WO1991011717A1

WO1991011717A1 - Rapid immunoblot diagnosis of acute mycoplasma pneumoniae

Info

Publication number: WO1991011717A1
Application number: PCT/CA1991/000037
Authority: WO
Inventors: Nevio Cimolai
Original assignee: The University Of British Columbia
Priority date: 1990-02-05
Filing date: 1991-02-05
Publication date: 1991-08-08
Also published as: AU7212091A

Abstract

An improved method for rapid diagnosis of the common etiologic agent of community acquired respiratory disease, Mycoplasma pneumoniae, utilizes an immunoblot analysis on ministrips prepared by transfer from SDS-PAGE of the major adhesin protein of low passaged and solubilized M. pneumoniae strain 185.

Description

RAPID IMMUNOBLOT DIAGNOSIS OF ACUTE MYCOPLASMA PNEUMONIAE

Field of the Invention

This invention relates generally to the field of enzyme immunodiagnosis . More particularly, it relates to a modified IgM immunoblotting assay system for the detection of acute Mycoplasma pneumoniae infection.

Background of the Invention

Mycoplasma pneumoniae is a common etiologic agent of community acquired respiratory disease. Laboratory diagnosis is based predominantly upon culture and complement fixation (CF) serology although IgM serology, direct antigen detection, and direct genetic probing have also been described.

Assay systems which require culturing are not generally desirable because of the problems inherent to medium preparation (i.e., growth inhibitors) and because of the retrospective value of positive cultures which may require up to several weeks for finalization. Diagnosis by CF is also often of retrospective value, especially since paired sera are commonly required. Furthermore, the purported cross-reactivity of the CF test glycolipid antigen with human tissue is also believed to result in nonspecific positive tests i.e., false positives. Antigen detection by immunological techniques has been attempted but a sensitive and rapid method is not widely available. A recently commercialized assay (Gen-Probe, San Diego, California) uses I 125-cDNA hybridization . The use of radioisotopes and the potential cost of performing such a test, however, may hamper widespread use in communities where the majority of M__;_ pneumoniae disease is treated.

IgM serology for M^_ pneumoniae infection has been previously reported and a variety of methods have been employed including complement fixation (after serum pretreatment) , indirect hemagglutination, indirect immuno- fluorescence, radioimmunoassay, indirect ELISA, and u-capture ELISA. None of these methods has gained wide- spread use although some have been commercialized.

Although the more recent versions of ELISA methodology appear to have reasonable specificity, a detectable IgM response may not be apparent early in the disease. Furthermore, older patients (>20 years) are often reported to lack an IgM response, possibly because of repeated infection, i.e., anamnestic rather than primary response.

Immunoblotting is another diagnostic and serologic tool which could potentially be useful since the immune response to a number of specific bacterial polypeptides may be assessed. Others have examined the IgG immunoblot response to M__;_ pneumoniae polypeptides and have documented a consistent fingerprint pattern of polypeptides that are recognized by M^ pneumoniae positive sera.

A polypeptide of approximate molecular weight of 160-170 kilodaltons, now recognized as the putative adhesin protein and the major immunogenic protein, is consistently recognized in convalescent sera. Others have also examined the IgG immunoblot response and their data concur. In addition, it has been confirmed that the acute immune response (IgM) consistently includes reactivity to the adhesin protein. In a small number of patients, serial examination of the IgM response after infection revealed immunoblot positivity by the third week of disease.

Published European patent application 0,254,384 published January 27, 1988, discloses a monoclonal antibody which binds to a protease sensitive, periodate insensitive epitope on Mycoplasma pneumonia membrane polypeptide. The polypeptide has an approximate molecular weight of 43 k. A method of using the antibody in order to carry out the diagnostic for detecting M__;_ pneumoniae in clinical specimens is disclosed.

Despite significant advances in biotechnology, the rapid diagnosis of __;_ pneumoniae infection by the laboratory is still problematic. Direct antigen detection and genetic probing studies have not yielded a diagnostic kit which could be transportable to small community laboratories who are likely to be at the forefront of com¬ munity diagnosis. The present invention is based on developing a practical alternative which meets the criteria of rapid performance, moderate technical requirement, and minimization of requisite laboratory equipment.

