CA1127536A

CA1127536A - Identification of viral cell infections by antibody and bacteria

Info

Publication number: CA1127536A
Application number: CA337,922A
Authority: CA
Inventors: Alice S. Huang
Original assignee: Harvard College
Current assignee: Harvard College
Priority date: 1978-10-19
Filing date: 1979-10-18
Publication date: 1982-07-13
Also published as: FR2439233A1; GB2034464B; FR2439233B1; SE7908656L; GB2034464A; DE2941575A1

Abstract

Abstract of the Disclosure Viral infections are diagnosed and the infecting virus identified by immunoreaction of an antibody against a known virus with a sample of cells infected in vivo, followed by separation of free antibody from the sample and incubation of the sample with bacteria capable of binding to the antibody and removal of unbound bacteria. An enhanced number of bacteria bound to a cell surface as compared to a control free from antibody identifies cells infected with the known virus.

Description

7~3~

This invention relates to diagnosis of viral infections and pertains more specifically to a kit and a method of using it to identify viral infections in vivo.
It has previously been reported that bacteria such as Staphylococcus aureus contain surface protein which recognizes the Fc portion of immuno-globulin (Kronvall et al., J. Immunol., Vol. 104, pages 140-147, 1970).
This has led to widespread use of S. aureus for immunoprecipitation of antigens from cell lysates (Kessler, J. Immunol., Vol. 115, pages 1617-1624 (1975);
Goding, J. Immunol. Methods, Vol. 20, pages 241-253 (1978)).
It has also been found by Austin et al., Lab. Investig., Vol. 39, pp. 128-132 (August, 1978) that binding of Staphylococcus aureus to cultured rabbit cells infected in vitro with a strain of influenza virus is enhanced when the infected cells are treated with anti-influenzal serum. The authors also point out that anti-influenzal antibodies are elaborated quite early in the course of this viral infection in vivo, and postulate that because of this, bacteria are localized to the site of infection in vivo by binding to the infected cells.
It has now been found that contrary to the suggestion of Austin et al., cells which have been infected in vivo by virus in many cases do not have all of the immunoreactive sites on their surfaces bound to host-generated antibodies and do not display greatly enhanced bonding to bacteria. It has further been found that cells infected by virus in vivo possess a capability, unlike other cells not so infected, of bonding to antibody or binding partner specific to the virus infecting the cells, after which such antibody-bonded infected cells are capable of bonding through the antibGdy to bacteria. The presence or absence of bacteria bonded to the antibody-bonded infected cells can be readily determined by simple observation using a light microscope, using as a control infected cells to which no antibody has been bound, thus 3~

making it possible, by employing a variety of antibodies, eaeh speeifie to a different viral infeetion, to identify the particular virus or viruses eausing the infection. The procedure can be carried out rapidly, in a matter of a few hours, using equipment readily available in test laboratories, and eonsequently provides a simple and rapid means for diagnosis of viral infec-tions.
The present invention provides a method of identifying a viral infeetion in a specimen of cells infeeted ln viv~ eharaeterized by providing a oantrol sample of said speeimen and at least one test sample, incubating with each test sample an antibody against a knDwn virus capable of binding to the surfa oe of a oe ll infeeted with said knewn virus to form a reaetion mixture having free antibody and bound pairs consisting of antibody bound to said infected oe ll surfaces, where required separating free antibody from said test sample, incubating said control sample and each said test sample with bacteria having the capability of binding with said antibody, separating from each sample any unbound baeteria, and determining the presen oe or absenee of bac~eria bound to the surfa oe s of oells of said control and test samples.
In a particular aspect, the present invention provides a method of identifying a predetermined speeifie viral infeetion if present in a specimen of eells of an individual, which speeimen of oe lls may be infected in vlvo, which method comprises colleeting from the individual a control sample of said speeimen and at least one test sample of said speeimen, both unknown as to the presen oe or absen oe of viral infeetion, ineubating with eaeh unkncwn test sample an antibody, against a kncwn virus, eapable of binding to the surfaee of a oe ll infeeted with said known virus form to a reaetion mixture having free antibody and bound pairs eonsisting of antibody bound to said infeeted eell surfa oe s, incubating said eontrol sample with non-immune globulin, and separating frnm said control sample non-immune globulin whieh is not bound to .~

