of the same species as was used to raise the antisera.