Summary of the Invention

The invention provides for a method for assaying for the presence of acute M^ pneumoniae infection, a mini- strip useful in the assay method and a method of diagnosing acute M__;_ pneumoniae infection in a human. The ministrip which is useful for the detection of M__;_ pneumoniae infection is comprised of a support base which has attached to its surface a membrane replica of an SDS-PAGE gel. The gel is prepared from low passaged, solubilized M. pneumoniae cells which contain the major adhesin protein of M. pneumoniae. The method of assaying for the presence of acute ^ pneumoniae infection comprises first contacting a serum or plasma to be tested with such a ministrip and then detecting the presence or absence of IgM reactive to the adhesin protein in the serum or plasma by determining the presence or absence of a binding of the IgM to the ministrip at the location of the major adhesin protein on the ministrip. It is possible to diagnose a patient as having acute M_ pneumoniae by using the ministrip in the assay of the invention and determining whether or not the patient's serum or plasma contain IgM reactive to the adhesin protein on the ministrip.

A primary object of the invention is to provide a ministrip which is useful in the detection of acute M^ pneumoniae infection which is comprised of a support base the surface of which is attached to a membrane replica of an SDS-PAGE gel prepared from low passaged, solubilized M. pneumoniae cells which contain the major adhesin protein of M. pneumoniae. Another object of the invention is to provide a method for assaying for the presence of acute M__;_ pneumoniae infection by contacting a serum or plasma to be tested with such a ministrip and then detecting the presence or absence of IgM reactive to the adhesin protein by determining the presence or absence of a binding of the IgM to the ministrip at the location of the major adhesin protein on the ministrip.

Yet another important object of the invention is to provide a method for diagnosing acute M__;_ pneumoniae in human patients suspected of having such an infection by contacting their serum or plasma with the ministrip and following the assay method of the invention.

An advantage of the present invention is that it provides for a rapid assay technique for detecting acute M^ pneumoniae infection.

A feature of the present invention is that it can be performed relatively easily and requires relatively moderate technical expertise.

Yet another feature of the present invention is that it requires a minimum amount of laboratory equipment. Other advantages of the present invention include its low cost, efficiency, accuracy and specificity.

Another feature of the present invention is that it makes it possible to readily localize a large amount of antigen without prior purification of the solubilized M. pneumoniae cells. Yet another feature of the present invention is that the assay means allows for visualization of reactivity to a relatively specific banding site on the ministrips.

Still other advantages and features of the present invention include a ministrip having a relatively long shelf life which can be economically produced, easily transported and readily used at remote locations .

These and other objects, advantages and features of the present invention will become apparent to those persons skilled in the art upon reading the details of the structure, production and usage as more fully set forth below.

Detailed Description of the Preferred Embodiments Before the present ministrips, assay methods and processes for making and using such are described, it is to be understood that this invention is not limited to the particular enzyme labels or assay procedural steps described as such labels and steps may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting since the scope of the present invention will be limited only by the appended claims. it must be noted that as used in the specification and appended claims, the singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a test serum" includes mixtures of serums, reference to "an IgM" includes mixtures of such IgM of the type described, reference to "the assay method" or "the assay method step" includes various combinations of such methods and steps of the type described, and so forth.