1~7~fi said oe lls, incubating said unknown contrDl sample and each said unkncwn test sample with bacteria having the capability of binding with said antibody, separating from each sample any unbound bacteria, and determining the presen oe or absence of bacteria bound to the surfaces of oe lls of said control and test samples by visual examination under a microscope.
The invention also is embodied in a kit for identifying viral infections in a specimen of cells infected in vivo characterised in that it ; includes in operational relation with each other for use in identifying a viral infection in a specimen of oe lls infected in vivo a supply of bacteria capable of binding to an antibody against the infectious agent, a supply of at least one antibody against a known viral infection, and a supply of non-im~une globulin.
In carrying out a diagnosis in accordan oe with the present invention, it is essential to include a control sample of the oe ll specimen; if the control sample exhibits bonding of bacteria to oe lls of the same order of magnitude as the test samples, the diagnosis cannot be made. However, if one or more test samples exhibit substantially enhanced bonding of bacteria to -2a-,7'~3~j cell surfaces as compared to the control, the infectious virus is identified as one against which the antibody used in the test is specific.
In practice, it is convenient to run a series of test samples simultaneously with a control sample, employing with each test sample a dif-ferent antibody which is specific for a different virus. Antibodies against a variety of different viruses are commercially available or can be raised, if desired, by infecting any desired animal such as rabbits, sheep, etc. with a known virus, bleeding the animal after a suitable time period, and harvest-ing the antiserum containing the desired antibody.
10Although the present invention is particularly adapted to identi-fication of viruses causing infections of the upper respiratory tract, it is useful in the case cf several different viruses such as vesicular stomatitis, herpes, and paramyxo including respiratory syncytial and parainfluenza.
The specimen of cells infected in vivo to which the present inven-tion can be applied can be obtained in any conventional manner as for example by taking a specimen of biological material from an infected individual, by taking drainage from a wound, by swabbing the throat, by col-` lecting nasal secretions, or by taking cervical tissue scrapings, or the like.
. The bacteria which can be used and which serve as a marker or label on the infected cells can be any bacteria which have surface protein reactingspecifically with antibody to a virus, that is, bacteria which possess surface immunoglobulin receptors. Preferred are bacteria containing protein A such as Staphylococcus aureus. The determination of the presence or absence of bacteria bound to the cell surfaces can be made most simply by examining the ; whole cells visually under a microscope. At a magnification of 400 to 800 X, the presence of infected cells among uninfected cells at a ratio of 1:1000 can readily be detected in a few minutes, the bacteria serving as prominent l~t~

visual markers on the cell surfaces.
The specimen of cells to be tested is preferably fixed by immersing it in a small volume of a suitable fixative, such as normal saline containing 1 % by weight of glutaraldehyde, avoiding excessive dilution of the specimen in order to facilitate subsequent microscopic examination.
A control sample and one or more test samples are then separated from the specimen and allowed to dry at room temperature or at moderately elevated temperature in the wells of a conventional microscope slide or other suitable containers, avoiding excessive heating which would, as is well known, destroy the morphology of the cells. Drying causes the cells to adhere to the container surface so that they are retained in position during subsequent rinsing steps. Each sample, after drying, is then briefly rinsed, either by immersion or with a squirt bottle, with normal saline containing a small quantity of non-ionic detergent. A small quantity of the desired antiserum or a dilution of the desired antibody is then added to each test sample and incubated. To the control sample is added nothing or a small quantity of normal saline before incubation; in a preferred embodiment there is added to the control sample before incubation non-immune serum from the same species of animal as the one in which the antiserum or antibody was raised. The amount of antiserum or antibody employed is preferably an excess over the amount required to react with all of the available immunoreactive sites on the cell surfaces. Incubation of control and test samples is carried out under identical conditions, ranging from about 2 minutes to about 60 minutes at room temperature; higher temperatures up to about 37C or even higher, as well as lower temperatures can also be used, as is well known for immunoreactions.
Generally, incubation is carried out by heating for 10 minutes or more at 37C., preferably for about 30 minutes.

5~3~

The immunorcaction occurring between the antiserum or antibody and the test sample of cells forms a mixture of cells having antibody specifi-cally bound to their surfaces and free antibody.
The free antibody is then separated from the bound antibody by rinsing or washing each test sample with a buffer having physiological pH and ionic strength, and the control sample is treated in the same way. However, when only a slight excess of antibody is employed, it is possible to omit this ` separation step.
There is then added to each sample, both control and test samples, a small amount of the desired bacteria such as Staphylococcus aureus, pre-r ferably in a suitable fixative such as a 1 ~ aqueous solution of formaldehyde, and the mixture is incubated. Conditions for incubation can be the same as described above for incubation of cells with antibody. There is preferably employed an excess of bacteria over the amount which reacts with all of the available immunoreactive sites on the bound antibody.
Any unbound bacteria are then separated from each sample by rinsing or washing the control sample and each test sample with a buffer having physiological pH and ionic strength.
The control sample and each test sample are then examined visually under a microscope to determine the presence or absence of bacteria bound to the cell surfaces. If necessary or desirable a suitable stain such as a Gram stain or Giemsa can be used, or phase contrast microscopy can be used to faciliate the determination.
It is also possible to react the bacteria first with a suitable antiserum or antibody to form an antiserum-bacteria complex, after which the complex is reacted or incubated with the whole cells to bond the bacteria to the surface of the infected cells.

A kit for carrying out such an assay contains as essential components:
1. A supply of bacteria, e.g., Staphylococcus aureus, preferably ; inaetivated, as for example, a 1% suspension of formalin-inactivated Staphylococcus aureus or a lyophilized supply of such inactivated bacteria capable of reconstitution to a 1~ suspension in normal saline or in 0.01 M
tris buffer at pH 7.4 containing normal saline.