A ministrip prepared in accordance with the procedures of this invention is a critical part of the invention and necessary in order to carry out the method of assaying and for diagnosing for the presence of acute M. pneumoniae infection. The ministrip is comprised of a support base which has certain antigens attached to its surface. More particularly, the surface of the support base is a transfer membrane replica of an SDS-PAGE gel. The SDS-PAGE gel has been prepared from low passaged, solubilized M__;_ pneumoniae cells which contain the major adhesin protein of pneumoniae. A polypeptide having a molecular weight of about 160-170 -kilodaltons is putatively the adhesin protein and the major immunogenic protein associated with M^ pneumoniae. The support base maybe comprised of a variety of materials which preferably include nitrocellulose, nylon, and charged-derivatized membranes . The assay method of the invention makes it pos¬ sible to assay for the presence of acute M^_ pneumoniae infection. The method is carried out by contacting a serum or plasma to be tested with a ministrip of the type described above. After contacting the serum or plasma to the ministrip one detects the presence or absence of IgM reactive to the adhesin protein in the serum or plasma by determining the presence or absence of a binding of these IgM to the ministrip at the location of the major adhesin protein on the ministrip. The IgM reactive to the adhesion protein will be present in the serum or plasma (having been generated by the subject's immune system) if the subject from which the serum was taken is infected with M. pneumoniae. The presence of the adhesion protein and IgM reactive to such proteins have been found in the sera of those who have been confirmed by other methods to have been infected with M^ pneumoniae. If the IgM reactive to the adhesin protein binds to the ministrip, this bound IgM can be detected by a variety of techniques such as by using a heterologous labeled antihuman antibody. The serum or plasma is preferably obtained from a human patient suspected of having acute M^ pneumoniae. The heterologous labeled antihuman antibody is preferably an antihuman antibody derived from an animal such as a goat. For example, labeled species of antihuman IgM and labeled species of antihuman IgG have been found to be useful. It is preferable to utilize a goat-derived antihuman antibody which is antihuman IgG. The label is preferably an enzyme label whose activity is easily monitored. Preferred labels include the enzymes alkaline phosphatase and horseradish peroxidase. Horseradish peroxidase can be coupled to goat antihuman IgM. The perpxidase enzyme will convert added hydrogen peroxide to oxygen free radicals. These radicals in turn react with a chromogenic precursor (3,3'- diaminobenzidine or 4-chloro-l-napthol ) to form an insoluble colored precipitate. However, other preferred labels include those having enzyme activity which can be monitored by color changes .

Another approach for attaching a label involves the use of a sandwich method. With this method after any antibody in the serum has attached to the ministrip a cognate multivalent antigen is applied. This antigen will attach to any antibody on the ministrip. Next a labeled antibody reactive to the multivalent antigen is added and one can observe for the presence of the label . Thus the multivalent antigen is sandwiched between two antibodies . In order to provide a more detailed description of the invention an explanation regarding SDS-gels should be given. Firstly, SDS-gels are commonly known as PAGE-SDS gels (polyacrylamide gel electrophoresis in sodium dodecyl sulfate) . The PAGE-SDS gel techniques are utilized for characterizing a variety of different types of proteins. The procedure involves denaturing the proteins with the detergent sodium dodecyl sulfate (i.e., SDS) . The method provides for high-resolution because it can describe the bands in the gel not just in terms of their relative mobility to each other, but also in terms of their molecular size. Dodecyl sulfate binds strongly to proteins. Accordingly, only 0.1 percent of dodecyl sulfate is suf¬ ficient to saturate a polypeptide chain with approximately one detergent molecule per two amino acid residues . Each dodecyl sulfate carries a negative charge, so a typical polypeptide of molecular weight of 40,000 acquires about 180 negative charges. This is far in excess of any net charge that might exist (at neutral pH) on the polypeptide chain originally. Consequently, the charge/size ratio is virtually identical for all proteins, and separation can occur only as a result of the molecular sieving through the pores of the gel.

Despite the fact that the potential of separating proteins of identical size is not possible with PAGE-SDS _C- gel systems, the system does appear to give the sharpest overall resolution and cleanest zones of any method for separating proteins. Further, by comparison with a mixture of standard polypeptides of known molecular weight, whole gels can be calibrated in terms of mobility against size. _Q A linear plot over a substantial range can be obtained if mobility is plotted against LOG (molecular weight) .

In order to prepare the SDS-gels used in connec¬ tion with the present invention, it is first necessary to obtain M__;_ pneumoniae whole cells . These cells are then ₅ solubilized in a sample buffer. The buffer can contain a variety of components known to those skilled in the art but would typically contain a surfactant, glycerol, sodium dodecyl sulfate and betamercaptoethanol. The whole cells are boiled in the buffer solution for about 5 minutes in ₀ order to sσlubilize the cells. A small amount of whole cell protein obtained from the solubilized cells is applied to a SDS-PA gel. After putting the protein through the preliminary SDS-PAGE methodology (known to those skilled in the art) electrophoresis is carried out. After completion ₅ of the electrophoresis, the proteins which have been separated out in the gel are transferred to a suitable substrate such as a nitrocellulose membrane. Successful transfer of the proteins from the gel to the nitrocellulose can be confirmed by techniques known to those skilled in the art such as utilizing an Amido black protein stain. After the proteins have been transferred to the substrate such as the nitrocellulose membrane, it is necessary to localize the position of the major adhesin protein on the substrate. This is preferably done by al¬ lowing for a binding reaction to occur with an alkaline phosphatase-conjugated affinity-purified goat antihuman IgG. After the precise location of the major adhesin protein on the ministrips is known, identical strips can be readily produced utilized to test serum or plasma for the presence of IgM having a specific affinity for the adhesin protein.

The most critical material necessary to carry out the present invention is the ministrip of the invention. The ministrip comprised of a support base which has antigens attached to its surface which are immunoreactive with antibodies in the serum or plasma being tested if the subject from which the serum or plasma was taken is infected with M^ pneumoniae. More specifically, the ministrips are comprised of a support base the surface of which is attached to a transfer membrane replica of an SDS- PAGE gel,prepared from low passaged, solubilized M. pneumoniae cells which contain the major adhesin protein of M. pneumoniae.

In a preferred embodiment of the invention an assay kit which is useful in the detection of acute M_^ pneumoniae infection is provided. The kit includes at least two components in the form of the ministrip and a labeled animal antihuman IgM. The ministrip is of the type described above and the labeled animal antihuman IgM is preferably a labeled goat antihuman IgM the label is preferably an enzyme label and useful labels include horseradish peroxidase and more preferably alkaline- phosphatase which is conjugated to goat antihuman IgM. The kit also preferably includes a chromogenic precursor. Such a chromogenic precursor will react with the enzyme label in order to provide for an insoluble color precipitate. Useful chromogenic precursors include

3, 3 '-diaminobenzidine or 4-chloro-l-napthol.

In order to carry out the assaying method of the invention, the kit which contains the ministrip, the labeled animal antihuman IgM and the chromogenic precursor, can be utilized. First, a biological fluid is obtained from the patient or subject to be tested which fluid will contain antibodies which had been generated by the patient, e.g. serum or plasma. The serum or plasma obtained is placed in contact with the ministrip and incubated. During the incubation, antibodies which are in the plasma or serum will attach to antigens on the surface of the ministrip. After the attachment is complete, the ministrip is washed in order to remove any antibodies or other materials which are not attached to antigens on the ministrip. The labeled animal antihuman IgM is then brought into contact with the ministrip. If the serum contacted to the ministrip did contain antibodies, then the labeled animal antihuman IgM will attach to the antibodies on the ministrip. The strip is again washed to remove any labeled antihuman IgM which did not attach to antibodies already attached to the ministrip. After the washing is complete, a chromogenic precursor is added. If labeled antibodies remain attached to the strip then the peroxidase enzyme will convert added hydrogen peroxide to oxygen free radicals . These free radicals in turn react with the chromogenic precursor to form an insoluble colored precipitate. The color change is noted and a diagnosis of acute M__;_ pneumoniae infection can be made.

The following examples are provided so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make the ministrips and how to carry out the assays of the invention and are not intended to limit the scope of what the inventors regard as their invention. Efforts have been made to ensure accuracy with respect to numbers used (e.g. amounts temperature etc . ) but some experimental errors and deviation should be accounted for. Unless indicated otherwise, parts or parts by weight, temperature is in degrees Centigrade and pressure is at or near atmospheric.

Examples

Materials and Methods

Bacteria Mycoplasma pneumoniae strain M185 was used as the antigen for immunoblotting. This strain was previously isolated from a symptomatic patient with respiratory disease. The isolate was subcloned previously but used in low passage (<5). It was confirmed as __;_ pneumoniae by both agar epi-immunofluorescence and demonstration of the adhesin protein at the molecular weight range of a 160-17- 0 kilodaltons in sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). The bacterium was grown in broth and harvested using standard procedures known to those skilled in the art.