2. One or more supplies of antiserum or antibody, each specific, although not necessarily monospecific, to a known virus. The antiserum or antibody supply may be raised in conventional manner in any suitable animal, e.g., rabbit antiserum containing immunoglobulins specific to a particular viral infectious agent; it may oe in the form of a dilution at the appropriate titer for use in the method, say 2 logs above Neutralization Potential of the antibody, or in the form of a lyophilized mass reconstitutable to such a dilution.
The kit may also contain, as optional additional components, a supply of microscope slides having appropriate wells, and supplies of the desired buffers. It may indeed include all of the desired or necessary facilities for carrying out the tests, including a supply of glutaraldehyde and stain as well as such equipment as pipettes, slide incubators, drying oven, and microscope.
The following specific examples will serve to illustrate more fully the nature of the invention without acting as a limitation upon its scope.
Examples A suspension culture of baby hamster kidney cells was infected with vesicular stomatitis virus (VS~ at a multiplicity of 20 by incubation for 3 hours at 35C. A control sample of cells was mock infected. After incubation, hj the cells were washed in isotonic buffer, fixed in 1% glutaraldehyde solution, and washed. Then the infected cells and the control cells were separately incubated with rabbit-anti-VSV serum (1:20) for ~5 minutes at 37C.
The cell suspensions were washed to remove unbound or free antibody, and then the infected cells and control cells were separately exposed to a suspension of formalin-inactivated Staphylococcus aureus (1:10, V/V) for 30 minutes at 37 C. The two cell suspensions were then washed to remove remaining free bacteria not bound to the cells, and samples of the two cell suspensions were placed on glass slides and examined at ~00 X using a binocular microscope.
It was found that 100~ of the infected cells were prominently marked by bacteria adherently bound to the cell surfaces, whereas none of the control cells displayed any bacteria bound to the cell surfaces.
When normal rabbit serum was substituted for the anti-VSV serum in the foregoing procedure, neither infected cells nor uninfected cells were marked by bacteria bound to the cell surfaces.
Similar results were obtained when in the foregoing procedure there were substituted for the infected hamster cells, human cells infected with herpes simplex virus, and there was also substituted the appropriate rabbit-anti-herpes simplex serum. Similar results were also obtained when in the foregoing procedure there were substituted for the infected hamster cells, . monkey cells infected with respiratory syncytial virus, and there was also substituted the appropriate rabbit-anti-respiratory syncytial serum.
Similar results can also be obtained using human cells infected in vivo with any of the foregoing viruses; the control sample of in vivo infect-ed cells, not incubated with specific antiserum, will display few or none of the bacteria bound to the cell surfaces while cells incubated with the appropriate antiserum will display large numbers of bound bacteria. In the

3~
'`.', same way, cells infec~ed in vivo with one virus and incubated only with an antibody against a different virus will display few or none of the bacteria . bound to the cell surfaces.

' ' - - 8 s .
.

Claims

The embodiments of the invention in which an exclusive property or privilege is claimed, are defined as follows:

1. Method of identifying a predetermined specific viral infection if present in a specimen of cells of an individual, which specimen of cells may be infected in vivo, which method comprises collecting from the individual a control sample of said specimen and at least one test sample of said specimen, both unknown as to the presence or absence of viral infection, incubating with each unknown test sample an antibody, against a known virus, capable of binding to the surface of a cell infected with said known virus to form a reaction mixture having free antibody and bound pairs consisting of antibody bound to said infected cell surfaces, incubating said control sample with non-immune globulin, and separating from said control sample non-immune globulin which is not bound to said cells, incubating said unknown control sample and each said unknown test sample with bacteria having the capability of binding with said antibody, separating from each sample any unbound bacteria, and determining the presence or absence of bacteria bound to the surfaces of cells of said control and test samples by visual examina-tion under a microscope.

2. Method as claimed in claim 1 including the steps, prior to incubating with bacteria, of incubating said control sample with non-immune globulin and separating from said control sample non-immune globulin which is not bound to said cells.

3. Method in accordance with the method of claim 1 wherein said control sample and test sample are each place d on a microscope slide and dried prior to said incubating steps.

4. Method as claimed in claim 1 or claim 3 in which one said anti-body is against a virus infecting the upper respiratory tract.

5. Method as claimed in claim 1 or claim 3 in which one said anti-body is against a virus selected from the group consisting of herpes, paramyxo, and vesicular stomatitis.

6. Method as claimed in claim 1 or claim 3 in which said antibody is against a virus selected from the group consisting of respiratory syneytial and para-influenza.

7. A kit for identifying viral infections in a specimen of cells infected in vivo which includes in operative relation with each other for use in identifying a viral infection in a specimen of cells infected in vivo a supply of bacteria capable of binding to an antibody against the infectious agent, a supply of at least one antibody against a known viral infection, and a supply of non-immune globulin.

8. A kit as claimed in claim 7 in which said bacteria comprise Staphylococcus aureus.

9. A kit as claimed in claim 7 or claim 8 in which one said anti-body is against a viral infection of the upper respiratory tract.

10. A kit as claimed in claim 7 or claim 8 in which one said antibody is against a virus selected from the group consisting of herpes, paramyxo, and vesicular stomatitis.

11. A kit as claimed in claim 7 or claim 8 in which one said anti-body is selected from the group consisting of respiratory syncytial and para-influenza.