Sera

One hundred eighty-eight (188) sera were obtained form the British Columbia Centre for Disease Control which included 116 patients from 1987-1988. Seventy-two (72) patients had paired sera submitted, whereas 44 patients had only a single serum submitted.

M. pneumoniae infection was defined serologically by the demonstration of a single CF titre (_> 1/128) or a fourfold or greater seroconversion between two sera. Control positive sera were obtained from patients experiencing active pulmonary infection where the diagnosis was achieved by CF, IgG and IgM immunoblotting, and M_;_ pneumoniae isolation from respiratory specimens . Control negative sera were obtained form patients with nonrespiratory diseases who did not have either positive CF serology (< 1/ 8) or positive IgM i munoblots to the adhesin protein.

CF Test

This serology was performed using standard techniques by the Virus Serology Laboratory, B.C. 's Centre for Disease Control. Glycolipid antigen (Behring Diagnostics., Hoechst) was employed.

Electrophoresis and Transblotting Mycoplasma pneumoniae whole cells were solubilized in sample buffer (0.25 M Tris, 50% glycerol, 10% SDS, 5% betamercaptoethanol) and boiled for 5 minutes. A total of 2 milligrams whole cell protein equivalent (Bio-Rad Protein Assay, Bio-Rad Laboratories, Richmond, California) was applied to a SDS-PA gel measuring 1.0 mm by 16 cm by 16 cm. SDS-PAGE was carried out essentially by the method of Laemmli and included a 3% stacking gel and 10% running gel. After completion of electrophoresis, proteins were transferred to a nitrocellulose membrane (0.45 micron, pore size; Bio-Rad) in an electrophoresis unit (Transphor, Hoefer Scientific, San Francisco, California) for 30- minutes at 1.5 A (direct current). Successful transfer of protein from the gel to the nitrocellulose was confirmed by total protein stain using Amido Black. Other methods known to those skilled in the art can be used to confirm the transfer and/or specifically characterize the proteins on the membrane at any given location.

IgM Immunoblotting Localization of the major adhesin protein was determined by an IgG immunoblot as follows. A strip of nitro-cellulose paper containing the separated M. pneumoniae protein preparation was blocked overnight at 4 C in PBS-1% skim milk (pH 7.2). After washing 3 times in PBS-0.05% Tween 20, the strip was incubated with a 1:100 dilution (PBS-1% skim milk) of serum (from a previously determined positive control) for one hour at 37 C. After further washing with PBS-0.05% Tween 20 (3 times), the strip was incubated with a 1:1,000 dilution (PBS-1% skim milk) of alkaline phosphatase-conjugated affinity-purified goat antihuman IgG (Bio-Rad) . After a final wash, the strip was reacted with 5-bromo-4-chloro-3-indolyl phosphate and 1-nitro blue tetrazoliu chloride substrates (AP Colour Development Reagent, Bio-Rad). Once the adhesin protein band was localized, the remainder of the nitrocellulose paper was cut vertically into approximately 1/8 by 1-1/4 inch "ministrips" with equal space above and below the critical band. Ministrips were stored dry at room temperature and used up to four weeks after preparation. On the day of performing the assay the ministrips were rehydrated and blocked in PBS-1% skim milk for 1/2 hour at 37 C. The immunoblot reaction was carried out essentially as for the longer strips except that human IgM reactivity was assayed. A 1:2,000 dilution of alkaline-phosphatase conjugated goat antihuman IgM (Jackson Immunoresearch, Pennsylvania) was utilized as the second antibody.

Positive serum controls were employed to localize the desired immunoreactive band on each subsequent transblot.

Rheumatoid Factor Testing and IgG Absorption Sera which were shown to be positive for IgM by the ministrip method were assayed for rheumatoid factor by a latex agglutination method (Rapi Tex RF, Behring) . The manufacturer's methods were followed. Subsequent sera which were positive for rheumatoid factor were treated by IgG absorption (RF Absorbent, Behring). After IgG absorp¬ tion, the patient serum was diluted to a final 1:100 concentration and the IgM immunoblot was re-assayed by the ministrip method.

Results A positive IgM immunoblot in this assay is vis¬ ualized as a band at the level previously demonstrated to be compatible with localization of adhesin protein. The test requires approximately 3-4 hours to perform. Variation in intensity between positive sera are appreciated and the de.gree of intensity often correlates with the absolute CF titre. Measurement of the reactive site in positive control strips facilitates localization in weakly positive reactions and furthermore facilitates ' recognition since a few sera may react with other unknown antigens above or below the adhesin protein site.

In this predominantly retrospective serological study, there was a strong correlation of positive IgM immunoblot with a diagnosis of M__;_ pneumoniae disease based on the CF serology criteria previously described (Figure 2). Of the total 188 sera from 116 patients, 161 (85.6%) were positive by the IgM immunoblot method. 2/133 sera (1.5%) with CF titres j> to 1/128 were negative in contrast to 25/55 (45.4%) sera with CF titres < 1/128. Where two serum specimens were submitted, the acute serum was negative in 20/72 (27.7%) patients. Only 5/116 patients (4.3%) who met the diagnostic criteria based on CF serology were negative by the IgM immunoblot for either the single serum submitted or both sera in the pair.

In analyzing the results by patient age, there was a similar distribution of IgM positive immunoblots for sera below and above the 1/128 dilution. The incidence of negative IgM blots for patients younger than 20 years or older than 20 years with titres < 1/128 dilution was 44.4% and 46.4% respectively. For sera with titres _> 1/128, the incidence of negative tests for patients below and above 20 years was 1.5% and 1.8%, respectively. Rheumatoid factor screening was positive for 14 patients with IgM positive sera. These sera remained positive by IgM immunoblot after IgG absorption and there was no change visually in the quality of intensity of banding except for one serum. Although the overall male: female ratio of patients in this study was 1:1, the patients with positive rheumatoid factor had a 2:5 male to female ratio. The age range included 5-67 yr. Three pairs of sera were positive for rheumatoid factor in the convalescent specimen only.

Of the 116 patients, 17 patients were admitted to a pediatric. hospital (British Columbia Children's Hospital) with a respiratory disease secondary to M__;_ pneumoniae based on culture or serologic diagnostic criteria. The IgM blot was positive in 16/17 patients on the basis of the first serum specimen submitted. These initial sera were submitted in range of 7 to 26 days (mean 14.5 days) after onset of the disease and CF titres varied form <l/8 to 1:2,048. The negative serum was an acute specimen of a pair where the acute and convalescent CF titres were <l/8 and 1/256 respectively. The convalescent specimen from this patient was positive.

The instant invention is shown and described herein in what is considered to be the most practical, and the preferred embodiments. It is recognized, however, that departures may be made therefrom which are within the scope of the invention, and that obvious modifications will occur to one skilled in the art upon reading this disclosure.

Claims

1. A method of assaying for the presence of acute Mycoplasma pneumoniae infection; comprising the steps of: contacting serum or plasma to be tested with a ministrip which is a transfer membrane replica of an SDS- PAGE gel, wherein the SDS-PAGE gel has been prepared from low passaged, solubilized M_ pneumoniae cells which contain the major adhesin protein of M^ pneumoniae; and detecting the presence or absence of IgM reactive to the adhesin protein in the serum or plasma by determining the presence or absence of binding of the IgM to the ministrip at the location of the major adhesin protein on the ministrip.

2. The method of claim 1 wherein the IgM reactive to the adhesin protein is detected by attaching thereto a heterologous labeled species of antibody.

3. The method of claim 2 wherein the serum or plasma is obtained from a human.

4. The method of claim 3 wherein the heterolo- gous antibody is a labeled goat antihuman antibody, selected from the group consisting of antihuman IgM and antihuman IgG.

5. The method of claim 1 wherein the ministrips are prepared by conducting SDS-PAGE on solubilized

Mycoplasma pneumoniae cells to provide an SDS-PAGE gel which gel is transferred to a supported selected from the group consisting of nitrocellulose, nylon, and a charge- derivatized membrane.

6. The method as claimed in claim 1, wherein the cells are solubilized in,0.25 M Tris, 50% glycerol, 10% SDS, 5% betamercaptoethanol by boiling for 5 minutes.

7. A method of assaying for the presence of antibodies to Mycoplasma pneumoniae infection, comprising the steps of: contacting a biological fluid possibly containing antibodies to acute M^ pneumoniae infection with a ministrip which is a transfer membrane replica of an SDS- PAGE gel which has been prepared from low passaged, solubilized M^ pneumoniae cells which contain the major adhesin protein of M__;_ pneumoniae; incubating the ministrip with the biological fluid at a temperature of about 37°C; washing the ministrip in a buffer solution to remove substantially all of the biological fluid not attached to the ministrip; incubating the ministrip with a heterologous labeled animal derived antihuman antibody; washing the ministrip to remove substantially all of the labeled antihuman antibody; and contacting the ministrip with a chromogenic precursor and observing for the presence or absence of a chromogenic reaction and thereby determining the presence of absence of antibodies to M^ pneumoniae in the biological fluid.

8. A ministrip useful in the detection of acute Mycoplasma pneumoniae infection, comprising: a support the surface of which is attached to; a transfer membrane replica of an SDS-PAGE gel prepared from low passaged, solubilized M_^ pneumoniae cells which contain the major adhesin protein of M^_ pneumoniae■

9. The ministrip as claimed in claim 8, wherein the support is comprised of nitrocellulose.

10. The ministrip as claimed in claim 8, wherein the support is comprised of nylon.

11. The ministrip as claimed in claim 8, wherein the support is comprised of a charge-derivatized membrane.

12. A method of diagnosing acute Mycoplasma pneumoniae infection in a human suspected of having the infection, comprising the steps of: contacting serum or plasma from the human with a ministrip which is a transfer membrane replica of an SDS- gel prepared from low passaged, solubilized M^ pneumoniae cells which contain the major adhesin protein of M. pneumoniae; and detecting the presence or absence of IgM reactive to the major adhesin protein of M^ pneumoniae in the serum or plasma by determining the presence or absence of binding of these IgM to the ministrip at the location of the major adhesin protein on the ministrip.

13. The method as claimed in claim 12, wherein the Mycoplasma pneumoniae cells are strain Ml85 and the IgM reactive to the adhesin protein is detected by attaching thereto a heterologous labeled antihuman antibody.

14. The method as claimed in claim 12 , wherein the major adhesin protein has a molecular weight in the range of about 160-170 kilodaltons and the heterologous antibody selected from the group consisting of labeled goat antihuman IgG and labeled goat antihuman IgM.

15. The method as claimed in claim 13, wherein the label is an enzyme whose activity is easily monitored.

16. The method as claimed in claim 15, wherein the enzyme is alkaline phosphatase and further comprises adding 5-bromo-4-chloro-3-indolyl phosphate and 1-nitro blue tetrazolium chloride.

17. The method as claimed in claim 15, wherein the activity of the enzyme is monitored by color change.

18. An assay kit useful in the detection of acute Mycoplasma pneumoniae infection in a human comprising: a ministrip comprised of a support base the sur¬ face of which is attached to a transfer membrane replica of an SDS-PAGE gel prepared from low passaged, solubilized M. pneumoniae cells which contain the major adhesin protein of M. pneumoniae; and a heterologous labeled animal derived antihuman antibody.

19. The assay kit as claimed in claim 18, wherein the labeled antihuman antibody is selected from the group consisting of a labeled antihuman IgM and a labeled antihuman IgG.

20. The assay kit as claimed in claim 19, wherein the labeled antihuman antibody is an alkaline- phosphatase conjugated goat antihuman IgM or IgG.

21. The assay kit as claimed in claim 18, further comprising a chromogenic precursor.

22. The assay kit as claimed in claim 21, wherein the chromogenic precursor is selected from the group consisting of 3 , 3 '-diaminobenzidine or 4-chloro-l- napthol .